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PHYSIOLOQICAL REVIEWS Vol. 54, No. 2, April 1974 kinted in I7.S.A. Chemical Nature of Synaptic Transmission in Vertebrates K. KRNJEVIC: Department of Research in Anaesthesia, McGill University, Montreal, 418 419 419 422 423 423 424 424 426 430 432 432 432 433 435 435 439 441 443 444 445 445 448 448 457 460 460 463 467 467 468 468 471 472 472 479 480 483 483 483 483 488 488 488 Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 ............................................... I. General Introduction. ...................................................... ... Historical. ............................. B. Identification of chemical transmitters. ...................................................... I I. Acetylcholine ................................................... ... Introduction. ................. B. Acetylcholine in skeletal neuromuscular transmission. 1. How does ACh act?. .......................................... ..................................... 2. Kelease of ACh in muscle. ..................... C. Acetylcholine in transmission to smooth muscle. D. Inhibitory action of ACh on heart .................................. ............................ E. Acetylcholine in ganglionic transmission. ..................................................... 1. Release. 2. Actions of ACh on ganglion cells. ............................... ....................... F. .4cetylcholine in central nervous transmission. 1. Excitatory action of ACh. ...................................... 2. Central inhibitory actions of ACh ................................. G. Other evidence indicating cholinergic transmission in CNS ............ .......................................... H. Acetylcholine receptors. ............................. 1. Cation movements and L4Ch system. III. Amino -4cids. ...................................................... ....................................................... A. General. .......................................... B. Inhibitory amino acids. 1. r-Aminobutyric acid. .......................................... ..................................................... 2. Glycine. .......................................... C. Excitatory amino acids. 1. Excitatory actions of dicarboxylic amino acids. .................... ........................................ 2. Other relevant evidence .................................................... IV. Catecholamines. ................................................... *4. Introduction. .......................... B. Catecholamines in peripheral transmission. ........................ 1. Norepinephrine actions on smooth muscle. .............................. 2. Catecholamine actions on ganglia. .............................. C. Catecholamines in CNS transmission. ............................................ 1. Depressant actions. 2. Excitatory actions of catecholamines in CNS ...................... 3. Function of catecholamines in CNS. ............................. Monoamines ................................................. V. Other .................................. .4. 5-Hydroxytryptamine (serotonin) ................................................. 1. Introduction. ................................. 2. Is 5-HT a central transmitter?. ............................................ B. Imidazole derivatives. ................................................... 1. Histamine. ....................... 2. Imidazole acetic acid and other derivatives. Canada April VI. VII. SYNAPTIC Some Other Putative Transmitters. ................................... A. ,4denosine derivatives (adenosine-5’-triphosphate) .................... 1. In CNS ...................................................... 2. At periphery. ................................................ B. Ergothioneine ................................................... C. Polypeptides.................................................... 1. Substance P. ................................................. 2. Other polypeptides............................................ 3. Antidiuretic hormone. ......................................... Some Special Aspects of Chemical Transmission. ........................ -1. Chemical transmission in retina. ................................... B. Chemical transmission at sensory endings. .......................... 1. Carotid body chemoreceptors. .................................. 2. Electrical receptors. ........................................... .................................. C. Presynaptic actions of transmitters 1. Acetylcholine. ................................................ 2. Presynaptic action of neurotransmitter amino acids. ................ ......................... 3. Other agents acting presynaptically. D. Denervation supersensitivity. ...................................... 1. Muscle fibers. ................................................ 2. Denervation supersensitivity of nerve cells. ........................ E. Role of glial cells. ............................................... General Consideration about Synaptic Transmission. .................... A. Origin and nature of chemical transmitters. ......................... ........................................... B. Electrical transmission. C. Chemical differentiation in nervous system. ......................... GENERAL 419 TRANSMISSION : ... 489 489 489 489 491 491 491 491 491 492 492 494 494 495 495 495 496 497 497 497 498 499 500 500 503 505 INTRODUCTION A. Historical That nerves may exert their effects by secreting specific agents is a very ancient belief. Until the 18th century, nerves were thought to produce movements spirits” ; by conveying from the brain to muscles a special “nerve fluid” or “animal according to a prevalent opinion, these “animal spirits” were distilled from blood by the heat generated in the heart (cf. 334). When animal electricity was discovered and nerves were shown to be electrically excitable, it was natural to suspect that the “nerve fluid” might be identical with electricity (53 1, 840, 919). After all, the discharges of electric organs were indistinguishable from electricity, and even muscles generated electrical currents that could excite nerve fibers (357, 358, 877, 896). Thus in 1863 Krause (725) placed much emphasis on the resemblance between the electroplaque and the muscle end plate-where he correctly observed the nerve ending outside the muscle fiber membrane-and suggested that the muscle fiber was excited by an electric discharge analogous to that of the electric organ. This idea was accepted by Hermann (564) and Kiihne (776), and later by many other electrophysiologists (cf. 83l), reaching a peak of popularity in the middle 1930’s (368). On the other hand, du Bois-Reymond, who discovered the Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 VIII. I. VERTEBKATE I974 420 K. KRNJEVIC Volume 54 Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 action current (or “negative variation”) of nerves, critically examined this hypothesis in a long and somewhat obscure article in 1874 (359), which is often cited as the first to propose that the nerve ending excites muscle by secreting a chemical transmitter (254, 295, 373, 776, 1061). But this interpretation is misleading: du Bois-Reymond believed that the nerve terminal penetrates beneath the muscle membrane, and he was really discussing the mechanism of intracellular transmission between the end plate and the contractile substance of the muscle. In his opinion, the nerve impulse was a self-propagating “molecular reaction,” of which the “negative variation” was probably only an external manifestation; at the end plate this “molecular reaction” would spread directly to the immediately contiguous contractile substance. However, he added that if the nerve ending remained outside the sarcolemma, the mechanism of neuromuscular transmission could only be electrical. This article thus can hardly be seriously held as proposing the chemical hypothesis in the modern sense. It is significant that its complex argument has also been quoted in support of the electrical hypothesis (564, 83 1). Junctional transmission could not become the subject of meaningful investigations, or even speculation, before general acceptance of the neuron theory (cf. 449, established the existence of a protoplasmic discontinuity 1021, 1226) securely across which the neural signal must be carried. At the turn of the century, there was little discussion of junctional transmission; it was simply and noncommittally accepted as an obscure physical process (cf. 450, 1073, 1086). Thus, although Elliott first proposed in 1904 (395) that adrenaline (epinephrine), the active principle in extracts of the suprarenal medulla (952, 1150), might be the chemical stimulant released by sympathetic nerve endings, and shortly after Dixon (343) presented some evidence that vagal inhibition of the heart is mediated by a specific chemical and suggested more generally that “excitation of a nerve induces the local liberation of a hormone which causes specific activity by combination with some constituent of the end-organ, muscle or gland” (344), the new hypothesis was almost completely ignored (cf. 77, 292, 532), curiously enough even by Elliott himself in his subsequent publications (cf. 396). It may have been kept in the background by Langley’s (797) strong advocacy of the idea that specific “receptive substances” are situated at the junction between nerve and effector organ, which are activated by the arrival of impulses at the nerve ending, and whose characteristics determine the quality of the effector response. Langley’s scheme required neither chemical transmission nor specific transmitters, being in some respects the converse of what has been called ‘CDale’s principle” (371, 373). Although in 1912 Weiland (1238) had obtained evidence that the isolated gut releases an agent that causes intestinal muscle to contract [this was later identified by le Heux (8 10) as choline] chemical transmission was really launched as a widely accepted working hypothesis and the subject of systematic investigations by Loewi’s discovery in 1921 of “Vagustoff’ in fluid perfusing the frog’s heart (835). The active substance was shown to be acetylcholine (ACh) (836), which was already known to have a particularly powerful biological action (293, 6 16). Acetylcholine was then found in extracts of the spleen (296) and in fluid perfused through sympathetic ganglia (426, 429, 699, 847), and good evidence was obtained that April 1974 VERTEBRATE SYNAPTIC TRANSMISSION 421 Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 ACh is the transmitter at the junction between motor nerves and skeletal muscle (172, 297). Later studies on the sympathetic postganglionic transmitter essentially confirmed Elliott’s original hypothesis, but the active agent released by adrenergic nerve endings proved to be norepinephrine rather than epinephrine (198, 1057, 1215, 1216). An electrophysiologist was one of the first authors to suggest that central synapses may operate by chemical transmission [ 10 yr before the publication of Dale’s well-known article (294)] : in a review of studies on inhibition written shortly after Loewi’s discovery, Adrian (6) in 1924 discussed the possibility that inhibition in the central nervous system (CNS) might be caused by an inhibitory substance; he also noted prophetically that the increase in cardiac demarcation current observed during inhibition “might well be due to an altered membrane permeability of the surface membrane to certain ions.” However, Adrian clearly favored a mechanism of inhibition by a Wedensky type of block, partly because of his own and Keith Lucas’ observations on decremental conduction in nerves. Most electrophysiologists were even more reluctant to believe that a chemical process of transmission could explain the rapid transfer of signals in the CNS (368, 369, 404, 460, 475, 842). A radical change in attitude came only about 20 yr ago, after the introduction of intracellular microelectrodes in studies of synaptic transmission in muscle (424), the CNS (167, 1271), sympathetic ganglia (938), and intestinal smooth muscle ( 176). The main considerations that led Eccles (370) to change his views and adopt the chemical hypothesis are essentially summarized by these two questions: How can a minute nerve ending generate enough current to excite the much larger postsynaptic cell? What purely electrical mechanism can account really satisfactorily for postsynaptic inhibition? Without some evidence as to the nature of the postulated transmitters, a strong advocacy of the chemical hypothesis of central synaptic transmission may have seemed somewhat arbitrary. But it was soon vindicated by the discovery that certain spinal cells innervated by recurrent branches of motor axons are strongly excited by ACh and that the synaptic activation of these cells is enhanced by anticholinesterases and blocked by ACh antagonists (375, 376). On the other hand, it was clear that ACh could not be the main excitatory or inhibitory transmitter acting on motoneurons, though little further progress was possible without more satisfactory methods of testing active substances. The introduction of the microiontophoretic technique (325, 925) in studies of central neurons (268-269) was therefore an advance of considerable significance, since it now became possible to examine the effects of even very brief applications of active chemicals to individual cells, in situ. However, even as recently as 1965, one of the principal experts in this field was quoted as saying that the main evidence for chemical transmission in the CNS was morphological ( 105 1). But subsequent shifts of opinion have led to a wide acceptance of the idea that excitatory and inhibitory amino acids as well as the better known peripheral transmitters-ACh and catecholamines -are probably transmitters at various central synapses, although there is still only suggestive evidence available for most pathways. The recent great advances in this field thus have resulted from the extensive 422 K. KRNJEVI(? Volume 54 use of microelectrode techniques (both intra- and extracellular) as well as from the enormous development and refinement of biochemical and histochemical techniques permitting ever more precise investigations on the cellular and subcellular localization of possible transmitters, their metabolism and liberation, and the identification and properties of essential enzymes or receptors (46, 80, 113, 202, 229, 291, 403, 464, 513, 549, 582, 621, 661, 710, 717, 902, 1036, 1211, 1264). They have been greatly helped by the parallel development of studies on synaptic transmission in invertebrates (446, 48 1, 1160). of Chemical Transmitters The accent in this review is principally on the different kinds of transmitters in vertebrates and the variety of mechanisms by which they operate. No attempt is made to analyze exhaustively the transmitter mechanisms of all the known central and peripheral junctions. The criteria by which transmitters are identified have often been listed and discussed in detail: for some recent sensible views on this subject, the reader is referred to the articles or monographs by Werman (1250), McLennan (890), and Phillis (983). To prove that a substance is a transmitter or the transmitter at a particular junction requires the demonstration that its action on the postsynaptic cell is in every respect identical with the synaptic action and that it is released in adequate amounts by activity of the presynaptic nerve endings. Such a complete proof is not available for any postulated transmitter, not even at the neuromuscular junction (in vertebrates or invertebrates), which approaches most closely to this ideal. Although it is usually assumed that only one transmitter is released at a given junction, it is conceivable that two or more substances having significantly different actions may be released, in which case the criterion of identity of action (1250) could not be easily applied. In practice any substance that is a normal constituent of nervous tissue and has a strong excitatory or inhibitory action on nerve or muscle cells is potentially a transmitter: the probability that it is a transmitter increases with the amount of supporting information about the characteristics of its action, its metabolism and turnover in the tissue, its liberation during activity, and the possibility of blocking synaptic transmission by inactivating postsynaptic receptors with either an excess of the supposed transmitter or some more or less specific antagonist. The degree of general acceptance of a newly postulated transmitter depends not only on the amount and quality of the available information, but also on whether it conforms to current views about the characteristics of transmitters. Excessive emphasis tends to be given to certain arguments that later turn out to be of little or no significance. For example, many opponents of the were convinced that a chemical process could never be fast enough synaptic transmission because of the slowness of diffusion (368). based on a serious misunderstanding of the kinetics of diffusion distances that was not entirely excusable since this topic had been chemical theory to account for This view was over very short very lucidly dis- Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 B. Zdent$cation April 1974 VERTEBRATE SYNAPTIC TRANSMISSION 423 II. -4CETYLCHOLINE A. jntroduclion Ever since Dale’s (293) description of the effects produced by choline esters on various preparations, it has been customary to classify the actions of ACh as nicotinelike on the one hand, or muscarinelike on the other. Nicotinic actions are typically quick in onset and short lasting; they are blocked by an excess of nicotine or by curare and curarelike agents. By contrast, muscarinic actions tend to be slow in onset and prolonged; they are blocked by atropine and related compounds. In general, the parasympathetic system acts on its effector organs by muscarinic transmission, whereas nicotinic actions are seen characteristically at the skeletal neuromuscular junction and in autonomic ganglia; but ACh may act in both ways on the same cell, as in sympathetic ganglia. This classification has also proved useful for distinguishing cholinergic actions in the CNS: however, as seen below, many neurons show mixed effects that are not compatible with such a simple scheme. Systematic studies of nicotinic and muscarinic agents (cf. 14, 65, 973, 1085) suggest that the two classes of substances interact with distinct membrane receptors, which are activated by different portions of the ACh molecule. The nicotine receptors apparently react with the carboxyl side of ACh and the muscarine receptors with its methyl side (2 16). The effects produced by the activated receptor may be either excitatory or inhibitory. The character of the receptor seems to depend principally on its situation (the type of cell on which it is found), but more than one type of receptor can be present on the same cell: for example, Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 cussed by A. V. Hill in 1928 (472). S imilar misconceptions are no doubt obscuring our present thinking. Fortunately throughout the animal kigdom nervous tissue appears to contain relatively few substances that have powerful, rapid, and reversible actions on nerve cells. And since most nerve cells are not equally sensitive to all naturally occurring excitatory or inhibitory agents, the task of identifying possible transmitters is not so formidable as it might have been if every species or class had its own variety of neurotransmitters. In fact, certain substances seem to be used as transmitters through all the phyla where chemical transmission has been demonstrated with any certainty (446,48 1) : ACh, dicarboxylic amino acids, short-chain omega-amino acids, dopamine, and 5-hydroxytryptamine (5-HT) are the putative transmitters in animals as varied as vertebrates, molluscs, arthropods, and annelids. But the way in which they are used at different sites or in different phyla are strikingly and unpredictably varied. Hence, although it is instructive to know that a certain agent is probably the transmitter at a given junction, until there is a better understanding of the principles underlying the chemical specificity of various parts of the nervous system of different animals, one cannot argue by analogy that the same agent is necessarily the transmitter at other junctions in the same species or in a corresponding system of another phylum. 424 K. KRNJEVIC Volume 54 sympathetic ganglion cells (380) and spinal Renshaw cells (283) have both nicotinic and muscarinic receptors, whereas depolarizing and hyperpolarizing effects of ACh can be elicited at different sites on some neuroblastoma cells (929). A change of innervation can alter the receptor properties (795). B. Acetylcholine in Skeletul Neuromuscular Transmission 1. How does ACh act? After the first demonstration that normal skeletal muscle can be excited by close-arterial injections of ACh (172), the most significant developments have been the discovery that ACh greatly increases the end-plate membrane conductance (424, 1153, 1154). S ince the frog end-plate potential or current has a reversal level close to 0 [actually at about - 15 mV, according to Fatt and Katz (424), de1 Castillo and Katz (323), and Takeuchi and Takeuchi (1155)], it was first assumed that ACh made the membrane freely permeable to all ions (424); on closer analysis, however, the ACh-activated membrane was found to be permeable only to cations, the ratio of Na conductance to K conductance (GNa/GK) being 1.29 (1155). The reversal level is similar in frog tonic fibers (184) and possibly in mammalian muscle (cf. 51). No systematic comparison appears to have been made between the reversal levels for the action of applied ACh and for the end-plate potential observed in the same muscle fiber. However, the similar values (near - 15 mV) obtained in different experiments suggest that these two reversal levels are identical, at least when ACh is applied to the immediate junctional region (323, 364, 424, 433, 855, 115 1, 1152, 1155). The same remarkably constant reversal level is also seen when the membrane receptors are activated by different cholinomimetic agents or when they are partly blocked by curare (364, 433, 1155). It does not appear to be altered by tetrodotoxin (433, 677). These observations suggest that the activated ACh receptors do not open up separate Na+ and IS+ channels. However, the end-plate current has a different time course when measured at membrane potentials close to the Na+ equilibrium level (about +50 mV) or the K+ equilibrium level (about - 100 mV) (466), and under these conditions it shows a differential action of procaine and other local anesthetics that prolong the end-plate potential (320, 850, 851). This seemed to indicate that independent channels are responsible for movements of Na+ and K+ [like those involved in the generation of the spike (574)] and that the Na+ channels are particularly sensitive to local anesthetics. However, it is more likely that the interaction between the receptors and ACh or procaine is itself a function of the Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 This has been the most extensively studied transmission process, and it is the only one for which we have almost conclusive evidence with regard to the identity of the transmitter (ACh) and its mechanism of action (295, 326, 423, 549, 550, 612, 670, 671, 771). April 1974 VERTEBRATE SYNAPTIC TRANSMISSION 425 Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 membrane voltage (7 19, 720, 856, 857, 1125, 1126). The situation is quite complex: the effects of different local anesthetics, such as lidocaine and procaine, are by no means identical (320), and it appears that in the presence of local anesthetics postsynaptic hyperpolarization reduces the quanta1 content of the end-plate potential (85 1) as it does in the presence of excess K+ (1156). The reversal level for the action of ACh is not totally invariable. An increase in external Ca 2f lowers the G&Gg ratio at the end plate by selectively reducing AG,, (1152). The depolarizing action of ACh (and other cholinomimetics) on extrajunctional receptors in normal muscle (cf. 899, 432) and on all receptors in denervated muscle has a relatively negative reversal level (at about -40 mV), consistent with a GNa/GK ratio of on1.y 0.60 (433, though cf. 853). Separate nicotine and muscarine receptors are not likely to be present in muscle since curare and atropine do not specifically antagonize nicotinic and muscarinic agents respectively (854). It is curious that atropine makes the reversal potential for the end-plate potential more positive but leaves unaltered the reversal level for the action of applied ACh (855), particularly since atropine depresses equally both types of end-plate responses (100). A possible explanation is that atropine selectively reduces the relatively large AGNs of the junctional receptors, but has the same action on AG Na and AGK at the extrajunctional sites. Like procaine (85 1 ), atropine may also cause the release of ACh to become sensitive to postsynaptic currents. A very promising new technique has been introduced recently by Katz and *Wiledi (682) for the study of the molecular events elicited in the end-plate membrane by ACh. The membrane potential changes induced by single molecules of ACh may be revealed by a spectral analysis of the ‘%hot” noise recorded during continuous applications of ACh. The results suggest that the voltage fluctuations are produced by elemental increases in conductance of about 0.1 nmho lasting 1 ms. If this interpretation is correct, only 1000 molecules of ACh are needed to produce the quanta1 responses observed electrophysiologically (see below). Another, somewhat unexpected, feature of interest is that molecules of carbachol appear to evoke much briefer changes in conductance. According to more recent experiments (Katz and Miledi, personal communication) the elemental conductance changes produced by other cholinergic agonists are also relatively brief; this may explain the lower efficacy of various cholinomimetics (cf. 65). The failure to observe any effect of neostigmine on the duration of the elemental action of ACh shows that this is largely independent of cholinesterase activity. How the activated ACh-receptor alters the membrane permeability is still mostly a matter of speculation (367). Summary. ACh depolarizes skeletal muscle by raising the cation conductance of the end-plate membrane, with particular emphasis on GNa, SO that GNJGK is 1.3 (reversal potential - 15 mV). Extrajunctional receptors, whose density may vary seasonally, activate the cation conductance SO as to give a GNa/GK of only 0.6 (reversal potential -40 mV). The balance of evidence suggests that ACh does not open up separate Na+ and K+ channels in the membrane. According to studies of the electrical noise generated by ACh, the unitary conductance change produced -426 K. KRNJEVIC rby single molecules of ACh lasts about 1 ms, independently tivity; other cholinergic agonists evoke even briefer unitary account for their lower efficacy. Volume of cholinesterase events, which 54 acmay 2. Release of AC% in muscle Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 Acetylcholine is manufactured by the acetylation of choline. This process requires a specific enzyme, cholineace tyl transferase, found inside certain nerve cells and their terminals (presumably only, or at least mainly, in those that are cholinergic) and acetyl-CoA, produced by mitochondria. Choline is a normal constituent of extracellular fluid, from which it is taken up by nerve endings, by a mechanism specifically blocked by hemicholinium. A further important source of choline is made available by the rapid hydrolysis of released ACh through the action of the acetylcholinesterase highly concentrated at cholinergic junctions [for extensive recent reviews see Potter (1008) and Hebb (548)]. The ACh stored in nerve endings is released continually, as a spontaneous process, and at a much accelerated rate during depolarization. The released ACh can be detected by chemical techniques or bioassay (total release) or by the changes in potential that it evokes at the muscle end plate (quanta1 release). a) Total release. The original observation of a release of ACh induced by indirect stimulation (297) has been confirmed repeatedly (149, 399, 746, 1007, 1142). It has been claimed that an undiminished amount of ACh is obtained by stimulating long-denervated muscle, so that ACh cannot be released mainly from nerve endings (543) ; however, these results could not be confirmed by several groups of investigators who have attempted to repeat the experiment (15, 149, 768, 1134). The isolated phrenic nerve-diaphragm preparation has been particularly useful for studies of release, because it is thin enough to permit reasonable oxygenation and quick outward diffusion of ACh, the total number of nerve endings is known, and there is plenty of information about its electrophysiological properties. Using classical methods of bioassay, Krnjevic and Mitchell (746) were able to show that the mean quantity of ACh released per impulse by a phrenic nerve ending has the same order of magnitude as the minimal amounts of ACh previously found necessary to produce depolarizations comparable to end-plate potentials (743, 898). The validity of these observations was confirmed by Potter (1007), who exposed phrenic nerve-diaphragm preparations to labeled choline and obtained similar yields of [14C]ACh when he stimulated the phrenic nerve, and also by Schmidt et al. ( 1076), who developed a technique combining pyrolysis and gas chromatography to prove that the released material is indeed ACh. Such good quantitative agreement between ACh release and the amounts required at the junction seemed to complete the evidence that ACh is the mediator of neuromuscular transmission. Unfortunately, on closer examination, it is clear that some of these observations cannot yet be explained by such a simple interpretation. For example, the leakage of ACh in resting muscle (908, 1007, 1142) is much too great to be accounted for by the quanta1 release causing the spontaneous April 1974 VERTEBRATE SYNAPTIC TRANSMISSION 427 Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 miniature end-plate potentials (see below) (828). Mitchell and Silver (908) further observed that this leakage is very much less sensitive than the quanta1 release to changes in external Kf concentration (and also responds differently to a lowering of temperature) and that it is very little diminished after chronic denervation (see also 1007, 1142), when quanta1 release is practically abolished (904). Although the quanta1 release may not contribute more than l-2 % to the observed leakage, ACh is evidently coming from the region of the nerve terminal, since there is no significant release of ACh from the noninnervated area of the muscle fibers (908). This agrees with the fact that ACh and cholineacetyltransferase are concentrated at the junctional area, both in the normal and the denervated muscle (552, 1007). The appreciable amounts of enzyme that are found after denervation-sufficient to account for the observed ACh release -are presumably located in Schwann cells (cf. 537, 828, 904). The high rate of leakage in resting muscle may take place from the terminal by a diffuse, nonquantal flux, which might be practically undetectable electrically but could have a significant effect on the sensitivity of the end plate to ACh (683). Alternatively, ACh may be released -in a quanta1 or nonquantal manner-from the axons of the motor nerve (cf. 832), although ACh, like other ions, should not readily diffuse out of the intact nerve trunk (cf. 730, 745). It is unlikely that much ACh could escape from the synaptic gap at the end plate without acting on receptors. The junction seems to be constructed remarkably efficiently for the maximal utilization of any released ACh. The high density of receptors [3 X 10’ per end by Katz plate (66, 1005), which is enough for > lo4 quanta of ACh as estimated and the minuteness of the synaptic gap seem to ensure that and Miledi (682) most of the ACh molecules would collide with a receptor site within a few ms of their release. If preterminal or nonquantal release accounts for a major portion of the ACh collected in experiments, the reasonably good agreement between the amounts of ACh released and the amounts required for excitaticn may be fortuitous. Clearly, further evidence is needed on this point. b) @antal release. Both the spontaneous and the evoked release of ACh are manifested by quanta1 changes in membrane potential ( 150a, 322, 326, 424, 828). This is not a peculiarity of muscle, since the quanta1 character of transmitter release in ganglia and even at some central synapses is now well established (777). Katz’s original hypothesis that these quanta1 events are caused by the release of packets of ACh has been confirmed by subsequent studies. It appears that these packets consist of at least 1000 molecules of ACh. They are released at a very low-and usually random-rate even from resting nerve endings; however, the probability of release is enormously, though transiently, increased when an impulse reaches the terminal (326, 6 12, 669-67 1, 870). The frequency of release is progressively raised (along a steep logarithmic slope) when the terminal is depolarized, whether by an applied current or by excess K +*: this has led to the proposal that the release triggered off by an action potential is the direct result of the sharp, transient depolarization (829). The important observation that K+ potentiates the quanta1 release elicited by electrical depolarization (830, 1156) has recently been confirmed 428 IS. KRNJEVIC: Volume 54 Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 and further analyzed by Cooke and Quastel (239). Another interesting finding is that the amount of ACh released at a given end plate is a relatively simple function of terminal size (779). This seems to imply that the terminal membrane has a rather constant density of release sites. The release of ACh evoked by an action potential is very much dependent on the presence of Ca2+ and is depressed by Mg2+ (321, 326, 440, 612, 637, 676, 678). Katz and Miledi (674, 681) have been able to show that end-plate potentials are obtained only when the nerve impulse invades the nerve terminal; the release process is a separate event that takes place 0.75 ms or later (depending on the temperature) after the arrival of the spike (or brief focal depolarization) (675, 679). A similar potentiating action of Ca2+ can be demonstrated when the nerve terminals are depolarized by focal stimulation in the presence of tetrodotoxin (236, 678) or by excess KS- (236, 612, 794). Th e most plausible explanation for these findings is that Ca2+ enters the depolarized nerve terminal, and triggers off the process of ACh release. A direct demonstration of the entrance of Ca2f into nerve fibers as a result of depolarization has been made with squid axons (53, 54). Although in their initial experiments Miledi and Slater (903) failed to evoke a release fibers of the squid ganglion, of transmitter by injections of Ca2+ into presynaptic similar, more recent experiments have produced unequivocal evidence that intracellular injections of Ca 2+ do cause a release of transmitter (901 a). In addition to its immediate effect in triggering ACh release, intracellular Ca2f by a residual action probably contributes significantly to short-term facilitation of synaptic transmission (680, 1017). The mechanism of action of Ca 2f has not yet been explained. The quanta1 release may involve the cooperative action of four (346) or five molecules of Ca2+ ( 1251) in the frog or three in the rat (794); alternatively, according to the most extensive study to date (236), the probability of release of a quantum is a continuous function of the amount of Ca 2+ forming a hypothetical complex inside the nerve ending. The following simple scheme could explain these observations. The probability of release is reciprocally related to the magnitude of an internal boundary potential generated by negative charges fixed at the inner surface of the membrane (cf. 211). As Ca2+ enters the terminal, it is bound to the negative charges; the boundary potential is progressively reduced, and the probability of release is correspondingly raised. A similar mechanism may account for the increase in K+ permeability of spinal motoneurons caused by intracellular injections of Ca2+ (740). It is not clear whether Mg2+ acts by competing with Ca2f at sites of entry or of intraneuronal binding. Quastel et al. ( 1014) believe that some release may take place independently of Ca2+ > but their evidence is not very convincing in view of the large intracellular stores of Ca 2f (200 > 8 11) that probably contribute to the release of ACh (126a) ; these intracellular stores of Ca2+ are not readily depleted even by strong complexing agents (483). It has been suggested that Ca2+ may become fixed to, and so April 1974 VERTEBRATE SYNAPTIC TRANSMISSION 429 Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 There is no need to discuss here in detail the role of vesicles in cholinergic transmission. The suggestion that quanta of ACh are released from vesicles helped to explain many features of quanta1 transmission (326), and it has not lost its validity. However, it is still not supported by very strong, direct evidence [though compare very recent morphological data (569a)]. Some correlation has been found between vesicle counts in nerve endings and efficiency of release, at least under some conditions: for example, after treatment with black widow spider venom (220) or after intense activity (6 12). However, Birks’ (112) finding that vesicle counts can be greatly changed by the conditions of fixation raises some doubts about the significance of such counts. Another serious difficulty is that vesicles isolated from nerve endings have consistently been proved to contain ACh that exchanges only verv. slowly with the relatively free (cytoplasmic) pool of ACh from which the more recently formed (and therefore more strongly labeled) ACh released by nerve stimulation evidently originates (363, 863, 1037). It is significant that chemical transmission becomes fully established in the embryonic ciliary ganglion before synaptic vesicles appear in appreciable numbers (796). As pointed out by Ginsborg (490), a quanta1 release could take place even from a nonvesicular pool of ACh. Acetylcholine release has been much studied by observing changes in amplitude of end-plate potential evoked by repetitive stimulation of the motor nerve of the nerve endings (237(cf. 170, 322, 612) or more recently by focal stimulation 239, 677-679). Although this technique can measure neither the total release nor the absolute amount of ACh acting on the end plate, it is the only direct way of estimating changes in the synaptically effective release. It thus was used by Elmqvist and Quastel (397) to estimate the total amount of ACh available for transmission by recording end-plate potentials in the rat diaphragm in the presence of sufficient hemicholinium to block the synthesis of new ACh (cf. 113). This was found to correspond to nearly 300,000 quanta [in the frog, a comparable figure of 450,000 is obtained by counting the quanta released by black widow spider venom (839)]. However, the rapid fall in quanta1 content observed during tetanic stimulation (398) suggested that only a relatively small fraction of this total amount was readily available for release, just as in sympathetic ganglia (cf. 113). It may be noted that if a quantum consists of only lo3 molecules of ACh (682), 3 X lo5 quanta per terminal are equivalent to only 2-5 % of the ACh actually present in the muscle (552). After a closer look at quanta1 release, Christensen and Martin (2 17) have concluded that repetitive stimulation leads to a reduction in probability of release (assumed by previous authors to remain constant) as well as in the available store. They point out that the probability consistently has a finite positive value, as would be expected of a binomial as opposed to a Poisson process. Summary. The manufacture of ACh in motor nerve endings and its release by impulses are well established; both processes are greatly reduced by denervation (any residual release being probably from Schwann cells). Although there is reasonable, order-of-magnitude agreement between the amount of ACh needed for an appreciable postjunctional effect and what is released during motor nerve stimulation (in the phrenic nerve-diaphragm preparation), the rate of spontaneous 430 K. KRNJEVIC Volume 54 C. Acetylcholine in Transmission to Smooth Muscle The structure and electrical properties of smooth muscle have been studied and reviewed extensively (88, 188, 195, 590, 591, 1182). The most characteristic feature is the functional coupling between cells at areas where the separate membranes combine to form a highly permeable “nexus” (337). Like the gap junctions of the CNS, the nexus can be reversibly uncoupled under some conditions-for The extent to which the tissue behaves as a example, in hypertonic solutions. functional unit is therefore potentially variable. Another feature of interest is that an electrogenic Na-K pump (1179) probably contributes significantly to the resting potential (205, 782). The parasympathetic actions of ACh on different kinds of smooth muscle are too complex for a systematic analysis in this review (for reviews cf. 299, 482, 485, 1074). One action, however, the excitation of intestinal longitudinal muscle, has been much investigated in recent years, so that there is substantial information for a comparison with muscarinic effects observed in nerve cells. Parasympathetic nerves elicit a depolarizing junctional potential that resembles the slow ganglionic excitatory postsynaptic potential (EPSP) in having a very long latency and correspondingly long duration (484, 485) and in that it is blocked by atropinealthough the amount of atropine required is lOOO-fold greater than is needed to block the depolarizing action of topically applied ACh (485). The depolarization induced by parasympathetic stimulation or by the application of ACh induces a discharge of spikes, but, as in most types of excitable tissue, the spikes tend to disappear if the depolarization exceeds a certain level ( 144, 172, 186, 484). At the same time, there is a change in the shape of the spikes, which last longer because the phase of repolarization slows down (177, 186). This tends to promote repetitive firing, especially in pairs of spikes (177). The action of ACh on this smooth muscle is thus in several respects similar to its muscarinic excitatory action on some central neurons (749, 754). However, the most general opinion is that ACh excites smooth muscle as it does skeletal muscle by greatly increasing the membrane permeability to cations (89, 144, 177, 178, 186, 570), and perhaps even anions (365). This opinion was originally based largely on an analogy with the mechanism of excitation in skeletal muscle. In a recmt Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 release is very much greater than would be expected from the electrically observed spontaneous quanta1 release: one possible explanation is that ACh is also released by a nonquantal process. Quanta1 release is a steep (logarithmic) function of terminal depolarization and is independent of Na+ influx through the tetrodotoxinsensitive channels. It is probably triggered by an influx of Ca2+, after a delay of nearly 1 ms, by an action that may be analogous to the increase in cation permeability induced by intracellular Ca 2+. Although morphological evidence suggests that ACh is released from synaptic vesicles by exocytosis, other experiments indicate that only the most recently synthesized and probably extravesicular ACh is released by stimulation. April 1973 VERTEBRATE SYNAPTIC TRANSMISSION 431 Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 study, Bolton (144) found that ACh or carbachol causes a maximum depolarization to -9 mV. This was assumed to be close to the reversal level for the underlying change in ion fluxes, which gave a lOO-fold maximum apparent increase in conductance. This level was altered as expected by variations in external Na+ concentration, but it was very insensitive to changes of K+ or Cl-. The author concluded that the main action of ACh is to increase greatly the membrane permeability to both Na+ and K+. The muscarinic excitatory action in the gut thus seems to differ fundamentally from the muscarinic action on vertebrate neurons, which is probably initiated by a reduction in K conductance (500, 754, 1236). It is possible, however, that this difference is more apparent than real. The technique used for measuring membrane conductance in the gut is indirect, being based on the assumption that the smooth muscle always behaves as a linear core conductor, so that the electrotonic potential evoked at a distance accurately reflects changes in membrane resistance (144). But the electrical coupling through the nexus connections may be altered by the conditions of the experiment, as in the comparable systems of coupled cells in the salivary epithelium of insects (833) or in the crustacean nervous system (965). Loewenstein (833) has shown particularly that a small increase in internal Ca2+ has a marked uncoupling effect. Since the excitatory action of ACh on smooth that this might muscle is associated with an influx of Ca 2f (365), it is conceivable reduce intercellular coupling sufficiently to lower sharply the space constant and Similar complications may explain therefore the apparent membrane resistance. Hidaka and Kuriyama’s (570) observation that the fall in resistance evoked by ACh precedes depolarization by about a second. Thus, the possibility has not been entirely excluded that, as in neurons, the initial effect of ACh in the gut is a reduction in K permeability [as is suggested by the prolongation of spikes (cf. 177, 186)]. The depolarization initiated by ACh (through a reduction in GK) may itself lead to a marked increase in conductance to Na+, K+, and even Cl- (and probably Ca”+). The observations of a marked increase in 42K efflux from gut muscle under the influence of carbachol (182, 365, 366) are often cited as evidence that ACh directly increases the cation conductance; however, this K+ efflux tends to disappear in the absence of external Ca 2+ (366) and it is very poorly correlated with the excitatory action -according to Burgen and Spero (I 82), 1000 times more ACh is required to augment Kf efflux than to initiate a contraction-which appears to indicate a secondary rather than a primary increase in K permeability. It would seem premature to decide that the muscarinic excitatory actions in smooth muscle, on the one hand, and on central and ganglionic neurons, on the other, are totally different. Summary. ACh probably mediates the parasympathetic excitation of smooth muscle by a muscarinelike action. According to studies on intestinal smooth muscle, this depolarizing action of ACh is associated with a large increase in K+ fluxes; but it is not yet clear whether a general increase in ionic or cationic permeability is indeed the primary action or a secondary effect after depolarization and the entry of Ca2+. 432 D. Inhibitory IS. KRNJEVIC Volume 54 Action of ACh on Heart E. Acetylcholine in Ganglionic Transmission 1. Release The concentration of a large number of nerve endings in a very small volume of tissue, which is moreover readily perfused through its blood supply (426, 699, 847), has made the cat’s superior cervical ganglion exceptionally useful for investigations on the synthesis, storage, turnover, and release of ACh (90, 113, 227-229) (for a study on the amphibian isolated ganglion see 940). The released ACh is normally not taken up by the nerve endings. It is hydrolyzed by cholinesterase, and about half the choline thus formed is immediately absorbed into the nerve endings by an active process that is blocked by hemicholinium-3. This choline is then resynthesized into ACh. The most recently formed ACh is preferentially released by stimulation (227). Even when exogenous ACh is accumulated (in the presence of anticholinesterases) it is probably not taken up by the nerve endings, since it cannot be released by preganglionic stimulation (683a). Although there is evidence of a slow excitatory process in ganglionic transmission (800), the initial excitation is mediated by fast EPSP’s (with corresponding spontaneous miniature junctional potentials) whose principal features are similar to those of the end-plate potentials (EPP’s) recorded in muscle (12 l-l 24, 330, 409, 777, 871,938). The EPSP’s have a quanta1 composition, and the number of quanta Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 Although ACh has some complex actions at various sites in the heart (for example 138, 817) that cannot yet be simply explained, the cholinergic inhibitory effect of the vagus on atria1 muscle is a well-established muscarinic action [readily blocked by atropine (18, 474, 835)] w h ose mode of operation is quite distinct. Gaskell’s (474) original finding that vagal stimulation increases the atria1 demarcation current was fully confirmed by intracellular recording. Vagal impulses and ACh have a clear hyperpolarizing effect (183, 324). Further studies by Trautwein and his collaborators revealed a large increase in membrane conductance (1188) and a reversal level for the inhibitory junctional potential at about - 100 mV ( 1187). These are the characteristics of a typical chemical inhibitory process, such as is seen in many central neurons (see below), but it has proved to be quite exceptional in being generated solely by movements of K+ rather than Cl-. Trautwein and Dude1 (1187) were able to show a clear dependence of the reversal potential on the external K+ concentration, as would be expected of a process determined by a change in G, (964). Summary. The inhibitory effect of the vagus on the heart is mediated by a muscarinic action of ACh. It is manifested by a fall in membrane resistance and a hyperpolarization with a highly negative reversal level ( - 100 mV), sensitive to changes in external K +. It is therefore very probably due to a large increase in K+ conductance. April 1974 VERTEBRATE SYNAPTIC TRANSMISSION 433 2. Actions of AC% on ganglion cells It has long been known that ganglionic transmission is blocked by an excess of nicotine (799), but there is now a good deal of evidence that in addition to the rapid, nicotinic mechanism of cholinergic transmission there is a slower excitatory process that appears to act on muscarinic receptors, since it is blocked by atropine (380, 427, 825, 867). Th e nicotinic and muscarinic actions of ACh in ganglia are therefore considered separately. a) Nicotinic actions. Fast ganglionic EPSP’s can be mimicked by direct applications of ACh or nicotine ( 12 1, 330, 492). Like the muscle EPP, they reverse at a level between 0 and - 20 mV (121, 330, 705, 938, 1236). There is only indirect evidence for a conductance change during the fast EPSP (12 1), but ACh certainly causes a large increase in conductance (492, 706). The ganglionic response differs from the EPP mainly in having a substantially longer active phase, possibly owing to the absence of postsynaptic acetylcholinesterase (12 1, 938). In an illuminating study of postganglionic parasympathetic neurons in the atria1 septum of the frog, Harris et al. (536) were able to show that, as in muscle, the sensitivity to ACh is highest at the synapses, but the maximum effect of ACh was much less than the maximum observed in muscle. Although the fast excitatory process in ganglion is nicotinic, the most effective blocking agents are not curare and its derivatives, but hexamethonium and tetraethylammonium (909, 97 1). b) Muscarinic actions. Eccles and Libet (380) found that the slow components of the ganglionic response could be blocked with atropine. Intracellular recording later demonstrated a corresponding slow EPSP, which is also sensitive to atropine and is evidently caused by a muscarinic cholinergic transmitter (824, 825, 1184). This EPSP has a very long latency (over 100 ms) and it can last for several seconds. It is evoked particularly effectively by repetitive stimulation. The fast and slow EPSP’s are generated in the same cell and, at least in some cases, by the activity of the same preganglionic fibers. The most interesting aspect of this slow EPSP is that, like the comparable muscarinic excitatory effect of ACh on cerebral cortical neurons (750, 752, 754, 763), it appears to be generated by a special kind of transmitter action. This was Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 released by an impulse is a function of the extracellular Ca2: Mg2 ratio. In the frog the quanta1 content of an EPSP appears to be in the order of 100, but individual nerve endings probably release only 2-3 quanta per impulse (940); in mammalian ganglia, only l-2 quanta may be released by a single preganglionic fiber, presumably having several endings on a cell (124). Summary. Most features of the synthesis, storage, and release of ACh in preganglionic terminals are similar to the corresponding processes in motor nerve endings of skeletal muscle. Acetylcholine is hydrolyzed after release, and choline: is taken up for the resynthesis of ACh. The newly synthesized transmitter is preferentially released. As in other systems, the release is dependent on Ca2+ and blocked but the number of quanta released bY Mg 2+; the EPSP has quanta1 characteristics, per terminal is very low. 434 IL KRNJEVIt? Volume 54 Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 first suspected when Kobayashi and Libet (705) found that the slow EPSP recorded in the frog’s sympathetic ganglion is not associated with a decrease in the membrane resistance. Moreover, the slow EPSP was diminished by hyperpolarization and increased by depolarization, and it proved to be readily blocked by certain metabolic inhibitors such as !2,4-dinitrophenol. These features seemed to indicate a depolarization caused by a change in the activity of an electrogenic ion (possibly Cl-) pump; although neither ouabain nor variations in internal or external Cl- had much effect on the EPSP (705). In further experiments by Nishi an increase in resistet al. (941) and Kobayashi and Libet (706) on frog ganglia, ance was regularly seen during slow EPSP’s, and there was a linear relation between the change in resistance and the amplitude of this EPSP. Moreover, the muscarinic depolarizing action of ACh (revealed after curarization) had similar anomalous properties, being abolished or even reversed by strong hyperpolarization. Such a clear increase in resistance is not seen in mammalian ganglia, possibly owing to the greater difficulty of eliminating the fast nicotinic action. Since the frog ganglion cells show no evidence of anomalous rectification, the increase in resistance during the slow EPSP, the diminished amplitude of the EPSP with hyperpolarization, and its reversal at a highly negative level are all simply explained by a reduction in membrane conductance to an ion having a very negative equilibrium potential (1236). Since the EPSP proved to be insensitive to changes in Cl- concentration, Weight and Votava (1236) concluded that a reduction in K+ conductance (G,) was the most likely mechanism, in agreement with the independent observations on cortical neurons (500, 752, 754). Arguing against this idea, Libet (826) has pointed out that the slow EPSP, when it is reversed, has a much shorter time course than normal and, further, that this EPSP is highly susceptible to block by metabolic inhibitors. An asymmetrical reversal of synaptic potentials is by no means uncommon (cf. 38, 240 691) and it can be explained partly by a nonuniform distribution of the currents or ions injected into the cell and partly by a voltage-sensitive interaction between transmitter and membrane receptors (cf. 7 19, 856, 857). Another possibility is that the EPSP is followed by a third, even slower synaptic process, having a different mechanism (1237). Studies on cortical cells have shown that applications of 2,4-dinitrophenol probably cause a sharp increase in G ]Ec,which blocks the muscarinic depolarizing mechanism particularly effectively (498, 500): a similar action could well explain the block of slow ganglionic EPSP’s by metabolic inhibitors. According to Weight and Votava (1237), the reversal level of the slow EPSP can be varied by changing the extracellular K+ concentration, as would be expected if the underlying process is indeed a fall in GK. Summary. Ganglion cells differ from skeletal muscle fibers in having well-defined functional nicotine and muscarine receptors. The former are responsible for the early fast EPSP evoked by preganglionic stimulation. When activated they cause a sharp rise in conductance and depolarization-with a reversal level between 0 and - 20 mV-presumably due to a large increase in GNa and GK. These nicotine receptors are readily blocked by hexamethonium. The muscarine receptors are responsible for the slow EPSP (lasting several seconds), which is associated April 1974 VERTEBRATE SYNAPTIC TRANSMISSION 435 with a rise in resistance, and has a highly negative reversal level, sensitive to changes in K+. The simplest explanation for these unusual features is that activation of the muscarine receptors causes a fall in GI(. F. Acetvlcholine in Central Nervous Transmission 1. Excitatory action of ACh a) Nicotinic actions. 1) ON RENSHAW CELLS. The main characteristics of the excitation of Renshaw cells by activity of the recurrent branches of motor fibers are the very brief latency of onset of the discharge and its very high frequency, even in response to a single stimulus (375, 376, 452). The initial peak frequency is commonly in the order of 1000 set-l and is relatively independent of the intensity of the stimulating volley, an increase of which tends mainly to prolong the discharge of the cell. This synaptic action can be almost completely blocked by certain ACh antagonists, especially by dihydro-beta-erythroidine (376) and mecamylamine (I 195). Microiontophoretic studies with ACh and related compounds (117, 268, 269, 282) have shown that ACh has a very quick and rapidly reversible excitatory effect on Renshaw cells, which can be obtained also with nicotine and other nicotinic agents; however, the action of nicotine is somewhat slower in onset and it lasts much longer, presumably because nicotine is not broken down by cholinesterases [in the presence of anticholinesterases, the effect of ACh is markedly prolonged (269)]. The most potent blocking agents, in addition to those mentioned above, are hexamethonium and tetraethylammonium (283). Tubocurarine and related agents are not only relatively ineffective as blockers, but they may even increase excitability. It is evident that in their pharmacological characteristics, Renshaw cells are much more like sympathetic ganglion cells than muscle end plates (cf. 65); a common cholinergic innervation, by the very same motoneurons, therefore does not preclude quite distinct postsynaptic receptor properties. A further similarity Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 Many authors suggested that ACh might act as a transmitter in the CNS as well as at the periphery, before any direct evidence became available (cf. 294, 295, 425, 1265), although Dale (295) was careful to emphasize the importance of ob-. taining sound evidence before making any serious claims. The earliest suggestions were prompted mainly by the finding of ACh and cholinesterase in various parts of the CNS and the observation of changes in activity induced by injections of ACh. The first really decisive progress in the study of central transmitter mechanisms came with the demonstration that recurrent branches of motoneurons are probably cholinergic and that they excite certain interneurons (Renshaw cells) by a nicotinic action not unlike that seen in skeletal muscle (376). The evidence for the existence of distinct Renshaw cells was recently comprehensively reviewed by Willis (1268). 436 K. KRNJEVIC Volume 54 Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 between ganglion cells and Renshaw cells is that both also have well-differentiated muscarinic receptors. So even in this case -which is the paradigm of the applicability of Dale’s principle to central synapses (37 1, 373)-the characteristics of the transmission process are by no means fully determined by the nature of the presynaptic fibers. It is generally presumed that here, as in other nicotinic junctions, the mode of operation of ACh is by an increase in membrane permeability to cations. But since only very limited intracellular recording has been possible from Renshaw cells, there is no reliable evidence about the membrane potential and conductance changes induced either by ACh or by the synaptic action. The fact that the initial spikes evoked by a ventral root stimulus can be driven at very high frequencies and that they are not abolished by even the strongest postsynaptic blocking agents has led to the suggestion that the initial firing may be caused by electrotonic transmission (1252). The demonstration by Quastel and Curtis ( 1013) that a prolonged application of hemicholinium-3 (HC-3) blocks the first spike does not prove conclusively that even the first spike must be generated by ACh, since HC-3, in addition to depressing the synthesis of ACh (cf. 113), can block impulses in nerve fibers (454). On the other hand, there is no compelling reason for supposing that one should be able to block completely every chemically mediated synaptic transmission by specific antagonists. The cholinergic nature of the recurrent branches of motor fibers is also indicated by evidence that antidromic stimulation of the ventral roots causes a release of ACh in the spinal cord (778, 907), although these observations did not exclude the possibility that ACh was released from some other neurons or even from the motoneuronal cell bodies or ventral root fibers (1072). 2) OTHER CENTRAL NICOTINIC ACTIONS. Since the introduction of the microiontophoretic technique, ACh has been tested on a large number of cells of many different kinds in all parts of the CNS. Many have proved sensitive to ACh [for systematic reviews, see McLennan (890) and Phillis (983, 984)], but so far almost none has given responses strictly comparable to those of Renshaw cells. The excitations are practically never so rapid and so quickly reversible, although some unusually sharp responses have been observed in the lateral geniculate of the cat (988) and in the brainstem of the rat (155). In several areas the actions of ACh can be reproduced with nicotine, and it is sometimes blocked quite effectively by dihydrofl-erythroidine -for example, in the medulla ( 1066), the lateral geniculate (26 1, 988), the thalamus (29), the cerebellum (880), and the supraoptic nucleus of the hypothalamus (63, 350, 351). Although the most striking excitatory actions of ACh in the cortex are muscarinic (see below), some nicotinic excitations have been observed in the cingulate gyrus (732) or in very superficial layers (1136). Nicotinic excitations as a rule have not been very easily reproduced and authors are apt to disagree over the supposed efficiency of various blocking agents (cf. 253, 880). Furthermore, in almost no instance has it been possible to block convincingly a given synaptic input with a nicotine antagonist. One therefore cannot with great confidence point to any central pathway, other than the motor fiber collaterals, as very probably cholinergic and nicotinic. Nevertheless, it is clear that many cells in April 1974 VERTEBRATE SYNAPTIC TRANSMISSION 437 Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 the CNS have comparatively well-defined nicotine-type receptors, which are predominantly excitatory when activated: it would seem rash to dismiss them all as nonspecific and of no functional significance, particularly in view of the difficulty of stimulating selectively all the various pathways that may be afferent to a given cell. Stlmmary. Although some nicotinic excitatory actions of ACh have been observed in most regions of the CNS, the only well-defined system is that of the spinal motoneurons’ axon collateral, the Renshaw cell synapse. The characteristic feature is a quick excitation by nicotinic agonists, which is rapidly reversible and is prevented by ganglionic blocking agents. Tubocurarine and related compounds are not useful ACh antagonists in the CNS, because they strongly excite many neurons. b) Muscarinic excitation. A relatively slow and prolonged excitatory action of ACh and other muscarinic agents, blocked by atropine and hyoscine, is seen with many central neurons, not infrequently even neurons that show a nicotinic effect (983). An almost pure muscarinic excitation is seen in the cerebral cortex, where this kind of action was first observed (749, 750). This therefore is described in some detail, since it has been studied rather extensively and substantial evidence is available about its likely mode of operation. 1) OF CORTICAL NEURONS. Excitatory effects of intracarotid injections of ACh in the cerveau isole’ preparation had been described by Bonnet and Bremer (145) and Bremer and Chatonnet (163), who noted that they could be blocked by atropine. But more precise information about the site and mechanism of action of ACh could not be obtained without a more direct and precise method of application. The first convincing demonstration that certain cortical cells can be excited by ACh was made with the microiontophoretic technique (748, 749, 1115). Th e excitation is usually slow in onset (not infrequently being preceded by reduction in spontaneous firing) and it always outlasts the application by some tens of seconds. It is seen most clearly with cells relatively deep in the cortex (below layers 2 and 3); in the sensorimotor areas, nearly all pyramidal tract cells are clearly sensitive to ACh. A systematic pharmacological investigation (750) revealed the remarkably unambiguous muscarinic character of this action [later confirmed by Crawford and Curtis (251)]. Further studies combining intracellular recording with extracellular iontophoresis (763) made clear that this depolarizing effect is not associated with the expected fall in membrane resistance. This was confirmed in more detailed investigations (500, 75 1, 754), in which it was shown that the membrane resistance tended to increase during application of ACh and that the depolarizing action had a reversal level close to - 100 mV. Since there was no evidence of anomalous rectification, these results could best be explained by a reduction in Gel or GK (both Cland K+ normally have an equilibrium potential close to - 100 mV), either by a direct action on the cell membrane or by an indirect process of disinhibition. The latter could occur if ACh inhibited adjacent inhibitory neurons, but this should result in changes in Gel, which are predominantly responsible for cortical inhibitory postsynaptic potentials (IPSP’s) (691, 753). But intracellular injections of Cl- causing large positive shifts in Cl- equilibrium potential and IPSP reversal level did not 438 K. KRNJEVIC Volume 39 Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 obviously change the character of the response to ACh. It therefore was concluded that ACh probably acts by reducing GIc. As supporting evidence it was pointed out that Ba2+, which is known to interfere rather specifically with movements of K+ in a variety of excitable tissues (cf. 1257), also tends to excite cortical cells in a rather similar way (753). It should be noted that tetraethylammonium, an antagonist of G, in nerve fibers (574), does not excite cortical cells like ACh or Ba2+; its effect seems to be mainly to prolong the spike (753), either because it blocks the K+ channels only when swept in by an outward current (cf. 39) or because different K+ channels-having specific pharconcerned respectively in delayed rectification and macological properties -are in the G, predominating during the interspike intervals (cf. 233, ‘234); the role of the additional channel in the generation of repetitive activity has been emphasized, and it is possible that ACh tends to inactivate particularly a similar component of G, in cortical neurons. A significant aspect of this action of ACh is its tendency to enhance responses evoked by other means. This seems to happen in two ways: the reduction in steadystate GK removes the stabilizing effect of the outward K+ current generated by any depolarizing tendency; the slowing of repolarization and diminution of delayed rectification may facilitate repetitive firing. There is reason to believe that repetitive activity is essential for the fixation of memory traces in neurons or neuronal circuits and therefore the elaboration of conscious processes (cf. 479, 556, 823). This action of ACh thus could be of special importance in determining a certain kind of cerebral function rather than the general level of activity. 2) MUSCARINIC EXCITATION IN OTHER PARTS OF CNS. After the discovery of the muscarinic excitation of neocortical neurons, comparable responses were obtained from many other kinds of central neurons, though seldom in such a relatively pure form. For example, slow atropine-sensitive excitations have been seen in the ventrobasal thalamus (29), hippocampus (118), pyriform cortex (809), caudate nucleus (894), cerebellar cortex (253, 880), lateral (988) and medial geniculate ( 1163 1166), and in the medulla and pons (154). Even Renshaw cells proved on closer examination to have muscarinic receptors, with properties similar to those of cortical cells (283). Thus, like sympathetic ganglion cells, Renshaw cells are provided with at least two kinds of ACh receptors, one rapidly acting and one slowly acting. The mixed muscarinic-nicotinic character of ACh sensitivity often observed when testing different neurons may be the result of having-variable amounts of the two types of receptors on the same cell. The ease with which muscarinic excitations can be demonstrated varies greatly from experiment to experiment (cf. 749, 809). This peculiarity may be partly due to a specific interference with the effects of ACh by general anesthetics, which has been observed in the cortex (207, 749, 1024), caudate nucleus (132), and thalamus (986); however, some authors have failed to detect such a specific action of anesthetics (29, 249, 25 1, 282). 3) BLOCK OF MUSCARINIC EXCITATION BY METABOLIC INHIBITORS, HYPOXIA, AND GENERAL ANESTHETICS. Studies on cortical neurons have shown that the excitatory effect of ACh can be blocked specifically but reversibly by 2,4-dinitrophenol (DNP) and probably some other uncouplers of oxidative phosphorylation, as well A/v-ii VERTEBRATE 1974 SYNAPTIC TRANSMISSION 439 2. Central inhibitory Central by Chatfield actions of ACh cholinergic and Lord inhibitory pathways have been postulated (2 15)] t 0 explain some effects of topical [for example, applications of Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 as by hypoxia (498, 500, 686). The mechanism of action of DNP appears to be by an increase in membrane permeability to K+ (498, SOO), which may be caused by a rise in intracellular free Ca2+ after the slowing down of mitochondrial activity (cf. ZOO, 81 i). This interpretation is supported by recent experiments in which Ca2f was injected in to motoneurons : this caused a fall in both excitability and membrane resistance that could be ascribed mainly to a rise in GX (43 1, 740). Although it is not immediately evident why a rise in internal Ca2+ should enhance GK, a simple explanation may be that Ca2+ neutralizes a negative boundary potential at the inner surface of the membrane (cf. 211) and in this way reduces the effective transmembrane potential that normally keeps the membrane cationic conductance at a relatively low level. If this is correct one might expect the membrane properties to be altered in a similar way by a rise in internal free Ca2+, membrane depolarization, or a reduction in external Ca2+, since all of these would reduce the effective transmembrane potential (cf. 453). It seems significant that all three lead to a fall in excitability associated with a high GK. Variations in metabolism must change the efficiency of Ca2+ sequestration by mitochondria and thereby the level of neuronal excitability. General anesthetics depress mitochondrial activity ( 10 15, 1016), and therefore narcosis may be brought about at least partly through this mechanism (737, 738). If cerebral muscarinic pathways play a special part in determining conscious processes (see above), they would indeed be particularly susceptible to depression by an increase in G,. Although there is as yet no direct evidence that anesthetics specifically raise GK in central neurons, their effects on the pattern of firing and sensitivity to ACh of cortical neurons can be remarkably similar to those of DNP orhypoxia (207). If other types of muscarinic transmission also operate by lowering GK, one would also expect them to be rather sensitive to DNP. This appears to be true for sympathetic ganglia (705, 941), but not for the spinal Renshaw cells (270). &mmary. The most common excitatory action of ACh in the CNS has muscarinic characteristics; it is relatively slow in onset and very prolonged and is readily blocked by atropine. Some neurons (e.g. Renshaw cells) resemble peripheral ganglion cells in having both nicotine and muscarine receptors and show corresponding fast and slow responses. According to intracellular studies in the cortex, the depolarizing effect of ACh is associated with a rise in resistance and it has a reversal level close to the expected value of K+ equilibrium potential. The mechanism of depolarization is probably a reduction in GK, which may also explain the characteristic repetitive afterdischarges. This interpretation receives some support from the comparable excitatory action of Ba2+, a general blocker of GX. The repetitive firing induced by ACh may be of importance for the laying down of memory traces (and possibly consciousness). This action of ACh is particularly susceptible to depression by general anesthetics and other inhibitors of mitochondrial activity, which permit intracellular free Ca 2+ to accumulate and so cause a rise in G,. 440 K. KRNJEVIt? Volume 54 Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 atropine on evoked cortical potentials. Substantial evidence has now accumulated from microiontophoretic experiments that ACh can depress the firing of certain neurons. The first demonstration of such an effect was made by Randic et al. (1024), who observed a depression of some cortical cells by ACh that could not be explained simply as a current artifact, especially since it was abolished by atropine. Although an indirect action -through the excitation of a neighboring inhibitory cell-could not be excluded, this was unlikely in view of the predominance of depressant effects in the superficial layers of the cortex, where excitation by ACh is seldom seen (248, 251, 749, 1024). Comparable depressant effects have been noted in several subsequent studies (418, 754, 809, 891, 993, 994). Phillis and his collaborators (65 1, 993; see also 617, 1209) h ave found that stimulation of several areas of the brainstem, as well as of the cortex itself, may cause a characteristic very prolonged inhibition of the superficial cortical cells depressed by ACh. The inhibition and the effect of ACh are both blocked by atropine (and, curiously enough, by strychnine), and Phillis therefore believes that there must be a cholinergic inhibitory pathway acting mainly on cells in the upper layers of the cortex. This inhibitory system appears to be at least partly intracortical, since the characteristic inhibition can be elicited even in chronically isolated slabs (65 1). The significance of this postulated inhibitory system is not clear at present. Even in the superficial layers only a small percentage of neurons are strongly depressed by ACh (754,891, 1024, 1121). N evertheless, it seems that cells that tend to discharge spontaneously in high-frequency bursts are consistently very sensitive to this action (K. KrnjeviC, personal observations). There is some evidence that neurons of this type may themselves have an inhibitory function ( 1034, 1130). It therefore is possible that cholinergic systems afferent to the cortex facilitate the activity of deeper cortical (pyramidal type) neurons both by direct excitation and by disinhibition. A further point of interest is that even those central neurons that are mainly excited by ACh are not infrequently initially depressed (29, 749, 984, 1065, 1166), especially when ’ %pon taneous’ ’ firing is at a very high rate (498). Most other regions of the CNS also contain cells that are depressed by ACh (29, 63, 132, 154, 285, 988, 1163, 1166, 12 13, 1235). In general these depressant effects are most easily blocked by atropine and other muscarinic antagonists (63, 154, 285, 854, 994, 1024). Thus, with some exceptions (984, 994, 1166), the inhibitory action of ACh has a rather clear muscarinic character. This is seen especially well when a given cell has both nicotine and muscarine receptors: for example, hypothalamicneurosecretory cells (63, 35 1) are excited by nicotinic agents and inhibited by muscarinic ones; the excitation is blocked by the nicotine antagonist dihydro-beta-erythroidine and the inhibition by atropine (63). In the medulla and pons, excitatory actions of ACh show a mixed nicotinic-muscarinic character, but inhibitions are purely muscarinic (153, 154). So far a clear change in membrane permeability has not been observed during the depressant action of ACh on central neurons (754), but one cannot yet dismiss the possibility that this effect is similar to the muscarinic actions that operate by April 1974 VERTEBRATE SYNAPTIC TRANSMISSION 441 G. Other Evidence Indicating Cholinergic Transmission in CNS The presence of ACh-sensitive neurons is necessary but not sufficient evidence of cholinergic transmission. However, there is plenty of supporting information about the presence of ACh, acetylcholinesterase (AChE), and cholineacetyltransferase (ChAc) (the enzyme needed for acetylation of choline) in many regions of the CNS ( 18 1, 196, 254, 425, 430, 547, 549, 550, 554, 560, 659, 848, 977, 1062, 1263). In several areas the AChE and ChAc activities are well correlated : for example, they are both at an exceptionally high level in the striatum, the interpeduncular nucleus, and the hippocampus (766, 820, 1137), and both tend to diminish in parallel after lesions that cut off the presumed cholinergic input to the cerebral cortex (551) or the hippocampus (820). But in other areas, such as the cerebellum, the correlation is much less satisfactory (660, 820). The commonly used histochemical techniques that demonstrate AChE-containing cells and fibers (480, 709, 7 12, 766, 767, 8 19, 953, 1088, 1089, 1095-1097)-though convenient for selectively tracing certain pathways-cannot by themselves give proof of cholinergic transmission. The release of ACh is obviously an important aspect of cholinergic transmission, which has now been demonstrated unequivocally in various areas, particularly at the surface of the cerebral hemispheres (73,8 1, 208, 230,361,562,635,655, 849, 906, 977, 982, 1148), even in the absence of anticholinesterase ( 1110). It increases with the amount of cerebral activity and with the degree of arousal but Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 raising GK, such as vagal inhibition of the heart (1 187) and secretory potentials in salivary glands ( 1079). Alternatively, the relevant receptors may be an .alogous to those responsible for the atropine-sensitive hyperpolarizing action of ACh seen with some neuroblastoma cells in tissue culture (929, 975) and even in L cells that are of fibroblastic origin (928). This response reverses at a highly negative potential and therefore at first sight appears to be analogous to the vagal inhibitory effect, but there is some evidence that it is associated with a rise rather than a fall in resistance (929). Summary. Depressant effects of ACh have been seen in many regions, and they may be shown even by cells predominantly excited by ACh. They appear to be exclusively muscarinic, but it is not known whether they are caused by a rise in Gx or by some other mechanism. General conclusions about membrane efects of ACh. At least three different actions of ACh have been identified in vertebrates: a rapid nicotinic excitation, caused by a general increase in cation permeability; a slow muscarinic excitation, probably caused by a specific reduction in K permeability; and a muscarinic inhibition, caused by a specific increase in K permeability. These three mechanisms, operating singly or in various combinations, may account for most of the observed actions of ACh. It is of interest that muscarinic actions seem to operate principally on K+ conductance, except perhaps in the gut, and that more generally all the identified actions of ACh in vertebrates probably lead to changes in cation permeability exclusivelv. 442 K. KRNJEVIC Volume 54 Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 diminishes during sleep [except during REM sleep (635)] and particularly in deep anesthesia. Acetylcholine is released particularly effectively by stimulation of the reticular formation (208, 655, 977, 982). Acetylcholine is found in synaptosomes extracted from the brain (33 1, 1262, 1264). Suspensions of such synaptosomes release ACh when they are stimulated by electrical pulses or excess K+ (309). Yamamura and Snyder ( 128 1) have further shown that synaptosomes possess a high-affinity, Na-sensitive mechanism for the uptake of choline. These observations thus are in reasonable agreement with the idea that cholinergic pathways from the phylogenetically older regions of the brain (e.g. mesencephalic reticular formation, hypothalamus, striatum, and septum) have a facilitatory (“arousing”) action on the neocortex of the cerebral hemispheres (459, 733, 766, 767,977, 1088, 1116, 1118, 1129, 1148) as well as on several of the sensory relay nuclei (e.g. 164, 876, 984, 988, 1166). Although this relatively simple picture may well be correct, it has not yet been possible to prove in a really convincing way that the arousal system is essentially cholinergic. The difficulty is partly because there is no certain method of stimulating this pathway selectively and exclusively and partly because its fibers appear to be very small and diffusely distributed, so that the responses evoked by stimulation have a long latency and are not well synchronized. Moreover, the effects of ACh antagonists have not been easy to interpret: for example, atropine causes a marked increase in the release of ACh from the cortex (849). It is still not known whether this is due to the removal of an inhibitory feedback from ACh-sensitive cortical neurons to the reticular formation (361; cf. 457, 565) or whether atropine enhances ACh release by a direct action on the cholinergic nerve endings (1003). The operation of such an inhibitory feedback may help to explain the relative inefficiency of atropine as an inducer of sleep (156, 1264), although an even more important factor may be that these cholinergic synapses, like those in the bladder ( 18) and the colon (488), are blocked only by very high doses of atropine. The situation is not made any simpler by the fact (noted above) that many central neurons appear to have mixed nicotine and muscarine receptors and that reticular stimulation also has a marked inhibitory action on many cortical neurons (cf. 609, 703, 992, 1120, 1129), although if many of these neurons should prove to be inhibitory interneurons (cf. 1130), the reticular arousal system could operate by a push-pull process. It is not yet possible to state unequivocally that there is cholinergic transmission even at such sites of outstanding AChE and ChAc activity as the striatum and the ha benuloin terpeduncular pathway ( 79 1, 890). A large number of behavioral studies with drugs have given some indication of a significant involvement of cholinergic mechanisms in sleep and arousal, memory, learning, and various other aspects of behavior (336, 410, 654, 701, 704, 1009). Phylogenetically ancient groups of neurons synthesizing and releasing specific transmitters (cf. 17) could be responsible for distinct aspects of behavior. Several basic drives are believed to be generated in the limbic system and hypothalamus, which can be considered as central components of the autonomic system. The high concentrations of ACh, ChAc, and AChE in this part of the brain seem to indicate April 1974 VERTEBRATE SYNAPTIC TRANSMISSION 443 H. Acetylcholine Receptors There has been a marked upsurge of interest in the macromolecular membrane components with which ACh is presumed to interact when it causes excitation. The very localized action of ACh in muscle and the fact that it is not sensitive to tetrodotoxin (677) strongly suggest that ACh does not act at the sites of Na+ entry during the action potential (574). Specific identification is the essential problem in the isolation of receptor macromolecules. How can one be certain that the material in question is the functional receptor in situ? 0ne approach is to bind to the receptor a slowly reversible or irreversible antagonist that can act as a label. But attempts at identifying or separating ACh receptors by using curare have not been very successful (210, 385, 1229), mainly because curare tends to bind to membranes rather unspecifically. A recently discovered irreversible and more specific antagonist, cr-bungarotoxin, has been used to much greater effect for labeling or isolating the probable receptor protein from electric organs and muscle (66, 101, 2 14, 420, 902, 1005). Another technique, affinity labeling, utilizes some characteristic reaction to tag the receptor: for example, by alkylating selectively thiol groups released from the disulfide bond that is typically closely associated with the ACh binding site (658). An interaction between the macromolecule and ACh itself can be revealed more directly by the XhlR spectrum of ACh (664, 666) or by equilibrium dialysis (389). Acetylcholine (and other) receptors have been the subject of several recent extensive reviews (333, 1026, 1027, 1105). A possible criticism of these studies is that they do not necessarily prove that the macromolecules in question control ionic movements. In an attempt to provide some relevant evidence, Parisi et al. (967, 968) h ave incorporated into artificial membranes a “receptor” proteolipid extracted from electric organs and have obtained some evidence of a transient increase in membrane conductivity on applying Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 the presence of some important cholinergic pathways. But the evidence obtained so far from behavioral studies has been too indirect and often insufficiently rigorous to identify with any precision the postulated cholinergic synapses or their mode of operation. The relatively tenuous neural links between such ancient regions and the more recently developed neocortical areas-the site of the more highly evolved intellectual functionsare certainly consistent with the manifest autonomy of different aspects of behavior. Summary. There is much indirect evidence for cholinergic activity in the CNS, such as the presence of ACh and enzymes involved in its synthesis and hydrolysis, and it is clear that ACh is released, probably from cholinergic nerve endings, in various regions of the brain, e.g. cerebral cortex, hippocampus, striatum, and hypothalamus. Studies of the effects of various drugs also indicate a significant role of excitatory or inhibitory cholinergic pathways in various aspects of behavior, such as arousal and wakefulness, drinking, aggression, etc. But so far no specific cerebral pathway has been conclusively shown to act by the release of ACh. 444 K. KRNJEVIC Volume 54 1. Cation mouements and AC/z system As pointed out above, all the known transmitter actions of ACh in vertebrates apparently involve changes in the permeability of cations only. Acetylcholine may increase both G, and GNa (1155) or GK alone (1187) or it may reduce GK (752, 754, 1236); there is no evidence that it ever affects anion permeability. This suggests that some component of the ACh system may have a basic and widespread (perhaps universal) function linked to cation movements through cell membranes. An obvious candidate for such a role is cholinesterase. The remarkably wide distribution of cholinesterase activity throughout the nervous system (17, 923), even in what are manifestly noncholinergic pathways, has long been a puzzle. In many areas there is a good correlation between ACh content, cholineacetyltransferase activity, and cholinesterase (especially AChE) activity, but often the correlation is very poor -for example, in the dorsal root fibers or the cerebellum (1095). On the other hand, Nachmansohn’s (923) hypothesis that ChE is present in all nerve fibers because ACh is directly involved in the conduction of the nerve impulse has received little or no support from a variety of experimental tests (668). Moreover, cholinesterase activity is found in many organisms, tissues, or cells where it cannot be related to neurotransmission (42, 709, 7 12); obvious examples in vertebrates are the frog skin (707), the kidney (451), the cornea (980), and red blood cells (709). There is some indication that ChE may be linked to the active transport of Na+ (451, 707, 716), but an alternative or additional possibility (754) is that ChE may Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 ACh. By fractionating the innervated face of electric organs, Kasai and Changeux (661, 662) h ave obtained a suspension of “microsacs” consisting of membrane rich in AChE and receptive to ACh (and other cholinomimetics or ACh antagonists); after loading the microsacs with labeled ions, they succeeded in showing that ACh induces a specific increase in cation permeability, comparable with the effects observed in intact muscle (for example, AG&AGK was 1.20). These relatively isolated receptors therefore control ionophores in an approximately normal way. The remarkably similar number of receptor and AChE sites at various junctions (661, 1005) lends some support to the notion that subunits of a single macromolecule are concerned in these two distinct functions (86, 8 14, 1287). It is clear that these subunits must be largely independent since the ACh receptor property is essentially unaffected by total block (67, 71) or even removal of the esterase activity. Most of the recent studies have dealt with the “nicotinic” ACh receptor, and there is much less information about the “muscarinic” receptors (422, 970). It has been suggested that the muscarinic effects of ACh are mediated intracellularly by guanosine 3,5-cyclic monophosphate (478, 808). Summary. There has been a rapid advance in studies aiming to isolate or identify in situ functional membrane receptors for ACh, partly thanks to the use of radioactively labeled cu-bungarotoxin, an irreversible nicotine antagonist. Relatively pure protein has been obtained in solution having many of the binding characteristics of the nicotine receptor in situ. April VERTEBRATE 1974 SYNAPTIC TRANSMISSION 445 III. AMINO ACIDS A. General Amino acids have only recently come to be considered as possible neurotranswas discovered by Ritthausen in 1866 mitters, though glu tamic acid -which ( 1038)has since the early years of this century (2) been known as an important constituent of nervous tissue; it is present in the brain in higher concentration Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 be directly involved in the passive movements of cations (especially K+), either as a carrier molecule or by providing a negatively charged hydrophylic channel for the passage of cations-in any case, effectively functioning as an ionophore. The anionic chain of cholinesterase has binding sites for curare and atropine (86, 665667, 1180) that have been proposed as nonhydrolytic receptors for ACh. These or comparable sites may act as K+ ionophores. It thus could be significant that the high ChE activity of motor nerve fibers is associated with a relatively high resting membrane GK in these fibers (102). Similarly the ChE activity observed in the transverse tubular system of muscle fibers (68, 901) may be connected with the high apparent G, of the tubular membrane (16, 388, 577). In the CNS the ChE activity of neuroglia (480, 709) is associated with a high relative GIc (329, 775). In red blood cells either PK or PNa (according to species) can be reduced by ACh and some related compounds, and this effect is blocked by anticholinesterases (5 16, 589). The GK of many excitable cells is specifically blocked by tetraethylammonium and several other quaternary ammonium compounds more or less closely related to choline (40, 574, 753). The apparently nonspecific convulsant effects of topical applications of curare and strychnine in areas such as the cerebral cortex (52, 212, 759) may be due to a reduction in membrane GK (cf. 37, 456). Microiontophoretic applications of curare and various related compounds (especially gallamine) excite many central cells that are not sensitive to ACh (285, 472, 1066); a striking feature is a marked tendency toward repetitive discharges (472), very similar to the effect of excess Ba2+ (753), which also lowers GH in a variety of tissues (1256, 1257). Strychnine inhibits cholinesterase activity (922), and in some peripheral nerves it prolongs spikes and reduces afterhyperpolarizations (24 1,645, 1230). All these facts are consistent with the possibility that some anionic portion of the cholinesterase molecule may have a very general, primordial function in providing sites of transmembrane cation movement, a function that may well have long preceded its adaptation for the hydrolysis of free ACh [and perhaps for use as an ACh receptor ( 1287)]. Summary. The widespread distribution of enzymes related to ACh (especially cholinesterase) even in neural tissue indicate some more general function, unrelated to synaptic transmission. Various items of evidence are reviewed that suggest the possibility that cholinesterase may have a primordial function in the regulation of transmembrane movements of cations, possibly as an ionophore. 446 K. KRNJEVIC Volume 54 Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 ( 10m2 M) than in most other organs. It seemed to be closely involved in cerebral metabolism, as indicated by its striking effect on the oxygen consumption and accumulation of potassium by isolated brain or retinal slices ( 1174, 1239). The presence of free glutamate and glycine could also be ascribed to the requirements of protein manufacture. Gamma-aminobutyric acid (GABA), on the other hand, which was not discovered as a brain constituent until 1950 (45, 1044), in vertebrates is found in hardly any other tissues and is not a component of protein-although it is by no means a “new” compound, being a very common product of widespread photosynthetic and other metabolic reactions in microorganisms and plants (11, 1228). Its relatively high concentration ( 10e3 M) in the brain suggested some special function, presumably related to neural activity, but there was no clue as to what this function might be. Later studies on metabolism showed that an appreciable proportion of the glucose-utilizing system of the Krebs cycle in the brain was partly shunted via a pathway passing through GABA. One function of GABA therefore appeared to be to provide an alternative route of cerebral metabolism (884, 1040). The possibility that glutamate and GABA might be concerned in the control of neuronal excitability was first proposed by Hayashi (541, 542). Although Brooks et al. (169) had observed a strong excitatory action of dicarboxylic acids, including dicarboxylic amino acids, on the brain of winter frogs, Hayashi’s experiments (541) demonstrated a much more specific and direct excitatory action of glutamate on the mammalian brain. By making injections of small volumes of glutamate solutions into the cortex through a fine metal tube, Hayashi anticipated the microiontophoretic technique and was thus able to show that glutamate acted exclusively on gray matter. In other experiments Hayashi (542) discovered the marked depressant action of GABA and other short-chained a! ,o-amino acids. Although his method of evaluating the depressant power may seem crude by later standards-it was based on the effectiveness of a given agent in reducing convulsions evoked by various excitants -nevertheless it enabled him to describe quite accurately the relative potencies of the short-chained CY+-amino acids. His conclusion that the most effective depressants are those with five or six carbon atoms in the main chain and that compounds with more than seven carbon atoms have little or no depressant activity has been confirmed by practically all subsequent investigators. However, Hayashi did not think that either glutamate or GABA was likely to be a natural transmitter, but suggested that GABA was probably the parent substance of both the excitatory and the inhibitory transmitters; his favored candidate for the latter was ,&hydroxy-y-aminobutyric acid. At about the same time, by a curious combination of circumstances, GABA was shown to have a strong inhibitory action on the crayfish stretch receptor cell; however, further attempts at finding the significance of this action led to a prolonged and singularly unhelpful controversy. Florey’s discovery (445) that extracts of mammalian brain strongly inhibit the crayfish stretch receptor indicated the presence in the brain of an unknown inhibitory agent, which was named factor I. A splendid example of interdisciplinary collaboration between Florey and Elliott (80) led to the identification of GABA as the main component of brain that could April I974 VERTEBRATE SYNAPTIC TRANSMISSION 447 Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 account for its factor I activity. The authors therefore suggested that GABA may be an inhibitory transmitter in mammals, even though they had very little evidence that GABA inhibits neuronal activity in the mammalian brain. However, serious consideration of GABA as an inhibitory transmitter in mammals proved to be short-lived. It came under strong attack from several quarters. On the one hand, it was claimed that, when tested on a variety of excitable preparations, the action of GABA differed in several essential respects from the action of factor I (447, 888); an even more serious objection was that the inhibitory activity and the GABA content could be completely dissociated, since it was possible to make fully potent preparations of factor I that contained no GABA (886, 887). But in spite of all efforts, the unknown postulated inhibitory agent could not be identified. Experiments on the effects of GABA on neural activity also failed to support its proposed role as inhibitory transmitter in vertebrates. Several groups of authors applied GABA and some related substances to the surface of the cerebral or cerebellar cortex while recording field potentials evoked by direct or indirect stimulation. They all observed marked effects, but these were interpreted as inconsistent with a general inhibitory function of GABA. For example, an elaborate analysis by Purpura et al. ( 1011) led to the conclusion that GABA specifically blocked axodendritic excitatory synapses by a curarelike action, not associated with changes in membrane polarization or conductance. Comparable experiments by Iwama and Jasper (627) were interpreted differently: GABA clearly had a direct depressant effect, but only on neural elements in the most superficial layers of the cortex, because deeper injections were ineffective. This conclusion was also not consistent with a general inhibitory role of GABA. A very different new technique -release from multibarreled micropipettes by microiontophoresis -was used by Curtis et al. (279) to apply GABA and @-alanine to single neurons in the spinal cord of the cat. The results were quite conclusive: all spinal interneurons tested were markedly depressed by GABA or fl-alanine. By the use of concentric double micropipettes the authors also recorded intracellular potentials while applying GABA extracellularly. Again very marked effects were seen regularly: there was a sharp reduction in cell excitability and also in the amplitude of synaptic responses evoked by excitatory or inhibitory pathways, but no sign of a membrane hyperpolarization. However, the depressant effects of both GABA and @alanine appeared to be quite impervious to strychnine, which had long been known to block spinal inhibitory processes (162, 958). Citing the lack of hyperpolarization, the resistance to strychnine, and the depression of both EPSP’s and IPSP’s, Curtis et al. (279) firmly concluded that GABA could not be the inhibitory transmitter in the spinal cord nor probably in the brain. Thus arose a wide consensus that, in the vertebrate CNS, GABA has a nonspecific (modulator?) depressant action and that the natural inhibitory transmitter(s) must be some other substance(s) (110, 250,258, 372, 392,888). Similarly, any speculation that glutamate might be a significant excitatory transmitter for some time was strongly discouraged by the results of experiments that suggested, on the one hand (101 l), that glutamate has a slowly reversible de- 448 K. KRNJEVIC Volume 54 B. Inhibitory Amino Acids 1. y-Aminobutyric A) ACTION ceptions [cf. the in every part of action of GABA of the CNS (see transmitter. acid 0N NERVE CELLS IN DIFFERENT PARTS OF CM. With almost no exfirst sensory cells in the lamprey’s spinal cord (872)], all neurons the CNS of vertebrates have been found sensitive to the inhibitory (259, 279, 735, 748, 1065). Since GABA is found in every region below), GABA is likely to be the most extensively used inhibitory Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 pressant action in the cortex and, on the other, that its marked excitatory effect on spinal interneurons (280) could not be a specific transmitter action, because it was unaffected by a variety of enzyme inhibitors that-it was argued-would have delayed the removal of a genuine transmitter. In spite of further results confirming that GABA could account for all the factor I activity of brain extracts (815), the belief that GABA, glutamate, and aspartate could not be natural transmitters (cf. 129) stimulated a continued very intensive search for other active compounds, but no further agents of comparable potency have ever been isolated (860, 887, 1059). However, after a systematic, wide-ranging survey of the chemical sensitivity of cortical neurons KrnjevX and Phillis in 1963 (748; see also 73 1, 732) were convinced that glutamate and GABA could be natural transmitters in the brain. This opinion was based largely on observations of the remarkably potent, and almost uniquely rapid, reversible and reproducible actions of these naturally occurring compounds, unequaled in all these respects by any other agents known to be normally present in the brain. The demonstration that both GABA and glutamate are released from the surface of the cerebral cortex (633), as well as further microiontophoretic studies in different regions of the CNS (1065), added increasing support for the notion that GABA and glutamate might be physiological transmitters. Compelling new evidence came with the demonstration that GABA has a hyperpolarizing action on cortical neurons (762, 764) and on neurons in Deiters’ nucleus (945). It was further shown that, like the natural transmitter, GABA greatly increases the chloride permeability of cortical neurons; and, under a variety of conditions, the cortical IPSP and the potential changes induced by GABA proved to have a similar reversal level (352, 762, 764). S’mce strychnine has little or no specific effect on inhibition in the cortex (759), the lack of antagonism between GABA and strychnine was no discrepancy. The principal remaining problem, how to explain the anti-inhibitory action of strychnine in the spinal cord was solved when Werman and his collaborators inhibitory action of glycine on spinal neurons, (253, 1255) d iscovered the powerful specifically and markedly susceptible to block by strychnine (273-275). Although evidence that glutamate may be an excitatory transmitter is still largely circumstantial (644), this idea has now received a wide degree of acceptance (259). April 1974 VERTEBRATE SYNAPTIC TRANSMISSION 449 Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 1) CEREBRAL CORTEX. The powerful inhibitory action of GABA on cortical neurons, first described by Krnjevic and Phillis (73 1, 748), has now been repeatedly confirmed (250, 1065). The main characteristics observed on extracellular recording are quickness and reversibility, so that it is very effective even when applied extremely transiently by very brief pulses of iontophoretic current ( < 1 ms). The pause in firing elicited by such transient applications of GABA is very similar to the silent period observed during natural inhibition (cf. 757, 758). The minute amounts of GABA required under these conditions ( < lo-l4 mol) provide further evidence of its great potency. A new technique of combining intracellular recording with extracellular microiontophoretic applications (762, 764) made possible a much more critical comparison between the actions of GABA and natural inhibition evoked by epicortical stimulation (757, 758, 822). These experiments revealed a hyperpolarizing action of GABA associated with a marked increase in membrane conductance. LMoreover, both the evoked IPSP’s and the effect of GABA could be reversed to a depolarization by injections of chloride into the neurons. Like ACh in muscle, GABA probably acts on surface receptors, since it was ineffective when injected intracellularly (cf. also 340). More complete tests (352, 690) confirmed these findings and in particular showed a highly positive correlation between reversal levels for IPSP’s and for the action of GABA over a wide range of membrane potential. They also demonstrated a new phenomenon: a striking fall in GABA potency during a prolonged application, so that after an initial lo-loo-fold increase the conductance rapidly declined to a very much lower level, which, however, was still well above the resting level, and showed no further decrement even after several minutes. These observations provide strong evidence that the action of GABA and that of the natural inhibitory transmitter in the cortex are identical. The predominant effect is a sharp increase in chloride permeability, as indicated by the great ease with which intracellular injections of chloride reverse the IPSP or the GABAevoked hyperpolarization. A variety of small inorganic anions and even some large organic anions can substitute for chloride, showing that the inhibitory current is carried through relatively unselective anionophores (691). It is unlikely that a flux of K+ contributes significantly to the inhibitory action, because several agents that block movements of Kf have no detectable effect on IPSP’s (753, 754). 2) DEITERS’ NUCLEUS. Some cells of this nucleus, situated in the medulla, are among the largest in the CNS. This feature makes them unusually suitable for intracellular studies of inhibition, especially as they receive a direct inhibitory innervation from the cerebellar cortex (377, 620). Systematic tests of the effects of GABA on Deiters’ neurons by Obata et al. (945) gave convincing evidence of a hyperpolarizing action and less direct evidence that this is associated with an increase in membrane conductance. Unfortunately, probably owing to their large size, Deiters’ neurons proved to be rather impervious to injections of chloride, which had little or no effect on IPSP’s or potential changes evoked by GABA. More direct evidence of an increase in membrane conductance during the action of GABA was obtained in later experiments (948) in which a similar reversal level was found for the IPSP 450 K. KRNJEVIt: Volume 54 Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 and for the effect of GABA. The careful independent observations of ten Bruggencate and Engberg (1172) in all respects confirmed the findings of Obata and his collaborators. Applications of GABA occluded IPSP’s by causing a clear and reproducible hyperpolarization and a large increase in membrane conductance. Both the effects of GABA and the IPSP’s could be reversed by membrane hyperpolarization, and the reversal levels were quite similar. 3) SPINAL CORD. As mentioned above, the first intracellular observations on the effects of GABA, by Curtis et al. (279), did not reveal any significant potential changes. After the demonstration of a clear hyperpolarized effect of GABA on some other central neurons, Curtis et al. (275) reexamined thoroughly the membrane effects produced by depressant amino acids on spinal motoneurons. They indeed found that here also GABA tended to hyperpolarize the cell membrane and greatly reduced the membrane resistance. Like the IPSP, the effect of GABA could be reversed by artificial hyperpolarization. Both phenomena had approximately similar reversal levels, and they were both also reversed by injections of small univalent anions but not by large inorganic or divalent anions. These observations indicate that, as in other parts of the CNS, the main action of GABA is to increase the membrane permeability to small anions. Quite similar effects of GABA have also been recorded in the CNS of fish, in Mauthner cells (340) and spinal interneurons (872). Although GABA tends to depolarize more peripherally situated neurons, such as those in sympathetic ganglia (5, 3 15) and some sensory ganglia (3 17, 3 18), there is now good evidence that this is also caused by an increase in membrane conductance (944), probably mainly to Cl- (5). The almost universal inhibitory action of GABA on vertebrate central neurons thus appears to be due to the same mechanism -that is, a pronounced increase in chloride permeability (275, 352, 690, 762, 764, 948, 1172). The great increase in membrane conductance effectively clamps the membrane potential at a relatively negative level and so prevents depolarization by excitatory influences. This effect is fully adequate to explain the marked inhibitory action of GABA. In most respects the tests described above have shown excellent agreement between the natural inhibitory effect and that produced by GABA, except possibly for hyperpolarizations that are often somewhat less conspicuous with GABA than during IPSP’s even when GABA produces much larger increases in conductance (cf. 352). This may well be an experimental artifact, which could be caused by one or more of several factors : electrical coupling between iontophoretic and recording electrodes, the acidity of the GABA solutions used for iontophoresis, excessive applications of GABA causing a rise in internal Cl- concentration or even a different change in membrane permeability (cf. 774), or activation of a partly different population of receptors, perhaps extrasynaptic receptors with distinct properties (cf. 433); some divergence could also be expected if GABA is normally released from nerve terminals in association with other substances that modify its action. Finally, one cannot totally exclude the possibility that the main transmitter is not GABA but some close, much less stable derivative. Summary. Investigations on the action of GABA in several parts of the CNS have shown a striking similarity with the effects of synaptic inhibition : the neuronal April 1974 VERTEBRATE SYNAPTIC TRANSMISSION 451 Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 membrane resistance is sharply lowered, the transmembrane potential becomes more negative, and there is a similar reversal level, which can be altered by injections of Cl- (or other small anions) inside the cell. These effects of GABA are seen even when it is applied in amounts < lo-l4 mol: they have a very quick time course, but there is some evidence of desensitization during prolonged applications. These observations are fully consistent with the hypothesis that GABA is thenatural transmitter released by many inhibitory neurons and that it acts by increasing Gor. A comparable action is seen even on more peripheral ganglion cells. 6) Actions of GABA on other elements of CNS. 1) NERVE FIBERS. There is no evidence that GABA can block conduction in myelinated nerve fibers (cf. 279, 471, 885). Of greater significance is the possibility that GABA might alter the excitability of the unmyelinated, terminal region of the fibers. This could be of great functional importance, either by changing the probability that nerve endings are invaded by afferent impulses or because it might reflect a change in polarization of the terminal, which may determine the amount of transmitter released by the nerve ending (373) [cf. the neuromuscular junction, where there is good evidence that transmitter release is a function of membrane depolarization (237, 829)]. Different tests of GABA on terminals have not given entirely consistent results. The most striking effect is a strong depolarization of dorsal root fibers in the amphibian spinal cord, first observed by Schmidt (1077) and repeatedly confirmed comparable depolarization is produced by by later observers (64, 30 1, 1170). A several other amino acids. One possible explanation, that the action of GABA is indirect, has not been entirely eliminated. The depolarizing effect is fully seen even in the presence of high concentrations of magnesium (64, 301), indicating that it is probably not mediated synaptically; however, this does not exclude the possibility that GABA releases a depolarizing agent from certain cells, e.g. neuroglia. According to other tests (64), this presynaptic action of GABA is not sensitive to extracellular chloride, but is affected by changes in sodium concentration. On the other hand, a more recent study (939a) has obtained strong evidence of an increase in Ger similar to that seen when GABA is applied to nerve cell bodies. Such marked presynaptic depolarizations have not been observed in the mammalian CNS (cf. 379). The effects of GABA on terminal excitability were examined by Curtis and Ryall (284)) who used a multibarreled micropipette for simultaneous electrical stimulation and release of GABA. The results indicated a marked depression of excitability. Comparable experiments on afferent terminals in the cuneate nucleus by Galindo (469) raised some doubts about the reliability of this technique. There was evidence that the stimulating current pulses were significantly diminished during the iontophoretic release of GABA. Davidson and Southwick (304) superfused the region of the dorsal column nuclei with solutions containing GABA and found mainly evidence of an enhancement of terminal excitability [cf. also some tentative observations on the spinal cord by Eccles et al. (379)]. 2) EFFECTS OF GABA ON NEUROGLIA. In contrast to the hyperpolarization and the very regular, large increase in conductance produced by GABA in cortical neurons, unresponsive cells in the cerebral cortex -defined as cells that generate 452 K. KRNJEVIC Volume 54 Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 no spikes or synaptic potentials, either spontaneously or in response to any kind of stimulation-consistently failed to show a hyperpolarization or a fall in resistance (762, 763). Indeed, large applications of GABA led to a slow and reversible depolarization, but without a change in membrane resistance. Such unresponsive cells were later positively identified as neuroglia by intracellular staining (5 18, 692). These observations suggested the possibility that GABA may be actively taken up into glial cells by an electrogenic pump and that glia perhaps play a significant part in the removal of neurotransmitters from extracellular fluid (763). This suggestion has received a good deal of support from recent autoradiographic studies of the uptake of labeled GABA and other transmitters (see below). Since the glial cell membrane is probably selectively permeable to K+, and its membrane potential is determined by the K+ diffusion gradient (cf. 329, 775), if the uptake of GABA is associated with some depolarization, this could cause an efflux of Kf from glia and a slow depolarization of neighboring cells and fibers. Summary. GABA appears to have little effect on nerve fibers, but depolarizes dorsal roots, especially in the amphibian spinal cord. This action is probably mediated also by changes in Gel. c> Other evidence pointing to transmitter function of GABA. 1) DISTRIBUTION OF GABA IN CNS. The discovery of GABA in the brain (45, 1044) initiated numerous studies on its distribution. It has been found in all partsof the CNS, but consistently more in gray matter than white matter (1 I, 76, 107, 390, 417, 458, 5 12, 576, 646, 95 1, 1040, 1046, 1100). Certain areas are strikingly rich in GABA : for example, the substantia nigra and parts of the striatum (417, 951). The cerebral GABA content is by no means constant; there is some evidence that it is reduced in certain convulsive states (700) [but cf. Elliott (39 l)]. The significance of the lowered GABA (and glutamate) content of human and animal epileptic cortex is not clear (1205). 2) SUBCELLULAR DISTRIBUTION OF GABA. Although some isolated nerve cells, such as Purkinje neurons of the cerebellum, have a relatively high GABA content (946) it is not certain whether this reflects a truly high level of GABA inside these neurons or a high content of GABA in inhibitory terminals attached to their surface. The experiments of Elliott showed that GABA, like factor I, is present in at least two forms in the brain : free GABA, which is released by homogenization, and occluded GABA, which is released by more severe treatment (393, 394). The second was likely to be intracellular. According to subfractionation studies, most of the occluded GABA appears to be situated in nerve terminals (860, 1244). Even synaptic vesicles can be shown to have small amounts of GABA associated with them (769, 784, 860), but most of the GABA content of the brain is probably in a relatively free form in the cytoplasm rather than bound to some subcellular particles (cf. 391, 860, 1059). Although GABA may not be greatly concentrated in nerve terminals, the amount present in cortical synaptosomes is sufficient to produce significant inhibition. This was shown by releasing GABA from suspensions of synaptosomes directly on to cortical cells (769) ; all the inhibitory effects observed could be reproduced semiquantitatively with an artificial mixture of GABA and some other amino acids. April 1974 VERTEBRATE SYNAPTIC TRANSMISSION 453 Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 3) METABOLISM OF GABA AND RELEVANT ENZYMES. The principal source of GABA is glutamic acid, which is itself readily available from glucose via the tricarboxylic acid cycle (76, 39 1, 1043). A surprisingly large fraction of glucose given systemically is rapidly converted to glutamate and GABA (467, 1222, 1283). Soon after the discovery of cerebral GABA, Roberts and Frankel (1045) showed that the brain contains significant amounts of an enzyme that decarboxylates glutamate to produce GABA. In most parts of the CNS, there is relatively good correlation between glutamic decarboxylase (GAD) activity and GABA content ( 12, 76, 783, 1040, 1043). Another enzyme removes GABA by transamination with a-oxoglutaric acid, producing succinic semialdehyde ( 1042, 1068). Both enzymes are found in most parts of the brain and their properties have been extensively studied (76, 1041, 1144). The return to the Krebs cycle is completed by oxidation of succinic semialdehyde to succinate, facilitated by a specific dehydrogenase (13, 1098). The tissue content of GABA is a function of the activities of GAD and GABA-transaminase, but the main determining factor is the amount of GAD activity ( 104 1, 1043). Glutamic decarboxylase has some interesting properties, including a high susceptibility to inactivation by anions, such as chloride; it has been suggested that changes in cytoplasmic chloride content, secondary to activity or any other cause, thus could significantly influence the supply of GABA ( 1041). Glutamic decarboxylase has been shown to be present in cerebral synaptosomes (448, 1063, 1244), especially in a fraction distinguishable from that of ACh-rich synaptosomes by its higher density (448). High 1evels of GAD activity have been found in regions where there is likely to be a high density of inhibitory terminalsfor example, in the hippocampus ( 1138) or in the cerebellum (783, 784). Attempts at producing predictable changes in inhibition by interference with the enzymatic removal of GABA have not been an outstanding success. Neither inhibition nor the action of GABA is greatly altered by various inhibitors of GABAtransaminase (cf. 279, 765). In the experiments of Obata et al. (945) there was some suggestion of an enhancement of inhibition by hydroxylamine. After administration of amino-oxyacetic acid, Gottesfeld et al. (506) have found in the cuneate nucleus evidence of a reduced sensitivity to glutamate and a progressive increase in sensitivity to GABA, as well as in the duration of evoked inhibitions. These changes, which may be due to a slower removal of GABA, are less impressive than might be expected in view of the very large increase in brain GABA content observed at the same time. It therefore is likely that GABA-transaminase plays a relatively minor and probably only indirect role in the immediate removal of GABA from the synaptic region. 4) GABA UPTAKE. Ever since the first proposal that GABA may be an inhibitory transmitter in the brain (393, 748), it has been clear that occlusion by intracellular uptake is the most significant method of removal of GABA from extracellular space. That brain slices accumulate amino acids has been known for a long time (113 1). The first systematic studies on GABA by Elliott and van Gelder (393) showed that slices of cerebral cortex, unlike slices of muscle or of some other tissue, could remove GABA very rapidly from the medium in which they were soaked; this GABA was not destroyed but was held in an occluded form, from which it 454 K. KRNJEVIC Volume 34’ Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 could be released again by suitable treatment. These experiments provided the first evidence that the mechanism of removal of GABA, like that of catecholamines in the autonomic system, was by active uptake into cells rather than by metabolic conversion to an inactive agent. There were further studies by Tsukada et al. (1191) and Strasberg and Elliott (1139); the latter pointed out (cf. also 39 1) that a certain fraction of GABA binding is very dependent on the presence of Naf, and they suggested that this might play a particularly significant role in the removal of extracellular GABA. Much of this uptake is probably into nerve endings, since isolated synaptosomes also accumulate GABA, at least partly by a Na+-dependent mechanism (3 13, 786, 873, 1208). A ccording to DeFeudis and Black (314), there is a more pronounced Na+-dependent uptake of labeled GABA by certain subcellular particles from the cerebral cortex and hippocampus than from other regions of the brain (including the cerebellum). Although many other amino acids are also absorbed by brain slices, it appears that only GABA and the other putative neurotransmitter amino acids are taken up by a high-affinity (as well as the more general low-affinity) type of uptake process (87). Systematic studies by Iversen and Neal (625) and Beart et al. (82) have shown that some fairly closely related amino acids (but not glutamate, aspartate, or glycine) can compete with GABA in this uptake system. It is of interest that differences in an animal’s social environment can be reflected in the degree of activity of these uptake processes (3 13). In recent autoradiographic experiments it was found that when slices or homogenates of the cerebral or cerebellar cortex are incubated in [3H]GABA, radioactivity is preferentially concentrated in interneurons (585) and in some nerve endings (623). From other studies, however, it is evident that there is an important component of GABA uptake into neuroglia (148, 507, 563) that is consistent with earlier evidence of a high glial GABA content (864, 1 ZOO). Summary. GABA is found in substantial concentration (> 10m3 M) in all regions of the CNS. In its subcellular distribution it differs from ACh, being found mostly free in the cytoplasm and relatively little in vesicles, but nerve endings contain sufficient amounts for appreciable physiological effects. It is produced from glutamic acid by a specific cytoplasmic decarboxylase, which is also widely distributed, but is in significantly higher concentration in areas where inhibitory neurons are concentrated. A specific transaminase, associated with mitochondria, accelerates the catabolism of GABA. There is a rapid turnover of GABA, shown by its early labeling after systemic injection of radioactive glucose. Interference with the GABA enzymes leads to changes in brain GABA level, and some corresponding behavioral manifestations (convulsions or drowsiness). All parts of the CNS have a strong ability to take up GABA, by both low- and high-affinity systems (the latter is Na+ dependent), GABA being accumulated in certain nerve endings and also in neuroglia. All those features are consistent with the postulated transmitter function. 5) RELEASE OF GABA. There is now substantial evidence that GABA is released by neural activity, especially in the cerebral cortex and Deiters’ nucleus. The first tentative report of this was made by Jasper et al. (633), who described a continual leakage of endogenous GABA from the cerebral cortex of cats at a variable rate apparently related to the state of consciousness, being greater during sleep than April 1974 VERTEBRATE SYNAPTIC TRANSMISSION 455 Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 during waking. In a much more extensive study Jasper and Koyama (634) essentially confirmed these observations, although they could not detect any GABA under normal conditions: but a release was clearly seen in the enct$haZe isole’ preparation, and it was greatly reduced by stimulation of the midbrain reticular formation; conversely, the output was much greater after a lesion in the midbrain reticular formation. In general there was a reciprocal relation between the release of GABA, on the one hand, and the release of glutamate or acetylcholine, on the other. According to very recent experiments, the output of GABA from the cortex is especially enhanced by stimulation of a very localized region of the central gray of the midbrain (724). It thus seems that the rate of GABA release in the cortex is at least partly under the control of ascending systems possibly related to the reticular ascending pathways. A different approach was used by Iversen et al. (624). They demonstrated an increased efflux of endogenous GABA when the visual cortex was stimulated electrically at 200/set; this type of stimulation (though at a much lower rate) has been shown to elicit a very powerful inhibition of cortical neurons (757). Distant stimulation of the lateral geniculate was also an effective way of raising the efflux. The possibility that GABA is released from inhibitory nerve endings was reinforced by abolition of the evoked release when the cortex was superfused with a Ca2+-free solution. Electrical stimulation of suspension of cortical synaptosomes causes preferential release of GABA as well as excitatory amino acids ( 15 1). Studies have also been made of the release of radioactivity from cortical slices or intact cortex loaded with [3H]GABA (533, 624, 1122). The efflux of label is much accelerated by electrical stimulation and by excess Kf and it is reduced by a combination of low calcium and high magnesium. Comparable results have been obtained from the spinal cord (23 1, 289, 534, 1049). Although the evidence obtained with labeled GABA is highly suggestive, it does not prove that GABA is released as the natural transmitter. There is some reason to think that most nerve fibers can be loaded with a variety of exogenous compounds and these may be released by direct stimulation or even during normal activity (cf. 7 18, 930, 93 1, 1176). Bowery and Brown’s (148) demonstration that labeled GABA is taken up by several peripheral nerves (and even skeletal muscle) is particularly relevant : GABA accumulated by sympathetic ganglia can be released by K+ or by direct electrical stimulation, though not by stimulating the pre- or postganglionic trunks or by injections of carbachol. Since the uptake was not markedly reduced after preganglionic denervation, Bowery and Brown reasonably concluded that GABA is probably accumulated by glial cells, from which it can be released by depolarization. They rightly advise great caution in interpreting the results of this kind of experiment. Cerebellar stimulation sharply increases the release of endogenous GABA into the fourth ventricle (947). This confirms the identity of GABA as the probable inhibitory transmitter released by Purkinje cell axons projecting onto Deiters’ neurons (620, 945, 946, 948). Summary. Further evidence that GABA may be a physiological mediator of inhibition is that it is released in the cerebral cortex and in the fourth ventricle by 456 K. KRNJEVIC Volume 5& Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 inhibitory neural activity. Comparable observations on the release of exogenous GABA are suggestive, but less convincing evidence since many compounds, including GABA, are taken up and released by a variety of cells and fibers and even satellite cells. 6) ANTAGONISTS OF GABA. In crustaceans the action of GABA is rather specifically and predictably antagonized by picrotoxin (cf. 1039). By contrast, in vertebrates such an effect of picrotoxin or any other specific antagonists has not been so easy to demonstrate convincingly. Thus, Curtis (274, 288) found that picrotoxin did not antagonize the action of GABA on spinal neurons, and Krnjevic et al. (759) could not demonstrate a specific block of the inhibition of cortical neurons by GABA. The lack of effect in the spinal cord was not entirely surprising, since postsynaptic inhibition there appeared to be predominantly strychnine sensitive (257, 371). However, when Eccles et al. (379) found that “remote” or “presynaptic” inhibition was depressed by picrotoxin, they proposed that GABA may act as transmitter of presynaptic inhibition. But how could one explain the associated primary afferent depolarization and increase in excitability in view of Curtis and Ryall’s (284) claim that GABA applied by microiontophoresis causes a depression of terminal excitability? Other authors took a very different view of the remote inhibition. Kellerth (689) found that various forms of afferent stimulation, which are said to evoke presynaptic inhibition, can in fact be shown to reduce the excitability of motoneurons and therefore must be really postsynaptic inhibitions, probably generated by somewhat remote dendritic synapses. Furthermore, he observed that this kind of inhibition was readily blocked with picrotoxin. He estimated that of all the varieties of inhibition that can be evoked on spinal motoneurons about half are blocked by strychnine and the other half by picrotoxin. The first direct claim that picrotoxin blocks a GABA-mediated inhibition in the vertebrate CNS was made by Galindo (469) based on observations made in the cuneate nucleus of the cat. In the last few years there has been increasing evidence that picrotoxin antagonizes the action of GABA at various sites: in Deiters’ nucleus (948, 1172), spinal interneurons (402), olfactory bulb (934), cerebellum (1273), cuneate (693, 694), hypoglossal nucleus (ll73), and even autonomic (3 15) and sensory ganglia (3 17, 3 18). Since picrotoxin also blocks neurally evoked inhibition in the cuneate (693, 696), Deiters’ nucleus (1172), spinal cord (689), vestibuloocular pathway (571), cerebellum, (119, 1273), substantia nigra (lOlO), and cochlear nucleus (1232), there is quite strong inferential evidence that many physiological inhibitory pathways act by releasing GABA. The alkaloid bicuculline was discovered by Manske (86 1) and first shown to have convulsant properties by Welch and Henderson (1246). It has been proposed as a specific antagonist of GABA in all regions of the mammalian CNS (262). The results of further experiments by the same authors (263, 264, 271) and also by other authors (119, 693-696, 892, 934, 1169, 1273) support the idea that bicuculline antagonizes relatively specifically GABA (and some “ GABA-like” agents, but not glycine and its relatives) as well as neurally evoked inhibition at several sites in the spinal cord, brainstem, and cerebellum. However, several inde- April 1974 VERTEBRATE SYNAPTIC TRANSMISSION 457 2. Glycine a) Action on nerue cells. Glycine can be considered as the first of the series of short-chain a! ,o-monocarboxylic amino acids and therefore a member of the family of inhibitory amino acids (see 286, 542). In their comprehensive survey of the effects of these agents on spinal neurons, Curtis and Watkins (286) rated glycine as only a weak inhibitor. Its inhibitory potency on cortical neurons was also much less than that of GABA and several other longer chain compounds (748). A renewal of interest in glycine was prompted by the discovery that its distribution in the various quadrants of the spinal cord was consistent with that of an inhibitory transmitter (36). Werman and Aprison therefore repeated the test of glycine on spinal neurons and found it to be comparable in potency with GABA (1253-l 255; see also 3 16, 1060). A relatively high potency of glycine has been observed in the cuneate nucleus (47 1, 693, 695) and in several other areas of the brainstem (307, 610, 1168, 1169, 1172, 1173). To obtain more Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 pendent groups of authors (116, 473, 501, 1143) have failed to observe an effective and specific block of the action of GABA or inhibition in the cerebral cortex. Curtis and Felix’s (27 1) more extensive report on tests of bicuculline in the cerebral cortex describes such weak and inconstant effects (far short of block) as to provide substantial support for the belief that bicuculline is not a universally reliable antagonist of GABA-mediated inhibitions (501). The variety of agents apparently antagonized [including such “glycinelike” compounds as P-alanine and taurine (cf. z%)] confirms the low specificity of action of bicuculline in the cortex. These findings seem to indicate that GABA receptors vary in their susceptibility to block by picrotoxin and bicuculline. The GABA receptors in the cerebral cortex are particularly insensitive, but even in other regions of the CNS, the effects of bicuculline and picrotoxin are evidently not as predictable as the specific block of glycine by strychnine (3 16, 402, 573, 6 10, 999, 1172, 1173). Both picrotoxin and bicuculline may have a direct excitatory effect on many neurons, which greatly complicates the analysis (501, 759) ; the precise mechanism of this has not been studied in vertebrates, but it may be analogous to the reduction of potassium conductance caused by these agents in some invertebrate peripheral nerve fibers (456). Several other convulsants have also given some indication that they may block the action of GABA. The most interesting is ben~ylpenicillin, which has been reported to reduce GABA effects and inhibition both in the cerebral cortex (222) and the spinal cord (272, 302). Summary. Picrotoxin, bicuculline, and some related agents antagonize the action of GABA relatively specifically in the spinal cord and brainstem, but much less effectively in the cerebral cortex. They also block some inhibitory synaptic actions that are probably mediated by GABA. Their usefulness is limited because they may not be competitive antagonists, do not act universally against GABA, and probably have some direct excitatory action. 458 K. KRNJEVIC Volume 54[ Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 critical evidence in support of their suggestion that glycine was an inhibitory transmitter in the spinal cord, Werman et al. (1254, 1255) performed a systematic analysis of its effects on the membrane potential and conductance of spinal motoneurons. These experiments showed that glycine causes a hyperpolarization accompanied by a large fall in membrane resistance, which could be attributed to a pronounced increase in chloride permeability. Further tests indicated a similar reversal level for the action of glycine and for IPSP’s, and the identity of action of the two processes was confirmed by showing that the reversal potentials of both were changed in a similar way by injections of two foreign anions (cf. 1249). The authors therefore concluded that glycine fulfilled several of the main requirements of a putative inhibitory transmitter. These observations were soon confirmed by Curtis et al. (275), who now also found a relatively high inhibitory potency of glycine and similar intracellular potential and conductance changes. Comparable observations were also made in spinal interneurons (1171) and in spinal neurons grown in tissue cultures (606) g Further tests on cortical neurons (116, 690) confirmed the earlier impression (748) that glycine is only a weak inhibitor of most cortical neurons : equal iontophoretic applications of glycine produced less than soth of the conductance increase evoked by GABA, and the reversal potential for the effect of glycine was usually more positive than the reversal potential for the IPSP. Cerebellar neurons are also much less sensitive to glycine than to GABA (687). Sumnzary. Thorough tests have shown that glycine produces a hyperpolarization and fall in membrane resistance of many neurons by an increase in Gor. This inhibitory mechanism is indistinguishable from that of synaptic inhibition and it appears to be identical to the action of GABA. It is best seen in the spinal cord and brainstem, where most cells are sensitive to both glycine and GABA, but is inconspicuous in the cerebral or cerebellar cortex. relevant evidence. 1) DISTRIBUTION, METABOLISM, AND UPTAKE. Glycine b) Other is found in free form not only in all parts of the CNS (33, 35, 1175) but, unlike GABA, also in cerebrospinal fluid, in serum, and generally in most tissues. It is in distinctly higher concentration in the spinal cord, where its inhibitory function is likely to be most important-particularly in the ventral gray matter (35, 36, 646); this is in good agreement with the recent demonstration that the interneurons that mediate direct inhibition are situated rather more ventrally than had been proposed earlier (632). Although there is a rapid turnover of glycine in the CNS, the precise metabolic pathway involved is not entirely clear; one probable source is glucose, via 3-phosphoglyceric acid and serine. In any case there does not seem to be any shortage of glycine required for inhibitory function. The supply of glycine is enhanced by an active uptake by central nervous tissue : this is especially efficient with slices of spinal cord and medulla (926). Like any other putative transmitter amino acid, glycine is taken up by both a lowaffinity and a sodium-sensitive high-affinity mechanism (87), but the high-affinity uptake process is only present in the spinal cord. These findings are consistent with other evidence already discussed that glycine probably plays only a minor role as mediator of inhibition in the upper regions of the CNS. Labeled glycine is concen- April 1974 VERTEBRATE SYNAPTIC TRANSMISSION 459 Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 trated in the gray matter (926) and, more specifically, in synaptosomal fractions (837). However, autoradiographic studies show also a significant uptake into glial cells (584, 608). The high-affinity uptake of glycine is relatively specific, being inhibited by only a few very closely related amino acids (926). Like the other amino acid uptake systems, it is not readily blocked, but there is a moderate antagonism by y-hydroxymercuribenzoate (926), which potentiates the effect of applied amino acids (266). 2) RELEASE OF GLYCINE. Attempts at demonstrating a specific release of endogenous glycine from central nervous tissue have not so far been successful (33). Labeled glycine can be released from preloaded slices of rat spinal cord (534, 599) or from the hemisected frog spinal cord (1049) by electrical stimulation or excess potassium. As has already been pointed out, this type of release could be an experimental artifact. 3) ANTAGONISTS OF ACTION OF GLYCINE. The first evidence that the action of glycine is blocked by strychnine was obtained by Curtis et al. (274). Since strychnine has long been known to abolish the inhibition of spinal reflexes (152, 162, 257, 371, 958), this observation provided further support for the hypothesis that glycine is the natural transmitter of inhibition in the spinal cord (36, 1253, 1254). A clear and remarkably specific antagonism (with moderate doses) has been observed wherever glycine has a significant inhibitory action: for example in the spinal cord (267, 274, 3 16,803) on medullar reticular neurons (610, 1168), Deiters’ nucleus ( 1172), and the cuneate nucleus (469, 573, 693, 695). Several strychninelike agents, such as thebaine and bruceine, also block the action of glycine (274). The striking insensitivity of cortical inhibition to moderate doses of strychnine (168, 252, 759) is evidently consistent with the paucity of glycine-sensitive cells. However, even the presence of a marked sensitivity to glycine, as in the cuneate and Deiters’ nucleus (693, 695, 696, 948, I 172), may not necessarily indicate physiological inhibition mediated by glycine; so far ncne of the inhibitory inputs into these nuclei has been found to be susceptible to block by strychnine. The action of strychnine is by no means entirely specific. Curtis et al. (274) have found that strychnine will block the inhibitory action of some other closely related amino acids, particularly those with a relatively short carbon atom chain, such as P-alanine and taurine, in contrast to the GABA-like longer chain amino acids, whose action is either totally unaffected by strychnine or only when it is given in exceptionally high doses. Strychnine also blocks the depressant action of several other compounds, such as norepinephrine (NE), ACh, and 5-HT (983), as well as some kinds of inhibitions that probably are not mediated by glycine (cf. 14 1, 375, 65 1, 994). There is evidence that strychnine lowers the membrane GK (37, 456); such an action may well explain some of these less specific antagonistic or excitatory effects. Summary. Glycine is found throughout the CNS, as in other tissues; however, its distribution in the spinal cord agrees with that expected of the transmitter released by the inhibitory interneurons activated by IA muscle afferents. Relatively little is known about its metabolism and any essential enzymes, but there is clear evidence of a potent uptake mechanism, with low- and high-affinity components, K. KRNJEVIC 460 Volume 54 especially in the spinal cord. The only successful release studies so far have utilized exogenous labeled glycine. The marked and apparently competitive antagonism of glycine and some inhibitory pathways in the spinal cord (and possibly the brainstem) by strychnine is further evidence that glycine is probably the inhibitory transmitter of the corresponding synapses. C. Excitatory Amino Acids 1. Excitatory actions of dicarboxylic amino acids a) L-glutamate. In small doses, glutamate predictably evokes neuronal firing; when applied in large amounts, the excessive excitation is rapidly converted into a depression of activity, which may be strictly localized to the neurons under observation or may even spread over wide areas of the cortex (spreading depression). Because the excitatory action is so quick and powerful, it is readily observed only when very small amounts of glutamate are applied locally from a micropipette. The most systematic studies therefore have been made using multibarreled micropipettes and microion tophoresis : but the effect can be demonstrated when glutamate is released from a micropipette by applying a suitable pressure (748). The first systematic microiontophoretic study was made on spinal neurons in cats by Curtis et al. (280). Comparable effects were seen in an extensive study of cerebral cortical and cerebellar neurons in several mammalian species by Krnjevic and Phillis (748). Further investigations in practically every region of the CNS have confirmed these observations and shown that glutamate can excite central neurons throughout the vertebrate series (153, 259, 644, 983). i) EXTRACELLULAR OBSERVATIONS. The principal characteristics of the action of glutamate are its quick onset and almost instantaneous cessation. When brief pulses are used to release glutamate, the time course of the discharge seems to correspond to the expected time course of change of concentration in the tissue (731, 748). U n d er optimal conditions, cells can be excited with as little as 10-14lo-l5 mol. The rate of firing elicited by a steady application can be varied according to the amount of glutamate released; it is certainly not an all-or-none action. Desensitization is not a very marked feature, so that firing can be maintained for prolonged periods. The pattern of firing elicited appears to be a characteristic of the cell (472). Although practically all cells are excited by glutamate, some functionally or topographically distinct cells can be shown to be significantly more sensitive (471, 893, 913). Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 The two principal, naturally occurring dicarboxylic amino acids, L-glutamic and L-aspartic, can be considered together since they both have the same kind of excitatory action. As a rule L-glutamate is somewhat more powerful, and of course it is found in much larger amounts in the CNS. It therefore seems likely to play the more important role, so the main emphasis here is on glutamate; however, much of the evidence pointing to a transmitter function for glutamate may apply equally well to L-aspartate. April 1974 VERTEBRATE SYNAPTIC TRANSMISSION 461 Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 Large applications of glutamate cause transient, strong firing and then a disappearance of activity, lasting as long as the release of glutamate continues; a brief period of firing may follow the end of the release. This kind of block by inactivation presumably explains the apparent depressant effect of topical applications (cf. 1011). As van Harreveld ( 1206) first pointed out, applications of glutamate to the cerebral cortex readily elicit spreading depression (807, 869); he therefore suggested that glutamate released from cortical cells may by itself initiate spreading depression. This has recently been questioned by Do Carmo and Leao (345), who have obtained evidence that glutamate only causes spreading depression if associated with a local mechanical disturbance. 2) INTRACELLULAR OBSERVATIONS. Curtis et al. (280) were the first to show, by intracellular recording, that the increase in membrane excitability caused by glutamate is associated with a clear depolarizing effect. The depolarizing action could be maintained during a 30-s application of glutamate with no evidence of marked desensitization; it was accompanied by a reduction in amplitude of EPSP’s and by larger IPSP’s. Intracellular studies on cortical neurons by Krnjevid et al. (73 1, 763) confirmed the depolarizing effect of glutamate and showed that it was associated with a marked fall in membrane resistance (763). In other experiments Curtis (258) compared the reversal levels for the action of glutamate and of EPSP’s. He found a consistent difference between these two, the reversal level for glutamate being always more positive than that for the EPSP’s, which he took as further evidence that glutamate was not the natural excitatory transmitter. The most systematic intracellular studies of the effect of glutamate have been performed by Zieglgansberger and Puil on spinal neurons ( 108, 1285). These authors showed that motoneurons and also other spinal neurons were readily depolarized by glutamic acid, but cells with a low resistance (particularly motoneurons) were not easily made to fire spikes. The depolarization was regularly associated with a fall in membrane resistance. The maximal depolarizations were by 22-30 mV and the maximal conductance increase about 75 %. The ratio of potential change to conductance changes is much greater than is observed when applying inhibitory amino acids (cf. 352, 1255), as might be expected from a permeability change to an ion whose equilibrium potential is very different from the resting potential. The potency of glutamate in producing conductance changes appears to be considerably less than that of GABA (cf. 352). This does not necessarily mean that glutamate is intrinsically less potent; it may simply reflect the greater size of most of the spinal neurons studied. This is also suggested by the relatively slow time course of conductance increase. Zieglgtisberger and Puil (1285) o b served a reversal level for the action of and 0 mV, but noted that these values were glutamate somewhere between -30 probably partly falsified by the distribution of current in the cell and that the true reversal level was probably substantially more positive. In some recent experiments Curtis et al. (265) showed that the depolarizing action of a closely related excitant amino acid, DL-homocysteic, was not affected by intracellular injections of chloride that reverse IPSP’s, so it is unlikely that a chloride permeability change is involved in the excitatory action. Furthermore, since the depolarizing effect was not blocked 462 K. KRNJEVIC by tetrodotoxin, it cannot be mediated propagated action potential. by the sodium Volume channels responsible 54 for the Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 3) MECHANISMOFACTION OF GLUTAMATE. The depolarizing action of glutamate is evidently caused by an increase in membrane permeability (108, 763, 1285) that permits an increased influx of sodium ions. Most of the estimates of the reversal potential obtained so far (108, 265, 1285) h ave been in the range between 0 and -30 mV. They therefore are consistent with a substantial increase in K+ permeability as well as P Na and a AGNa/AGK ratio comparable with that observed when ACh is applied near the muscle end plate ( 1155). However, it would be premature to conclude that similar changes in membrane permeability are produced by these actions of glutamate and acetylcholine, since we have very little information about the sodium equilibrium potential in motoneurons. If ENa is not at a very positive level, the effect of glutamate could involve mainly Z&. Although glutamic acid has a substantial chelating action, it is unlikely that its excitatory effect can be simply ascribed to the removal of Ca2+ from the neuronal membrane by a straightforward chelation. This is clear from experiments of Curtis et al. (277) that showed that much stronger chelators than glutamate were much less effective as excitants. This was also found to be the case in the cerebral cortex (748). However, some central neurons, such as the relay cells excited by hair afferents in the cuneate nucleus, are markedly sensitive to such relatively mild chelators as citrate and ATP, which excite some of these cells very much like glutamate (though less predictably) (472). Th ese observations suggest that the action of glutamate may involve the displacement of Ca2+ from critical sites on the membrane, but other factors such as the glutamate molecules’ shape and charge distribution are likely to play at least as important a role as its chelating power in aqueous solutions. Some confirmation of this has been obtained recently by in vitro experiments with synaptic membranes from the guinea pig brain that show that glutamate and other excitant amino acids specifically tend to mobilize membrane-bound Ca2+ (1159). It therefore is possible that the permeability change induced by glutamate is initiated by the displacement of Ca2+ from certain membrane sites that control PNa. It should be noted that, like acetylcholine and GABA, glutamate is only effective as an excitant when injected outside the cells (cf. 240, 691). Its essential interaction is clearly with some surface “receptor.” b) Actions of aspartic acid. In most respects the effects produced by L-aspartate are similar to, but often somewhat weaker than, those of glutamic acid (277, 748), though some lateral geniculate cells respond somewhat more vigorously to aspartate than to glutamate (913). The few intracellular studies performed with aspartic acid have shown the same kind of depolarizing action (277). There is no evidence of any mutual interference between the actions of these two amino acids. c) Other releuant confounds. Somewhat comparable excitatory effects can be obtained with a variety of compounds related to L-glutamic acid (287, 288, 748). D-Glutamic acid is consistently much less effective, but its action is similar in time course, and it does not interfere with that of L-glutamate. On the other hand, D-aspartic acid is not very different in potency from L-aspartic. Curtis and Watkins April 1974 VERTEBRATE SYNAPTIC TRANSMISSION 463 2. Other relevant evidence The significance of the “transmitter-like,” strong and quickly reversible, but by no means unique, excitatory action of glutamate is very much enhanced by the wealth of evidence that glutamate is not only a naturally occurring constituent of the CNS, but is also exceptionally readily available through metabolism and is very effectively accumulated by central neurons. a) Presence of glutamate in CNS. Glutamate is well known as the amino acid found in highest concentrations (5-10 pmol/g) in the brain and other parts of the CNS. Early reports (1, 2) gave rather high estimates of cerebral glutamate because they included amino acids hydrolyzed from protein. The first reliable measurebetween glutamic ments of free glutamic acid in tissues, *with a clear distinction Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 (286) showed that one of the carboxylic groups of glutamate can be substituted with a sulfonic group without reducing the excitatory efficiency; in fact, this may give even stronger excitant properties. Thus, both L- and n-cysteic and -homocysteic acids are more potent than glutamic. The strongest of the excitant amino acids, with a quickly reversible, glutamatelike action, is D-homocysteic. An even more potent but much slower excitation is seen with the N-methyl derivatives of aspartic : N-methyl-D-aspartic acid may be the strongest depolarizing agent of the whole group (287). A f ew less closely related compounds have also been shown to have strong glutamatelike excitatory effects; of these the most interesting are the naturally occurring ibotenic acid, commonly found in certain mushrooms (647), and a Lathyrus neurotoxin (895a, 1233a; K. Krnjevid and M. *&lorris, unpublished observations), both of which are significantly more potent than L-glutamate. As originally emphasized by Curtis and Watkins (286) the characteristic excitatory effect is seen only with compounds having two negative charges and one positive charge at the correct separation: hence little or no activity is observed when any of these essential requirements is missing. For example, the following compounds, though very closely related, are largely inactive: the simple dicarboxylic acids, such as ketoglutaric; the amide of glutamate, glutamine; the longer chain dicarboxylic amino acids, amino pimelic and amino adipic; and the dipeptide, y-glutamyl glutamate. Summary. Very small amounts (e.g. 10-l* mol) of L-glutamate and aspartate excite most central neurons quickly and reversibly; larger amounts cause inactivation, after transient firing. This excitation is associated with membrane depolarization and a fall in resistance, presumably due to a rise in GNa, which is not sensitive to tetrodotoxin. The reversal level for the action of glutamate appears to be between 0 and - 30 mV, probably indicating an increase in Gg as well as Gm. There is some evidence that the mechanism of excitation is initiated by a mobilization of membrane-bound Ca 2+. A comparable or even stronger excitation is also produced by several related compounds not normally found in the brain: the sulfonic derivatives, cysteic and homocysteic acids, N-methyl aspartic acid (especially potent but slow), ibotenic acid, and a Lathyrus toxin. 464 K. KRNJEVIC Volume 5# Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 acid and glutamine were made by Krebs et al. (728). This work marked the beginning of modern investigations into glutamic acid and its role in cerebral function (cf. 1225, 1240). Studies of free ammo acids in tissues were greatly accelerated and made more precise by the introduction of continuous column chromatography (1158) and of micromethods that permitted estimations in small samples of tissue (107). Surveys of different regions of the brain (107, 1100, 1157) show that glutamate is present everywhere, but is in higher concentration (10 pmol/g) in the forebrain and cerebellum: in various parts of the brainstem, such as the medulla and pons, the concentration is about 50 %I less. A comparable lower concentration is also observed in the spinal cord. However, there are systematic variations in distribution within the spinal cord that may indicate that glutamate is released by specific pathways (512). Thus glutamate is the only amino acid consistently at a higher concentration in dorsal roots than in ventral roots, in agreement with the possibility that glutamate is the excitatory transmitter released by primary afferent fibers (471). On the other hand, since the ventral gray matter contains less glutamate than does the dorsal gray matter, but has a relatively high aspartic acid content, it was suggested that aspartate might be the transmitter released by polysynaptic pathways. The fact that a relatively selective destruction of spinal interneurons by ischemia is associated with a well-correlated loss of aspartic but not glutamic acid has been interpreted as supporting this hypothesis. b) Subcellular locali<ation of glutamate. Glutamic acid, like most of the other free amino acids, including aspartate, GABA, and glycine, is widely distributed in neuron al cytoplasm (860, 1059) and there is no evidence of specific sequestration in nerve endings, particularly by binding to synaptic vesicles (cf. also 1032). Nevertheless, because of the quite high concentration of glutamate in cytoplasm, the amount of glutamate held in nerve endings-presumably by virtue of their cytoplasmic content-is quite appreciable (860); in fact it is adequate for substantial excitatory effects on nerve cells (769). c) Glutamate uptake. The active absorption of glutamic acid by brain slices was first observed by Stern et al. (1131), who found that glutamate can be removed from the bathing medium against a very high concentration gradient. This phenomenon has been confirmed and studied under various conditions by many authors (e.g. 56, 57, 837, 1149, 1191). The glutamate uptake into synaptosomal fractions has been studied particularly by Logan and Snyder (837), who demonstrated a specific high-affinity uptake mechanism, which is especially dependent on the presence of sodium ions. Much emphasis has been placed on the existence of high-affinity uptake processes as evidence for a significant synaptic function, but some doubts about this interpretation seem indicated in view of the presence of low- and high-affinity uptake mechanisms in a variety of nonneural tissues (218). Balcar and Johnston (57) have studied the susceptibility of the glutamate uptake mechanism to inhibition by closely related compounds or other agents. They were able to show that the uptake was not blocked by tetrodotoxin nor by several of the most potent amino acids, such as N-methyl aspartic and DL-homocysteic, and therefore concluded that the uptake was not simply related to de- April 1974 VERTEBRATE SYNAPTIC TRANSMISSION 465 Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 polarization and the entry of sodium ions, either through tetrodotoxin-sensitive or -insensitive channels. On the other hand, the uptake is not wholly specific since\ it is strongly inhibited by other agents, such as cysteic acid and aspartic acid. There have been relatively few studies on the cellular localization of glutamate taken up from surrounding medium. Studies on the retina suggest that glutamate and aspartate may be absorbed by glial cells rather than neurons (382) as in some invertebrate preparations (1067). Glutamate is also taken up actively by periph-era1 nerves (311, 1259) but it is not certain whether this uptake is mainly into% sensory or motor fibers (or both), or alternatively into sheath cells, such as Schwann cells. In any case, very powerful uptake mechanisms are evidently available for theremoval of any extracellular glutamate in the brain and other parts of the nervous system. d) Metabolism of glutamate. It is clear from the many reviews on the subject (1185, 1203, 1225, 1240) that cerebral glutamate is a very active metabolite. There is general agreement that the main source of brain glutamate is glucose, via the evident when labeled glucose is adKrebs cycle ( 1050, 1185). Th is is strikingly ministered systemically: a remarkably high percentage of the labeled carbon enters the brain within a few minutes, most of which is incorporated in a few amino acids, over half into glutamate (75, 467, 468, 1222, 1283). As Vrba et al. (1222) pointed out, the transformation of glucose into amino acids occurs much more rapidly in the brain than in any other organ. Clearly, the main pathway of glucose metabolism in the brain proceeds through the synthesis and oxidation of amino acids, particularly glutamate. By this kind of experiment it can be shown that glutamate is present in at least two different compartments in the brain. One compartment is very quickly and effectively labeled with systemic glucose and is closely linked with GABA; presumably this is the glutamate from which GABA is synthesized by decarboxylation. Another compartment of glutamate is labeled more effectively by injections of radioactive pyruvate and acetate, or by exogenous glutamate, and it is particularly closely linked with glutamine (105, 106, 197, 790, 1055). The cellular or subcellular localization of these compartments has not yet been worked out in any detail. e) Release of glutamate in CM. There is still only very limited evidence that -or any other excitatory amino acid -is L-glutamate released by the activity of nerve endings in any region of CNS. The most convincing data are those of Jasper and Koyama, who have made some systematic studies of amino acid release from the surface of the cerebral cortex and have been able to show that it is consistently correlated with the state of cortical activity (634). The leakage of endogenous glutamate into fluid superfusing the cortex of enct$haZe isole’ preparations of cats was greatly increased by stimulating the reticular formation so as to evoke electrocorticographic arousal. There is some evidence that labeled glutamate is released by electrical stimulation of cortical slices (533) or by stimulating suspensions of cortical synaptosomes (15 l)., With amphibian preparations an increase in the rate of release of both glutamate and aspartate can be evoked by electrical stimulation of the isolated hemisected spinal cord ( 1049) or sciatic nerves (3 12, 126 1). 466 K. KRNJEVIC Volume 54 Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 Although the evidence for a significant release of glutamate (or aspartate) is still hardly very conclusive, this should not be surprising, even if L-glutamate is a transmitter, in view of the high tissue capacity for uptake. f) Antagonists of glutamate and other excitatory amino acids. There is no convincing evidence at the present that the action of glutamate can be specifically blocked. A number of possible antagonists have been proposed in recent years, but none has been tested sufficiently critically to carry conviction. Many agents of course will block excitation -for example, local anesthetics, such as procaine, and a variety of agents that appear to have a similar effect on excitable membranes (cf. 278, 748, 75 1). It therefore is essential that the postulated antagonist should produce no change in postsynaptic excitability, either by a local anesthetic type of action, by a GABA-like inhibitory action, or by any other kind of direct depressant effect. The first suggestion of a specific antagonist was made by Boakes et al. (139), who observed a consistent block by LSD 25 of the excitatory action of glutamate on brainstem neurons susceptible to excitation by 5-HT. Since LSD has a quite marked local anesthetic action on peripheral nerve fibers (342), the effects on central neurons may well be nonspecific (75 1). *McLennan and his colleagues have recently proposed a number of glutamate derivatives as specific antagonists of the action of glutamate, in particular DL-amethyl glutamate and L-glutamic diethyl ester (528, 529). This claim, however, has not received much support from the experiments of Curtis et al. (265); it is made particularly questionable by the finding of Zieglggnsberger and Puil (1285a) that both these agents produce a large increase in the membrane conductance of spinal motoneurons and thus appear to block excitation rather like glycine. Some previous suggestions that L-methionine-Dr,-sulfoximine and Z-methoxyaporphine might be specific antagonists of the excitatory amino acids have been only partly substantiated by recent experiments (265). Much more thorough and extensive testing will be needed to find out whether another recently proposed specific blocking agent, 1-hydroxy-3-aminopyrrolidone 2 (146, 305), is a genuine antagonist and not another GABA-like agent (276). Summary. L-Glutamate is found in high concentration in cells throughout the nervous system, although with substantial regional variations. In the spinal cord its distribution is consistent with the possibility that glutamate is released by the primary afferent fibers. Its subcellular localization indicates relatively little bindbut nerve endings nevertheless contain functionally significant ing to particles, amounts. All nervous tissue examined has demonstrated a strong capacity for the uptake of glutamate; as in the case of the other putative transmitter -amino acids, there are low- and high-affimty components of uptake, the latter being especially Na+ dependent. Glutamate takes part in several important metabolic processes and can be shown to exist in two or more functional compartments, which may reflect its utilization by various kinds of excitatory or inhibitory neurons. There is some limited evidence that endogenous glutamate is released as a result of neural activity in the cortex, whereas labeled glutamate has been shown to be released from brain and spinal cord slices or synaptosomes. April IV. 1974 VERTEBRATE SYNAPTIC TRANSMISSION 467 CATECHOLAMINES A. Introduction Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 The presence of catecholamines in the nervous system has been known for a long time; in fact the first specific mechanism of neurotransmission ever suggested was an adrenergic one (395). Ever since, these compounds have kept busy a rapidly growing number of investigators (for extensive reviews see 4, 126, 198, 245, 726, 912, 1056, 1057, 1216, 1220). The most significant advances in knowledge have been the following: the transmitter released by adrenergic fibers is in fact norepinephrine (12 16) ; the two broad classes of adrenergic effects [thought to be mediated by different agents, sympathin E and sympathin I (cf. 198)] can be accounted for by two principal kinds of receptors, the cy- and ,&types, found on many cells, and having different sensitivity to various agonists and antagonists (10, 816, 933); reuptake by the presynaptic nerve terminal is probably the most important mechanism of removal of NE liberated at the synapse (47); histochemical techniques are now available that are capable of showing the localization of catecholamines in various tissues (202); finally, the development of a remarkably effective agent capable of destroying relatively selectively neurons rich in catecholamines has provided an outstandingly useful tool for studies of the function of adrenergic pathways (859). Although it seemed for a time that all adrenergic junctions operate by the release of NE, the discovery of dopamine (DA) in certain nerve cells of the CNS (201) led to numerous studies of the hypothetical dopaminergic pathways and of their chemical characteristics (cf. 62, 601, 602, 1113) including, m.ost recently, possible dopaminergic neurons even in sympathetic ganglia ( 120). Before considering in more detail the mechanism of adrenergic junctional action, it may be useful to summarize the principal known features of the synthesis, storage, and release of catecholamines. The pathway of synthesis is as follows. The immediate precursor of DA, L-dopa, is obtained from phenylalanine or tyrosine by hydroxylation; this ratelimiting step in the synthesis appears to be regulated by the concentration of its products and it is mediated by tyrosine hydroxylase, an enzyme found in soluble form in neuronal cytoplasm. A relatively unspecific-also cytoplasmic-decarboxylase converts the amino acid dopa to dopamine. The accumulation of DA in cytoplasm is limited by monoamine oxidase, which converts catecholamines to inactive compounds. However, DA can be taken up by intraneuronal granulesvariously known as dense-core, osmiophilic, or granular vesicles-by a process that In the storage granules requires Mg2+ and ATP and can be blocked by reserpine. DA may be converted to NE, through the action of dopamine-P-hydroxylase. Although NE is thus synthesized inside these intraneuronal vesicles, an efficient uptake mechanism makes possible the recapture of most of the NE released from nerve terminals, which otherwise would be disposed of by the predominantly extracellular catechol-o-methyltransferase. For detailed evidence on these various 468 K. KRNJEVIC: Volume 45 B. Catecholamines in Peripheral Transmission Adrenergic mechanisms (acting by the release of NE) have been the most extensively studied. Until recently DA was thought of principally as a precursor of NE, although DA appears to be the predominant catecholamine transmitter in invertebrates (1247). But there is now growing interest in the possibility that DA may itself be a transmitter, released by dopaminergic neurons (see below). The postganglionic sympathetic neurons are generally believed to produce their effects, whether excitatory or inhibitory, mainly through the liberation of NE (485, 983, 1104, 1146, 1215-1217). 1. Norepinephrine actions on smooth muscle There is a great deal of information about smooth muscle, especially in the intestine, where NE has an inhibitory action. The excitatory effects are best seen in vascular smooth muscle or in the vas deferens. The latter especially has been investigated in great detail (482, 590, 592, 1101, 1146). a) Excitation. The vas deferens is very rich in NE-containing nerve terminals. It is characterized by the presence on most of the muscle cells of closely apposed terminal varicosities, containing small and large granular vesicles (9 1, 188, 1146). Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 points, the reader is referred to the many available reviews (46-48, 242, 495, 496, 582, 601, 717, 858, 924, 1054, 1216, 1217, 1241, 1242). In adrenergic cells NE is probably stored in both large and small granular vesicles, but in their terminals it is present mainly in small vesicles, apparently bound to ATP and a specific protein, chromogranin. The number of vesicles in a terminal varicosity is approximately 1000, and there are about 15,000 molecules of NE per vesicle (25, 188, 477, 582, 1103). The release of NE and other catecholamines has also been studied, though less extensively (47,495, 525,612, 1056, 1104). A s in other kinds ofjunctions the release process depends on the presence of Ca2f and is inhibited by Mg2f. Although miniature junctional potentials (592) indicate a quanta1 release-which is consistent with a process of exocytosis from vesiclesthere is a marked discrepancy between the apparent vesicle NE content and the amounts released. If one assumes that every nerve impulse liberates NE from all the terminal varicosities of a given fiber, the mean release per varicosity (about 400 molecules) is only about 2.5 % of the estimated content per vesicle (527). Can vesicles release just a small fraction of their NE content? Perhaps the vesicles from which the release takes place contain untypically small amounts of NE. Of course NE might not be released directly from vesicles, but rather from some other compartment (497), possibly even a cytoplasmic pool. But then how can one explain the surprisingly wasteful simultaneous and proportional release of the normally intravesicular proteins, dopamine-Bhydroxylase and chromogranin (48, 1104, 1243) ? The most questionable assumption is that all varicosities necessarily contribute to the observed release. April 1974 VERTEBRATE SYNAPTIC TRANSMISSION 469 Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 Intracellular recording has revealed spontaneous miniature activity (193) in many ways comparable to that seen in skeletal muscle (cf. 669). Junctional potentials are also evoked by nerve stimulation (192, 780). They are depressed by phentolamine and other a-blocking agents, though only by relatively large doses (20, 194, 780). Since transmission is not fully blocked by doses of cY-antagonists 1000 times greater than are necessary to abolish the effects of NE, Ambache and Zar (20) have argued that transmission cannot be adrenergic. However, this does not necessarily follow. Many of the nerve-muscle junctions in this tissue have only a very small intercellular gap (2OOA), so the transmitter liberated from the terminal must be at a very high concentration (approaching 10m3 M, as in other junctions of comparable dimensions). Unless the receptors have a particularly high affinity for the blocking agents, it may well be impossible to obtain a total block without using extremely high doses of antagonists, which are liable to produce nonspecific effects (cf. 1146). On the other hand, when applied directly, NE interacts with a much wider population of receptors (probably including extrajunctional ones) : therefore strong effects are produced by much lower concentrations of NE, which are readily counteracted by moderate doses of antagonists. Neuromuscular transmission should be blocked more easily when the nerve terminals remain at some distance from the muscle fibers. This is indeed the case in the vas deferens of very young animals ( 1147) and, in the adult, in vascular smooth muscle, where phentolamine and other cy-antagonists readily block both nerve-evoked contractions and the excitatory effect of NE (593). The problem is by no means settled. According to the most recent study of Ambache et al. (19), tyramine, which releases endogenous NE, has a mainly inhibitory action on the vas deferens that can be blocked by a-antagonists. These authors therefore conclude that the motor innervation cannot be noradrenergic. There is no real evidence about the mechanism of the presumed adrenergic excitations (cf. 300, 592, 1114), but it has generally been assumed that it is similar to that of the cholinergic excitation of skeletal muscle (592). Reversal potentials and changes in membrane conductance are not easily studied: owing to intercell coupling, junctional potentials can be recorded far from their site of origin throughout a region that includes several cells. b) Inhibition. There is a greater amount of information about membrane phenomena accompanying inhibition than for excitation. The smooth muscle of the intestine shows predominantly inhibitory (both cyand /3-) adrenergic effects (485, 590, 592, 781), but uterine and vascular smooth muscle is clearly inhibited only by P-agents or by NE after blockade of a-receptors ( 147, 299, 300, 593, 1114). The mechanism of the inhibitory action on the intestine has been studied particularly in the taenia coli by Biilbring and her colleagues (49, 177-180, 187, 852, 1182). Th ese authors were at first inclined to believe that NE primarily activates metabolism and an electrogenic ion pump, thus causing hyperpolarization and inactivation of the spikes (e.g. 187; cf. 1109). An alternative hypothesis was that an increase in metabolism promotes Ca2+ binding in the membrane, thus reducing I& (178). 470 K. KRNJEVIC Volume 54 Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 After Jenkinson and Morton’s (638, 639) finding that NE causes a large increase in K fluxes in taenia coli (but little change in fluxes of Na+ or Cl-), Biilbring and Tomita (179, 180) made further experiments and indeed observed a large increase in membrane conductance that could be attributed mainly to a rise in PK (and, to a lesser extent, in P,r). Since the effect is so dependent on the external Ca2+ concentration, it was suggested that membrane-bound Ca2+ induces a high Kf permeability. They agreed with Jenkinson and Morton’s (639) opinion that this is an a-receptor action. But when this is selectively blocked, a P-receptor action is revealed, which also inhibits the generation of spikes or contraction, even of fully depolarized muscle. The ,&effect is believed to be due to an interference with the spike pacemaker potential and, presumably, the influx of Ca2+ (639). Catecholamines would thus affect membrane Ca2+ in two ways. In the a-mode they would promote membrane binding of Ca2+, which somehow enhances movements of K+. This effect is perhaps comparable to the potentiation of G, caused by intracellular injections of Ca2+ in spinal motoneurons (740) ; in both cases the critical step may be an interaction between Ca2+ and the inner surface of the cell membrane. In the P-mode catecholamines would immobilize membrane-bound Ca2+ or reabsorb free Ca2+ and so prevent depolarization and generation of the pacemaker potential (180, 852). It is of interest that comparable a- and @-membrane effects are seen with liver cells (544, 545). According to the observations of Kao et al. (619, 657) the P-adrenergic action causes a hyperpolarization without changing membrane conductances, apparently by increasing the negative level of &; this was thought to indicate a primary metabolic effect, which enhances the intracellular accumulation of Kf. The initial mechanism of the P-action is widely believed to be mediated by activation of a membrane adenyl cyclase and the consequent formation of cyclicAMP (CAMP) (161, 354, 575, 1052). The further steps, however, are not clear. Cyclic-AMP is known to promote the phosphorylation of various enzymes, especially those concerned with glycolysis in muscle. The P-inhibitory action could then be explained in several ways. The increased supply of glucose could accelerate outward pumping of Na+ (and therefore also initiate an “electrogenic” hyperpolarization). Alternatively, CAMP could directly, or through some metabolic reaction, alter Ca2+ levels in or near the membrane (cf. 179, 180). Finally, CAMP could regulate specific ion permeabilities by phosphorylating a membrane protein concerned in ion movements (688). However, as pointed out by Daniel et al. (300) and Polacek and Daniel (1002), there is little real evidence that CAMP mediates changes in membrane properties evoked by P-receptor actions. A fall in & (which may or may not be secondary to changes in Ca 2f binding) may be a direct effect of the catecholamine. The hypothesis that NE activates an electrogenic pump has not had much support recently (147, 300). Summary. Both excitatory and inhibitory adrenergic innervations have been described. The motor innervation of the vas deferens and of vascular smooth muscle may operate by the release of NE, but the evidence is not conclusive, particularly for the vas deferens, where junctional transmission is not readily blocked by NE antagonists. The mechanism of excitation has not yet been elucidated. April 1974 VERTEBRATE SYNAPTIC TRANSMISSION 471 Inhibitory actions are more common and they have been analyzed in much more detail. They appear to have two components. The first, which is blocked by cu-antagonists, is associated with an increase in membrane conductance, probably due to a rise in GK. The second is blocked by P-antagonists and is not associated with a conductance increase; its mechanism has been variously explained as involving stronger binding of Ca 2f to the membrane, a reduction in GNa, the liberation of CAMP and/or activation of Na-K pumping (probably not electrogenic). actions on ganglia Sympathetic ganglion cells show a late inhibitory effect of preganglionic stimulation, the slow IPSP, which is probably mediated by a catecholamine inhibitory transmitter (380, 705, 706,824,827). This IPSP has rather unusual properties : it is depressed by depolarization and increased by moderate hyperpolarization; it is not associated with a detectable change in membrane resistance; and it is relatively insensitive to changes in external K+ or Cl- concentrations (705, 939). Nishi and Koketsu (939) therefore suggested that this IPSP is generated by an Kobayashi and Libet (705) could not produce a electrogenic Na pump; however, selective block with ouabain and therefore considered the hypothesis of an electrogenie Na pump very unlikely. After demonstrating a close similarity between the slow IPSP and the hyperpolarizing effect of injections of NE, Kobayashi and Libet (706) concluded that it must be produced by adrenergic fibers, although they could not explain its mechanism. The finding that the small, intensely fluorescent ganglion cells-the probable -contain DA rather than NE (120) has led adrenergic inhibitory interneurons Libet and Tosaka (827) to reexamine the properties of the postulated adrenergic inhibitory system: DA proved to be as effective an inhibitor as NE. This action of which are blocked by phenoxybenzamine but DA is mediated by a-receptors, rather insensitive to phentolamine or P-blockers. Since a block of dopamine-/3hydroxylase activity (which converts DA to NE) potentiates the IPSP, it appears that the natural inhibitory transmitter is indeed more likely to be DA. The same authors made a further, unexpected observation: DA has a remarkably potent and prolonged facilitatory action on the slow, muscarinic EPSP. Kebabian and Greengard (688) have now found in these ganglia an adenyl cyclase that is stimulated by small amounts of DA, actz’ng as an a-agent (cf. 1052, 1053). They suggest that CAMP and a protein kinase phosphorylate a membrane protein involved in ionic permeability. These authors did not specify what change in permeability is actually produced, but a simple explanation could be a reduction in Na+ permeability (300,400, 1094). This would be consistent with the depression of the slow IPSP by depolarization and the absence of large changes in resistance. Everything else being equal, the change in membrane resistance needed for a given potential change should be proportional to the initial membrane conductance (G) for the relevant ion. Hence the resistance would rise much less during an IPSP generated by a fall in G Na than during an EPSP of comparable amplitude caused by Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 2. Cutecholamine 472 K. KRNJEVIC Volume 54 C. Catecholaminesin CNS Transmission There is a continual large output of literature dealing with the presence, synthesis, degradation, uptake, and other aspects of the metabolism of catecholamines in the CNS and its possible relation to cerebral function and general behavior (for some reviews see 126, 133, 465, 495, 497, 622, 697, 983, 984, 1197, 12 16, 124 1). However, since much of this cannot yet be related to specific mechanisms of synaptic transmission, the present discussion is concerned mainly with evidence demonstrating clear effects of catecholamines on central neurons, especially where they appear to be related to synaptic mechanisms. Both excitatory and inhibitory effects of catecholamines have been observed. As a rule the main catecholamines have comparable actions, but the order of potency or direction of action may vary greatly in different regions and perhaps under different conditions, e.g. of anesthesia. No attempt is made to enumerate the relative potencies at various sites, but in general norepinephrine appears to be more active in the spinal cord, lower brainstem, and cerebellum, whereas dopamine is often the more effective agent in various parts of the forebrain. Dopamine is considered in greater detail when dealing with areas such as the striatum, where it is present in particularly large amounts and where it is believed to be playing an especially important transmitter role. 1. Depressunt actions a) General. The most commonly observed actions are depressant. They have been seen in the lateral geniculate (260, 989), cerebral cortex (75 1), hippocampus (118), pyriform cortex (809), hypothalamus (137), olfactory bulb (1064), striatum (132,567), thalamus (986), medial geniculate (1164), spinal cord (401), red nucleus (687, 1090, 1091, 1279), and midbrain (308), medulla ( 140, 153, 247) ; cerebellum reticular formation and superior colliculus ( 114 1). Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 a fall in GK, because the resting G&GK is likely to be only about Ho. It might be difficult to demonstrate conclusively such a small resistance change with certainty. In support of this scheme, McAfee and Greengard (879) have recently obtained some evidence that both DA and monobutyryl CAMP hyperpolarize the ganglion cells and that these effects and the slow IPSP are potentiated by theophylline, which speeds up the accumulation of CAMP in the ganglion, probably by inhibiting phosphodiesterase activity. Summary. There is strong histochemical and pharmacological evidence that the slow IPSP evoked by preganglionic stimulation is mediated by small interneurons releasing either NE or (more likely) DA. This inhibitory action is mediated by a-receptors; it is associated with no marked change in resistance, and the IPSP The mechanism of action has been ascribed to is depressed by depolarization. activation of an electrogenic pump or a reduction in GNa. There is some evidence that the NE/DA receptor is an adenyl cyclase and that CAMP mediates the change in excitability. L4pril 1974 VERTEBRATE SYNAPTIC TRANSMISSION 473 Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 These blocking effects have a variable, though often relatively quick, time course, but they are seldom as sharp as the inhibitory actions of o-amino acids (see above). Although attempts have been made to correlate the sensitivity of various cells with the presence of pathways postulated to be catecholaminergic, largely on the evidence of specific histofluorescence (cf. 291, 465), such correlations have not been very conclusive. For example, Hongo and Ryall (598) did not find sympathetic preganglionic neurons to be particularly sensitive to NE, even though they appear to receive a rich innervation from descending NE-containing fibers. More recent findings have raised some doubts about a proposed adrenergic inhibitory system in the olfactory bulb ( 1065). The extensive studies of Lundberg and his associates, while providing strong inferential evidence for the involvement of a descending adrenergic pathway in the control of spinal activity, have not yet succeeded in demonstrating the precise site of adrenergic regulation (9, 103, 401). There is well-documented evidence that the hypothalamus contains particularly large amounts of NE (29 1, 1196, 12 11, 12 12), that direct applications of NE in the region of the hypothalamus lead to changes in body temperature (428) or in behavior [especially eating (cf. 443, 520, 8 12, 905)], that NE is released in the hypothalamus (1123), and that when applied by microiontophoresis NE excites some hypothalamic neurons (137, 727, 917) ; nevertheless no adrenergic pathways have been demonstrated in the hypothalamus with any certainty or even a high degree of probability. 6) Norepinephrine in cerebellum. Bloom and his collaborators believe that they have obtained good evidence for the operation of an adrenergic inhibitory system in the cerebellar neocortex. Yamamoto (1279) was the first investigator to observe a depressant action of NE applied microiontophoretically on neocerebellar neurons in the cat. A depression of the spontaneous firing of Purkinje cells has been seen by many later authors (502, 503, 578, 687, lOgO>, but it is not clear whether the same kind of depressant mechanism operates in all species: the effects of NE are quite rapid in the rat, being approximately synchronous with its application (578), whereas in the cat they are slow and prolonged and somewhat less obvious (687, 1279). Norepinephrine is not as strong an inhibitor as GABA; the typical effect is a partial depression of firing, even when NE is given in amounts several times greater in potency between than doses of GABA (578, 687). Th ere is no marked difference NE and other catecholamines, although the action of NE is diminished more effectively by a P-antagonist (MJ-1999) than by other adrenergic blocking agentsmost of which, however, are not readily tested on cerebellar neurons because of “local anesthetic” effects (578). According to the same authors, monononspecific amine oxidase (MAO) inhibitors enhance the action of applied NE. Does this depressant effect of NE bear any relation to an adrenergic inhibitory innervation of the cerebellar cortex? The histofluorescence technique of Hijkfelt and Fuxe (583) has revealed in the cerebellar cortex of the rat a widespread plexus of rather sparse, NE-containing nerve fibers and terminals that probably originate outside the cerebellum, presumably in the brainstem. According to further studies (954), the cells of origin are all in the nucleus of the locus coeruleus, situated in the 474 K. KRNJEVIC Volume 5? Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 floor of the fourth ventricle. Using a combination of histofluorescence and electronmicroscopic and radioautographic techniques, Bloom et al. (135) have shown that some terminals on Purkinje cells contain or take up NE and that these terminals tend to disappear after treatment with 6-hydroxydopamine. But the vesicles in these terminals are neither flattened [as might be expected of inhibitory fibers (143, 979, 1194)] nor of the small or large granular type characteristic of adrenergic terminals (cf. 188, 477, 1103). It is curious that, in the cerebellum, large granular vesicles are seen most commonly in the terminal of the powerfully excitatory climbing fibers (802). The morphological observations thus are not very consistent. But Siggins and Hoffer and their coworkers (580, 1093) h ave obtained some further evidence supporting the hypothesis of an inhibitory projection from the locus coeruleus : discrete electrical stimulation of this nucleus apparently causes a widespread inhibition of cerebellar Purkinje neurons (but having a very much slower time course than the effects of NE). This inhibition is said to be potentiated by inactivation of cyclic nucleotide phosphodiesterase [which would augment tissue levels of CAMP, the supposed mediator of the action of NE (see below)] and blocked specifically by drugs that may interfere with the supply of NE, such as reserpine and cr-methylpara-tyrosine, or the production of CAMP (prostaglandins) or that destroy noradrenergic pathways (6-OH-dopamine) (580). On the other hand, observations of the general behavior and motor activity of rats treated with sufficient 6-OH-dopamine to eliminate permanently most of the cerebellar NE have not revealed any prolonged marked impairment, even when the lesions are made in very young animals (127, 626, 1162). The contribution of the supposed adrenergic inhibitory system to cerebellar function therefore is not very clear. Summary. The most commonly observed effect of NE (and other catecholamines) in most parts of the CNS is depression of firing. Though variable, it is usually quick and reversible, but is seldom as striking as the action of inhibitory amino acids. Attempts to correlate NE sensitivity with histochemical evidence of adrenergic innervation have not been very successful, with the possible exception of a proposed adrenergic projection from the locus coeruleus to the neocerebellum. In the rat particularly, cerebellar Purkinje cells are quite sensitive to NE, which appears to inhibit firing by a P-action. There is some evidence that stimulation of the locus coeruleus also inhibits these cells, but the existence and the specificity of the postulated connection are not yet fully established. c) DoFamine in striatum and other parts of CNS. The presence of large amounts of DA in the brain, especially in the striatum, was discovered by Carlsson (201). It has been confirmed by extensive studies of DA and its metabolism (495, 497, 601603,636, 1197). These indicate the possibility that DA is a central neurotransmitter. A substantial action of DA was first demonstrated on cortical neurons by Krnjevid and Phillis (75 1). Several catecholamines, applied by microiontophoresis, depressed unit firing, the most potent being DA. Comparable depressant actions of DA were observed subsequently in some other regions of the forebrain: the hippocampus ( 118, 566) and particularly the striatum (132, 235, 437, 567, 895). Excita- April 1974 VERTEBRATE SYNAPTIC TRANSMISSION 475 Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 tory effects have been seen only rarely, except by York (1282), who observed mainly excitation when DA was applied in the putamen. In various thalamic nuclei, as in the cerebral cortex, DA is much more potent than NE (987, 1164, 1167); in the cerebellar cortex its potency is comparable to that of NE (578, 687). By contrast, in the lower brainstem and the spinal cord DA is consistently much weaker than NE (115, 153, 47 1, 611, 1128), though here as elsewhere its effects are predominantly depressant. Thus DA has several of the attributes of a neurotransmitter, most likely of inhibition, but perhaps even excitation if York’s observations on the putamen can be confirmed. The greater prominence of DA-containing pathways (463, 1197) and stronger effects of DA in the forebrain (including the hypothalamus and thalamus) suggest that dopaminergic pathways are more likely to operate in that area. Various specific pathways have been proposed, the most extensively studied being a possible dopaminergic connection between the substantia nigra and the striatum. The DA content of the striatum is greatly reduced after lesions in the substantia nigra or at sites between the nigra and the striatum (28, 84, 505, 586, 910, 1000, 1196), suggesting the presence of a direct nigrostriatal pathway. If this system is mainly inhibitory, its destruction by disease could result in excessive activity of extrapyramidal pathways that control movements and so account for various manifestations of parkinsonism (61, 111, 601, 1113). This scheme would explain the beneficial effect of treatment with L-dopa, the direct precursor of DA (cf. 62, 246, 1196). With only few exceptions (442, 708, 1028), classical anatomists believed that only descending pathways connect the striatum with the midbrain. Although Shute and Lewis (1087) found evidence of a direct nigrostriatal projection of AChEcontaining fibers, according to Oliver et al. (953) these fibers in fact project in the opposite direction, from the striatum to the substantia nigra. On the other hand, a recent study of terminal degeneration produced in the caudate of the monkey by nigral lesions has confirmed the presence of nigrostriatal connections (204); according to Golden (504), in the fetus one can readily observe a continuous system of nigrostriatal DA-containing fibers (which in the adult animal cannot be demonstrated without pharmacological manipulations). It therefore cannot be doubted that direct ascending nigrostriatal connections do exist and that at least some of the connecting fibers contain DA. The presence of a functional dopaminergic pathway is further suggested by some evidence that nigral or caudate stimulation evokes the release of DA or its metabolites from the striatum (109, 889, 1006, 1221). Unfortunately the only monosynaptic nigrostriatal pathway for which there is convincing electrophysiological evidence-the slowconducting nigrocaudate link carefully analyzed by Feltz (435, 438)-has proved to be excitatory and almost certainly neither dopaminergic nor cholinergic (434, 437), in agreement with Hull et al.% (615) conclusion that all inputs into the caudate are probably excitatory. Hence, the clear inhibitory effects of nigral stimulation may not be mediated by direct nigrostriatal inhibitory connections. The substantial persistence of this inhibition after destruction of the DA-containing 476 K. KRNJEVIC Volume 54 Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 fibers in the caudate argues strongly against a purely dopaminergic inhibitory link (436). If DA is neither an excitatory nor an inhibitory transmitter in the caudate, what is its function there? One could imagine that it has a general depressant effect on neuronal activity or on the excitations produced by other inputs [a kind of modulating action (437)]; h owever, some doubt is cast on even this suggestion by Buchwald et al.? (174) observation that lesions of the nigrostriatal pathway, which cause a large reduction in striatal DA content, do not alter significantly the rate of spontaneous neuronal firing recorded in the caudate-although such lesions appear to increase spontaneous firing in the putamen (950). As suggested by Buchwald et al. (174), one must seriously consider the possibility that interruption of a dopaminergic nigrostriatal pathway is not the only or perhaps not even the main factor responsible for the manifestations of parkinsonism. It is therefore of interest that McGeer et al. (882) have recently shown in parkinsonian patients a pronounced loss of glutamic decarboxylase, the enzyme responsible for the synthesis of GABA, to whose inhibitory action caudate neurons are particularly sensitive (434). The delayed beneficial effects of large doses of L-dopa may be due to an indirect mechanism, such as a general loading of neurons with dopamine and its release by pathways that are normally not dopaminergic (930). Summary. In the forebrain, upper brainstem, and cerebellum, the action of DA is rather consistently inhibitory, with a potency equal to or greater than that of NE. The presence of large amounts of DA in the striatum and hypothalamus has been taken to indicate the existence of important dopaminergic pathways, especially an ascending link between the substantia nigra and the striatum. Although there is considerable biochemical, histochemical, pharmacological, and even clinical evidence for such a pathwayincluding the demonstration that nigral stimulation causes the release of DA or its metabolites in the striatum and that most striatal cells are depressed by DA-there is still no convincing electrophysiological evidence of the operation of such a pathway at the cellular level. d) Mechanism of inhibition by NE. It is still not clear how NE depresses excitability. Phillis et al. (990) reported that NE (as well as 5-HT and histamine) causes a hyperpolarization of spinal motoneurons, associated with a reduction in excitability and in the amplitude of EPSP’s and IPSP’s. According to Engberg and Marshall (400), this hyperpolarizing action is accompanied by an increase in membrane resistance and is enhanced by a conditioning hyperpolarization and diminished by depolarization (if large enough, this can even reverse the effect of NE). These authors therefore conclude that NE may reduce the ionic permeability, especially Z& . Siggins et al. (1091, 1093) have observed a similar pattern of changes in Purkinje cells during applications of NE-although the reported increases in intracellular negativity and membrane resistance seem very much greater than might be expected to occur physiologicallyand they concluded that NE inhibits these neurons by inactivating &a or Pea. This effect was at least partly antagonized by a P-blocking agent. These features bear a certain resemblance to the P-receptor action in smooth muscle, which is also consistent with a reduction in membrane April 2974 VERTEBRATE SYNAPTIC TRANSMISSION 477 Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 permeability to Na+ or Ca 2+ (300). However, it will be necessary to eliminate the possibility of an indirect or presynaptic effect reducing an ongoing release of excitatory transmittei, as this would give an identical result. A very different mechanism has been proposed by Phillis et al. (985) to explain the inhibition of cortical neurons. They have found that this action of NE can be such as lanthanum and manganese, and thereblocked by some “Ca antagonists,” fore suggest that the inhibition is initiated by a change in membrane permeability in a rise in internal free Ca2+ and a fall in excitability to Ca2+, possibly resulting comparable to effects produced by intracellular injections of Ca2+ in spinal motoneurons (740). Without much more evidence, it is impossible to decide whether NE can inhibit central neurons by two distinct mechanisms-one by reducing GNa and the other by an increase in GK, roughly analogous to the P- and the a-receptor actions in the gut, respectively: it is also not clear whether the effects observed in different parts of the CNS are essentially of the same nature. 1) ROLE OF CYCLIC AMP. In support of Yamamoto’s (1279) suggestion that the inhibitory action of NE might be mediated by CAMP, Siggins et al. (1090, 1091, 1094) and Hoffer et al. (579) have presented evidence that CAMP inhibits cerebellar neurons like NE, that inhibitors of phosphodiesterase potentiate the action of NE, and that Purkinje cells are particularly rich in CAMP (134). They therefore believe that NE acts through the intermediary of CAMP. This hypothesis is consistent with the idea that in many tissues [including some identifiable nerve cells (SSS)] adenyl cyclase acts as a P-receptor ( 161, 300, 575, 1019, 1052, 1053, 1109). There are several serious objections to this interpretation. It is not easy to understand how the applied CAMP penetrates the cell membrane rapidly enough to produce the effects reported (cf. 787); if it does penetrate, how do these effects lead to a transient reduction in membrane pNB. There is little evidence that CAMP is directly involved in changes in membrane permeability of other excitable tissues (300, 1002). Furthermore, adenosine and some other derivatives such as AMP and ATP, which also greatly increase the CAMP levels of the brain tissue (10 18, 1019), were found to have no inhibitory action on Purkinje cells (579). There are serious doubts about the direct relevance of studies on brain slices, in view of the demonstration that NE greatly increases the accumulation of CAMP by neuroglia assumption (579, 688, 879, (221, 487). 0 ne wonders also about the unquestioning 1091) that methylxanthines have no other significant action in tissues than to inhibit phosphodiesterase activity (cf. 1001, 1018, 1019). It should be noted that Godfraind and Pumain (502, 503), who tried to repeat the experiments on the cerebellum of the rat, could not obtain a marked depression of Purkinje neurons with CAMP. Purpura and Shofer (1012) have seen mainly excitatory effects of CAMP in the immature cerebral cortex, and Lake and Phillis (792) observed little or no effect of CAMP on cortical neurons, as well as no relation between the inhibitory potency of NE in different species and the amounts of CAMP previously found to be produced by NE in the cortex of the same animals. On the other hand, Anderson et al. (31) have reported that CAMP depresses neuronal firing in the lower brainstem. Finally, the possibility of a mainly presynaptic 478 IS. KRNJEVIC Volume 54 Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 phenomenon should be kept in mind in view of the fact that adenosine and 5-AMP, which specifically increase the CAMP content of nerve terminals, both diminish the release of ACh from motor nerve endings in muscle (493, 494). Unless further, much more critical evidence is presented, the hypothesis that noradrenergic inhibition of cerebellar or other neurons is directly mediated by CAMP can only be viewed with reservation. Summmy. There are at present two principal explanations for the mechanism of inhibition by NE. One is based on preliminary observations that NE causes a hyperpolarization of spinal motoneurons and cerebellar Purkinje cells associated with a rise in resistance; since the hyperpolarization is reduced or even reversed by depolarization, it can be ascribed to a reduction in GNa (or possibly G&. A second hypothesis proposes that NE increases Ca2+ influx and therefore leads to a secondary hyperpolarization by a rise of GX. Strong claims have been made that the inhibitory effects of NE and DA in the cerebellum and sympathetic ganglia are mediated by CAMP, but the evidence is insufficiently complete for any definitive conclusions, particularly with regard to the action of catecholamines at other sites in the CNS. e) Role of prostaglandins. Prostaglandins are ZO-carbon fatty acids folded over in a double chain by an internal link forming a cyclopentane ring. They have a strong action on smooth muscle and occur naturally in various tissues (104, 605, 12 14). Since they are not only present in the CNS, but can be shown to be released from the brain and spinal cord (158, 223, 224, 1022), they have been proposed as potential neurotransmitters (360, 604, 605). However, the exceedingly slow and prolonged changes in spinal reflexes apparently produced by prostaglandins (360) hardly indicate a transmitterlike action (cf. also 223). Tests of prostaglandins E (especially EJ applied directly to nerve cells have shown either no clear effect, as in the cerebral cortex (732, 792) and the cuneate nucleus (223), or, in some other parts of the medulla, an excitatory action characterized by very rapid desensitization (44). According to S&gins et al. (1092), although prostaglandins E have variable effects on the Purkinje cells of the rat cerebellum, they consistently and specifically antagonize the inhibitory action of NE. This was interpreted as due to a reduction in CAMP formation, and therefore in keeping with the hypothesis that CAMP mediates the action of NE. However, this seems a weak argument without some evidence that prostaglandins E reduce adenyl cyclase activity in these cells [cf. a similar argument used by McAfee and Greengard (879) with regard to the action of DA in sympathetic ganglia], especially since the most common effect of prostaglandins is to speed up the formation of CAMP in various tissues ( 104, 1083), including some nerve cells (486). Although it is not clear what general role prostaglandins would have in synaptic transmission, the fact that they appear to be widely released during nerve terminal activity (225, 558, 559, 1023) does suggest a significant function : perhaps a postsynaptic one, leading to the mobilization of membrane-bound Ca2+ (225; cf. also 1030, 103 1, 1083), or a presynaptic negative feedback, limiting the release of the neurotransmitter (557-559, 1146). April 1974 VERTEBRATE SYNAPTIC TRANSMISSION 479 Summary. The available evidence does not appear to indicate a simple neurotransmitter function of prostaglandins at central or peripheral junctions, but their evident release in the tissue during activity may be of significance in the presynaptic control of transmitter release (probably inhibited by prostaglandins) as well as influencing excitability through changes in Ca 2+ binding in synaptic membranes. 2. Excitatory actions of catecholamines in CNS Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 Except perhaps in the putamen (1282), DA has not been found to have pronounced excitatory effects in the CNS. Norepinephrine, on the other hand, consistently excites many neurons, especially in certain parts of the brainstem. For example, more cells are excited than are depressed by NE in the medulla or pons ( 140, 153) and in the perifornical and vendromedial areas of the hypothalamus (727). The excitations seen in the medulla are unlikely to be simply disinhibitions, since they are produced by relatively small doses of NE and have a much slower time course than the depressant effects evoked by NE in the same area (153). Even better evidence of a specific excitatory action is Yamamoto’s (1279) finding that neurons in Deiter’s nucleus are only excited by NE. Moss et al. (917) give another example of a neuron-specific effect in the hypothalamus: NE inhibits most neurosecretory cells of the paraventricular nucleus but excites the great majority of the other, nonneurosecretory cells. Such observations are most easily explained by the presence of at least two kinds of NE receptors in central neurons, one (or two, see above) mediating inhibition of firing and another responsible for excitation; there is at present no evidence that these receptors are more readily blocked by a- or P-blocking agents (cf. 153, 643, 983). Are both excitatory and inhibitory receptors to be found on most neurons, in all parts of the CNS? Some observations might suggest that the answer is yes. Thus, although the predominant effect of NE is a depression of firing in most regions of the CNS ( 118, 132, 401, 75 1, 989, 1060, 1141, 1164, 1279) some excitatory responses have often been obtained from the same cells. For example, Krnjevie and Phillis (75 l), studying mainly barbiturate-anesthetized cats, observed in the cerebral cortex a delayed but strong excitation when epinephrine was applied in large doses. As smaller doses had a purely depressant effect, it was suggested that depression was probably the significant action and that the excitations might have been artifacts. But Johnson et al. (642, 643) reported mainly (slow) excitatory effects in cats more lightly anesthetized with halothane, or in the unanesthetized enciphale isol&, and so concluded that excitation was the more important effect, which was probably depressed by anesthesia. Although Johnson et al. (642) thought they had eliminated the possibility of an artifactual excitation by an excess of Hf released simultaneously from the acid solutions in the micropipette, Frederickson et al. (455) have recently found that the incidence of excitatory effects (also in the cat’s cortex) is simply correlated with the acidity of the NE solutions used. According to these authors, irrespective of the degree or kind of anesthesia used, only 480 K. KRNJEVIC; Volume 54 3. Function of catecholamines in CNS Although catecholamines are found throughout all parts of the CNS, they are especially concentrated in basal areas of the brain (201, 202, 29 1, 465, 582, 597, 1197, 1211, 1212). As Vogt (1211) p ointed out, the hypothalamic and midbrain regions of high NE content agree rather well with the distribution of sites from which pressor responses and other manifestations of sympathetic activity can be elicited by stimulation. This area therefore can be considered as a central component of the sympathetic system, and it seems fitting that it should be rich in NE. A further natural step is to assume that catecholamines may be used as neurotransrnitters, just as in the peripheral sympathetic system. This idea has been very extensively discussed and on the whole received favorably (133, 153, 654, 697, 865, 866, 868, 1058, 1075, 1211, 1275). In addition to the studies described above, which show that catecholamines can influence neuronal excitability, what other evidence supports a possible transmitter role of NE and other catecholamines? Catecholamines can be released by direct or indirect stimulation of central nervous tissue (27, 48, 58, 109, 685, 918, 930, 1006, 122 1, 1243). This evidence is somewhat limited, probably because of the active presynaptic reuptake mechanisms that very effectively remove catecholamines from the extracellular space (47, 621, 685, 1107, 1189, 1217, 1242). Other arguments for the involvement of catecholamines in CNS function are based on the effects produced by precursors or antagonists or other drugs that promote release or block reuptake. One example has been studied extensively by Lundberg and his colleagues. They have shown that intravenous injections of L-dopa, the precursor of DA and NE, cause pronounced changes in spinal reflexes. The balance of activity between extensor and flexor motoneurons or static and dynamic y-motoneurons is greatly altered; moreover, there is a changed distribution of primary afferent depolarizations also evoked by volleys in cutaneous nerves and high threshold muscle afferents (((flexor reflex afferents”) ( 103, 63 1, 844). These complex phenomena can be explained by the activation of descending noradrenergic pathways (of supraspinal Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 depressant effects are seen if NE is released from solutions at pH 2 4. Stone (1135) has proposed that NE causes a constriction of cortical arterioles, the neurons being secondarily excited by local hypoxia, but this is unlikely because cortical arterioles are rather insensitive to local applications of NE (805). These studies have not excluded the possibility that NE enhances transmitter release by acting presynaptitally, as it does at some peripheral junctions (150, 640, 742). Summary. Although some apparent excitatory effects of NE may well be technical artifacts (possibly resulting from a simultaneous release of H+ during iontophoresis), certain neurons in the hypothalamus, pons, and medulla are regularly excited by relatively small amounts of NE under conditions that seem to indicate specific excitatory receptors for NE. The mechanism of this action has not been investigated. April 1974 VERTEBRATE SYNAPTIC TRANSMISSION 481 Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 origin) that exert an inhibitory influence on certain spinal interneurons. However, although the effects of L-dopa are impressive, it has not been possible so far to identify with any precision either the postulated descending tracts or the target interneurons. Putative dopaminergic pathways have been much discussed. As stated earlier, in spite of the wealth of suggestive neurochemical evidence, the electrophysiological evidence for a dopaminergic nigrostriatal link is unsatisfactory. Excessive activity of dopaminergic systems has also been implicated in the etiology of schizophrenia, on the ground that neuroleptic drugs specifically block DA receptors (cf. chapt. 3 in 142; 878). This hypothesis is based on rather indirect pharmacological considerations and has not so far received any support from direct studies of DAsensitive cells (cf. chapt. 2 in 142). The activity of noradrenergic pathways is thought to underlie emotional states, particularly a tendency to euphoria or more generally behavior leading to “reward” (697, 1075, 1123). This is suggested by the effects of such drugs as amphetamine [which is believed to promote the release of NE ( 140, 5 lo)] and antidepressants [which reduce the uptake of NE by nerve endings and therefore would enough. It has potentiate a noradrenergic activity (12 17)]. Th’ is 1‘d ea is plausible received wide acceptance by students of behavior and has probably been a useful conceptual framework for interpreting and designing experiments. Unfortunately, it still has no more than a very tenuous basis of hard physiological facts. This should be kept in mind, as there is a danger that it may be taken too literally and may impede rather than stimulate wide-ranging studies. Some caution is needed in the drugs, when even such classical modulators interpretation of the effect of “specific” of adrenergic function as reserpine and MAO inhibitors can be shown to affect markedly levels of excitatory and inhibitory amino acids (1070). Another related hypothesis is that adrenergic pathways play an essential role in the control of hunger and eating (520, 812, 905, 1099, 1112, 1196) as well as in the maintenance of a constant body temperature (428, 921). Until now the result of investigations of hypothalamic neurons have not given much support to a simple neurochemical scheme of coding of behavior (727). Similarly, the idea that catecholaminergic hypothalamic pathways control the release of pituitary hormones (1078, 1181, 1275) is supported by only somewhat indirect evidence. The supposed participation of an ascending noradrenergic system in the mechanism of arousal or the maintenance of wakefulness has less substantial evidence in its favor than is suggested by the enthusiasm with which it is repeatedly publicized (e.g. 648, 653, 654, 9 14). Thus the available evidence indicates an inhibitory rather than an excitatory action of NE in the cerebral cortex; this is hardly consistent with an arousing action, unless it can be shown that the cortical projection of NE-containing fibers [presumably from the locus coeruleus (cf. 333a, 954, 1197)] innervates mainly inhibitory neurons. Alternatively, the adrenergic mechanism may operate primarily at the brainstem level (cf. 153, 157, 1058). There is little clear evidence of a reciprocal action between brainstem adrenergic and tryptaminergic centers or pathways (cf. 247). Perhaps the most serious argument against an essential involvement of norad- 482 K. KRNJEVIC Volume 54 Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 renergic pathways in the control of wakefulness is an apparently normal pattern and amount of motor activity shown by animals treated with sufficient 6-OHdopamine to destroy selectively most of the central NE- and DA-containing fibers (127, 130, 411, 626). In fact, these animals maintain their body temperature and they eat, drink, and put on weight at the same rate as normal controls (626, 628, 789, 1069, 1286). More generally, there is surprisingly little noticeable change in their behavior: apart from a temporary disturbance after injections, presumably at least partly due to a sudden large release of catecholamines, treated rats soon appear to behave very much like normal animals, even when tested by relatively sophisticated techniques ( 116 1, 1162); they survive in this condition for many months, even though there is no evidence of any recovery of NE-containing cell jibers ( 127, 626, 628). Similarly, there is no permanent change in the behavior of cats treated with 6-OH-DA, except when they are pretreated with chlorimipramine (789). The only suggestive long-term changes demonstrated so far are a possibly significant reduction in feeding evoked by injections of 2-deoxy-O-glucose (1286) and a gradual increase in aggressive behavior (1177), which, however, is only poorly correlated with the disappearance of cerebral NE (1178). Such early postinjection effects as a reduction in self-stimulation (1124) are hardly very significant, unless they can be shown to be persistent (cf. 411, 626), especially since the initial disturbance is probably not related to the destruction of catecholaminergic neurons. (1162). Much of this evidence seems to indicate a relatively nonessential role of catecholamines [which would not be very surprising in view of the low density of NEcontaining terminals in most regions; according to Lapierre et al. (799a) in the cerebral cortex of the cat such terminals account for only 1 in lo4 of all synapses] yet such a conclusion will not be really justified until it is certain that even a very small number of remaining catecholaminergic neurons may not be sufficient to maintain adequately whatever specific function is mediated by the NE-containing neurons. According to the results of some experiments, really marked changes in behavior do become evident when the monoamine-containing fibers are almost totally destroyed (1196). Unfortunately, to obtain such a drastic effect, 6-OHdopamine must be injected directly into the brain and there is a serious risk of nonselective destruction of all local cells (cf. 999a). Summary. Central noradrenergic pathways have been variously thought of as the central component of the sympathetic system, a CCreward” system, an important element of the cortical arousal mechanism, or some combination of these and other related functions. However, in spite of much data showing that NE and other catecholamines are found in various fibers, that they are released during activity and avidly taken up by nerve endings, and that drugs that alter their metabolism affect behavior, it is still impossible to make definitive statements about their involvement in synaptic transmission at clearly identified sites. Serious questions about the significance of adrenergic pathways for normal function have been raised particularly by the surprising lack of effects of destruction of central NE-containing neurons by 6-OH-dopamine. April 1974 VERTEBRATE V. OTHER A. 5-Hydroxytryptamine SYNAPTIC TRANSMISSION 483 MONOAMINES (Serotonin) 1. Introduction 2. Is 5-HT a central transmitter? The discovery of 5-HT a> Presence, synthesis, and release of 5-HT. 1) PRESENCE. in the brain by Twarog and Page (1193) and Amin et al. (24) was the trigger for numerous investigations of its central function. Detailed mapping of the distribution of 5-HT was done first by biochemical methods (406) and later by a histochemical technique utilizing the specific fluorescence of 5-HT-containing tissues that had been treated with formaldehyde (202). Cell bodies and nerve terminals rich in 5-HT thus have been discovered, forming relatively coherent pathways Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 5-Hydroxytryptamine is a powerful activator of some intestinal smooth muscle, which was discovered first in extracts of gastric mucosa by Erspamer (405) and later, independently, in serum by Rapport et al. (1029). According to subsequent studies [reviewed by Erspamer (406) and Page (SSO)], 5-HT in mammals is present mainly in the “enterochromafhn” cells of the gut and in the CNS. Significant amounts of 5-HT are also found in the blood-where it is taken up by the platelets -but this is probably of gastrointestinal origin. It is also present, together with histamine, in most cells of certain animals such as the rat. There are large amounts of 5-HT and other indolalkylamines in amphibian skin and in a variety of snake venoms. 5-Hydroxytryptamine has a wide distribution in nervous systems of invertebrates, where it is probably both a central and peripheral transmitter (446, 481, 1160, 1192). It is even found in many plants, including such fruit as pineapple and bananas, but its function there is quite obscure (406). There is no evidence that 5-HT is a neurotransmitter at peripheral junctions in vertebrates. Its release from the enterochromafhn cells is unlikely to be a normal phenomenon, so that it is probably not even a regulator of gastrointestinal motility or secretions (406). A wide variety of peripheral actions of 5-HT have been reported (407). The effects on blood vessels are quite complex, so that blood flow may be either reduced or probably more often increased. Some of the changes are directly caused by 5-HT, but others may be indirect, being due to the liberation of other agents, such as histamine. Injections of 5-HT may produce marked alteration in tissue metabolism, possibly related to some reported alterations in cellular permeability. The physiological significance of the excitatory action of 5-HT on several kinds of sensory nerve terminals or fibers (96 1, 1204) is aIso not at all clear : in species where mast cells contain 5-HT, its release by tissue damage may reinforce sensory phenomena, especially pain. 484 K. KRNJEVIC Volume 54 Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 quite distinct from those containing catecholamines. The most outstanding concentration of 5-HT is in cells situated along the dorsal midline of the brainstem, especially the raphe nuclei, and in fibers traveling rostrally along the brainstem and down the spinal cord; however, there is relatively little 5-HT in nerve terminals of the forebrain and thalamus (29 1, 463, 465). Further developments, including the use of radioactively labeled isotopes and radioautography, have made possible electron microscopic studies of 5-HT-containing neuronal elements (8, 128, 582, 1107), and brain synaptosomes have been shown to hold substantial amounts of 5-HT (33 1, 1264). A specific population of synaptic vesicles contains 5-HT rather than catecholamines (976). 2) SYNTHESIS. 5-Hydroxytryptamine is synthesized from the amino acid tryptophan, by hydroxylation in the 5 position of the aromatic ring, and then decarboxylation (203, 244, 526, 960). The rate-limiting step appears to be that of 5-hydroxylation, the required hydroxylase being much less abundant in the brain than the decarboxylase. The initial breakdown of 5-HT occurs through the action of MAO, and there is a subsequent conversion to 5-hydroxyindole acetic acid, which is excreted in the can be depleted of 5-HT by reserpine, preurine ( 125, 244). N erve terminals sumably because the drug prevents the accumulation of 5-HT in the granular vesicles, just as it prevents the accumulation of catecholamines. Although 6-OHdopamine can selectively destroy cells rich in catecholamines in the rat, it has a less specific action in the cat, where it also lowers the 5-HT content of the brain (98 1). The latter may be lowered fairly selectively by a comparable procedure, intracerebral injections of 5,6-dihydroxytryptamine (74, 243, 298). The almost total disappearance of 5-HT from the forebrain and striatum after lesions in the brainstem (for example, the medial forebrain bundle) has been accepted as evidence that all tryptaminergic pathways originate in the raphe nuclei (505, 594). However, some doubts have been cast on this interpretation by the absence of terminal degeneration, which is more consistent with a transynaptic loss of 5-HT and other monoamines (561, 911). 3) RELEASE. There is some limited evidence that 5-HT is released in the CNS, both spontaneously and as a result of neural activity. 5-Hydroxytryptamine or some related compound was first detected in fluid superfusing the frog spinal cord (32). Stimulation of descending pathways is said to promote the liberation of 5-HT from the mammalian spinal cord (26), and excitation of rostra1 raphe nuclei augments the release of 5-HT into the anterior part of the cat’s lateral ventricle-this 5-HT is likely to originate from the caudate nucleus (594). A spontaneous release of 5-HT in the monkey’s superior colliculus has been reported (85). Stimulation of the midbrain raphe nuclei also induces a release of 5-HT (38 1) or 5-hydroxyindole acetic acid from the cerebral cortex (959; cf. also 175). Summary. Most of the widely distributed 5-HT-containing nerve fibers appear to originate from the raphe nuclei of the lower brainstem. 5-Hydroxytryptamine has been shown to be released in various regions of the CNS, including the cerebral cortex, striatum, and spinal cord. The active metabolism of 5-HT, its rapid uptake by a mechanism inhibited by tricyclic antidepressant drugs, and its presence in April 1974 VERTEBRATE SYNAPTIC TRANSMISSION 485 Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 nerve endings and even in a specific population of synaptic vesicles are suggestive evidence of significant tryptaminergic transmission. b) Action of .5-HT in CNS. The results of systemic or intraventricular injections of 5-HT in low or moderate amounts have a mainly sedative effect, though larger doses may cause strong excitation [see extensive review by Mantegazzini (862)]. These observations are to some extent paralleled by the results of microiontophoretic tests on neurons in various parts of the brain and spinal cord. The most general effect of iontophoretic applications is a reduction in excitability. This is the case with neurons in the neocortex (650, 748, 751), paleocortex and olfactory bulb (131, 809, 1213), archicortex (118, 566), striatum (567), cerebellar cortex ( 136, 687), hypothalamus (63, 136, 137), and red nucleus (308). Some excitatory effects have also been seen, particularly when applying relatively large doses of 5-HT (748, 751) or as the predominant response in cats receiving little or no anesthesia (642, 1048). But there is some reason to believe that this excitation may be at least partly caused by an accumulation of protons in the tissue when strongly acid solutions (pH < 4) are used for 5-HT release by iontophoresis (650) [cf. also comparable excitatory action of acid solutions of catecholamines (455)]. There is more convincing evidence of a genuine excitatory action of 5-HT in certain parts of the thalamus, the brainstem, and the spinal cord. What seems particularly significant is that under the same experimental conditions (anesthesia, same micropipettes, etc.) different types of cells in these regions consistently give opposite responses to 5-HT. Thus, in the lateral geniculate, some of the nonrelay cells are excited (107 1), whereas the relay cells are almost uniformly inhibited (984, 989, 107 1, 1167). In a broad survey of thalamic neurons, Phillis and Tebecis (987) found that neurons in the upper and middle regions were predominantly inhibited by 5-HT, but in the deeper portion they were often excited. This distribution paralleled the superficial inhibitory and deep excitatory effects seen with catecholamines. Brainstem neurons can be excited or inhibited by 5-HT (159), but more specific tests of selected populations of neurons in the medulla (43, 611) and the raphe nuclei (247) have shown some consistent excitatory actions : for example, 5-HT regularly excites reticulospinal neurons (611). There are also striking variations in responses observed in the spinal cord. In random surveys of spinal interneurons, investigators have seen either mainly depressant effects (401) or both excitation and inhibition (990, 1235). But most sympathetic preganglionic neurons are rather clearly excited by 5-HT (3 19), whereas preganglionic parasympathetic neurons show either no effect or some depression (1061). There is little direct evidence about the precise mechanism of action of 5-HT. The first studies on the lateral geniculate seemed to indicate that 5-HT blocks synaptic transmission more effectively than cellular responses to various excitatory agents, either by a presynaptic action reducing transmitter release or a specific antagonism of the natural transmitter (260). Later tests in the cortex (748, 75 1) showed a clear, apparently nonspecific, depression of cell firing, whether induced 486 K. KRNJEVIfi Volume 54 Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 synaptically or by applications of glutamate or ACh; it was therefore suggested that 5-HT and inhibitory amino acids may interact with the same receptors. If this were indeed the case, the inhibition would be due to an increase in membrane permeability to small anions (352, 691, 764, 1255). Although the hyperpolarization of spinal motoneurons induced by 5-HT (990) is consistent with such a mechanism, other mechanisms may well be at work (cf. 400). The recent findings that Ca antagonists abolish the inhibitory effect of 5-HT in the cortex has led to the suggestion that 5-HT (like NE) increases the membrane permeability to Ca2+ (985). A similar general postsynaptic depression can be demonstrated in the lateral geniculate (984, 989, 1071), but the latest experiments by TebEcis and Di Maria (1167) indicate a more specific block of synaptic transmission by small doses of 5-HT, thus supporting the original suggestion of Curtis and Davis (260). This interesting action has not so far been observed at any other junctional site in vertebrates, though 5-HT is said to produce a specific block of glutamate-mediated neuromuscular transmission in insects ( 1199). Although peripheral actions of 5-HT are blocked by LSD, brom-LSD, and methysergide (524), there is no general agreement about any specific antagonists of the central actions. A number of authors have reported little or no specific antagonism by LSD ( 136, 260, 751, 984, 989, 990), but Roberts and Straughan (1048) and Boakes et al. (139) are convinced that derivatives of lysergic acid do antagonize the (mainly excitatory) effects of 5-HT they have observed in the cortex and the brainstem. According to TebEcis (1164) strychnine clearly prevents the inhibitory action of 5-HT on some thalamic cells. Systemic administration of LSD, MAO inhibitors, or tricyclic antidepressants (the latter strongly block the reuptake of 5-HT) markedly and apparently specifically reduces the firing of the 5-HT-containing neurons of the raphe nuclei (7, 1084). Th ese effects have been interpreted as due to increased activity of a tryptaminergic inhibitory neural feedback in which LSD would act as agonist rather than antagonist (7), but clearly other possible explanations cannot be eliminated without a great deal more information. Summary. Specific populations of neurons are consistently mainly depressed or excited by 5-HT, the former effect being probably somewhat more common. Thus, if 5-HT is indeed a transmitter, it could function both at inhibitory and excitatory synapses. Various mechanisms of action have been proposed, especially for the depressant actions: reduction of GNa, increase in Gc,, an action similar to that of inhibitory amino acids, or even a block of presynaptic release of transmitter. There is also no agreement about any specific antagonist of central actions of 5-HT: LSD and derivatives do not produce consistent effects. c) Function of 5-HT in CiW. As soon as 5-HT was discovered in the brain (24, 1193), it was proposed that it might be a neurotransmitter released by certain central pathways. Although the evidence discussed so far is consistent with this possibility, it is also by no means conclusive. The differential distribution of certain nerve cells and terminals is very suggestive. But then one knows that 5-HT is present in enterochromaEin cells and platelets, from which it is not likely to be April 1974 VERTEBRATE SYNAPTIC TRANSMISSION 487 minergic neurons (cf. also 327). As pointed out very pertinently by Oswald (956), in a sane and lucid review, everyone feels like an expert on sleep. This may explain why the most elaborate and speculative hypotheses are so freely developed in this field on the basis of the flimsiest and most indirect experimental evidence. Summary. On the basis of the evidence reviewed in the previous sections 5-I-IT may be released by some central nerve terminals; although much of this evidence is far from conclusive, tryptaminergic pathways have been postulated to play a significant role in the control of spinal reflex activity, body temperature through hypothalamic projections, and the initiation of slow-wave sleep. Disorders of such pathways are thought to give rise to hallucinations. Most of these suggestions cannot yet be considered as more than useful hypotheses. Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 liberated for purposes of communication, at least not in the normal course of events (406). Evidence that 5-HT is actually released by central nerve fibers during normal activity is circumstantial at best. The strong excitation of peripheral nerve fibers by 5-HT (444, 1204) cannot be related to a process of junctional transmission. It is with these reservations in mind that one should consider claims that various functions are mediated by tryptaminergic pathways. There is some impressive evidence for a possible involvement of 5-HT, released by bulbospinal pathways, in the control (especially facilitation) of the discharges of spinal motoneurons and the transmission of polysynaptic reflexes (9, 30, 844). Other studies indicate that 5-HT plays a significant role in the hypothalamic control of body temperature (83, 85, 428, 594, 921). Many other functions have been suggested (for some recent reviews see 34, 960, 1275). There is wide support for the idea that disturbances of 5-HT metabolism and, presumably, of the carresponding tryptaminergic pathways may be responsible for hallucinations, whether induced by various drugs or by neural malfunction in schizophrenia and some other mental disorders (160, 524, 1269, 1274). A particularly strongly promoted hypothesis is that tryptaminergic neurons of the rostra1 raphe nuclei, acting in opposition to the arousing influence of a pontomesencephalic system of catecholaminergic neurons, are mainly responsible for initiating the slow-wave stage of sleep (652654). The evidence consists principally of the following: lesions of the raphe nuclei [insofar as such lesions are strictly selective (cf. 1004)] reduce or abolish the need for sleep; and an inhibitor of 5-HT synthesis, p-chlorophenylalanine (which prevents the hydroxylation of tryptophan), greatly reduces the amount of slow-wave sleep of cats, the effect being reversed by injections of 5-hydroxytryptophan. This much publicized idea has received support from some investigators (e.g. 846, 1198), but not from others: one obtains mainly arousal by stimulating the raphe nuclei, even when using near-threshold stimuli ( 1004) ; p-chlorophenylalanine reduces REM sleep rather than slow-wave sleep in man (1277), and the sedative action of tryptophan is seen even when the synthesis of 5-HT is blocked byp-chlorophenylalanine ( 1276). When the cerebral 5-HT in cats is depleted by the administration of another drug, fenAuramine, there is an increase in slow-wave sleep (641), SO it is by no means certain that slow-wave sleep is only or even mainly determined by the activity of trypta- 488 B. Imidasole K. KRNJEVIC Volume 54 Derivatives 1. Histamine 2. Imidaeole acetic acid and other derivatives Early tests of imidazole-4-acetic acid on cortical neurons (751) had shown a strong inhibitory effect, comparable with but usually weaker than that of GABA [cf. the crayfish stretch receptor (881)]. Th is observation was confirmed and extended to other central neurons (3 1, 607, 946, 991). Moreover, several related compounds have proved almost as powerful as the acetic acid derivative (499, 739). Even amyl- and n-propyl esters of imidazole acetic acid may have quite a strong action [in contrast to esters of GABA, which are much less active than GABA @WI * Roberts and Simonsen (1047) have shown that imidazole derivatives potentiate brain phosphodiesterase activity and have therefore suggested that they may influence neuronal firing through changes in intracellular levels of CAMP and ATP. However, in their experiments, 1-methylimidazole acetic acid and imidazole4-carbolic acid increased phosphodiesterase activity almost as much as did imidazole acetic acid. Since the first two agents are only weak inhibitors of neuronal firing (499, 607), it is very unlikely that the inhibition has anything to do with CAMP. A more probable explanation is that these inhibitory compounds interact with the membrane receptors for GABA (cf. 31, 263) and therefore increase the membrane permeability to small anions (691, 764). Although there is no evidence at present that these potent imidazole derivatives occur in the CNS in really substantial amounts, they may be produced by the metabolism of histidine or histamine (cf. 1082, 1106) and therefore could have a functional significance. Summary. In spite of the presence and release of histamine in the various parts Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 The best known naturally occurring imidazole compound is histamine. Its presence in nerves and release from peripheral nerve endings was first suggested and then demonstrated many years ago (788, 82 1) ; it has therefore long been considered a possible synaptic transmitter (cf. 254, 890, 1108), but there is little real evidence that histamine is a significant transmitter at any known site. It is found in variable amounts in the brain and spinal cord (515) and also in nerveending fractions of the brain (663). Its action on central nerve cells, which is mainly inhibitory (471, 607, 751, 990, 991, 1165), is not particularly striking when compared with that of the inhibitory amino acids or, for that matter, that of some other imidazole compounds. The relatively large amount of histamine in dorsal root fibers and the evidence that histamine is released from their peripheral terminals led Kwiatkowski (788) to suggest that it might be the transmitter liberated by their central endings, but no excitatory effects of histamine have been detected at the first afferent junction in the cuneate nucleus (47 1). However, some excitatory actions in the cortex have been described by Phillis et al. (991). April VERTEBRATE 1974 SYNAPTIC TRANSMISSION 489 of the CNS, its weak, predominantly inhibitory, action on some neurons hardly indicates a very significant role as a synaptic transmitter. Some other imidazole derivatives, including imidazole-4-acetic acid, have a much stronger inhibitory action, comparable to that of inhibitory amino acids and quite likely operating by the same mechanism. However, there is only very indirect evidence that these compounds occur naturally in the brain or that they are actually utilized as neurotransmitters. VI. SOME OTHER PUTATIVE (Adenosine-5’- Tri’hosphate) 1. In CNS When Holton and Holton (595) discovered appreciable amounts of ATP in spinal roots, they suggested that ATP may be released as a neurotransmitter from both peripheral and central endings of sensory fibers. Holton (596) and Abood et al. (3) indeed demonstrated a release of ATP from peripheral endings excited antidromically and also from nerve trunks (as well as muscle). There is no evidence at present of a specific release of ATP from the central terminals, but McIlwain and Pull (883) have observed a leakage of adenosine from isolated brain tissue that is accelerated by electrical stimulation. Tests of ATP on most central neurons have not revealed a powerful excitatory (or inhibitory) action : for example, neither in the spinal cord (28 1) nor in the cerebral and cerebellar cortex (75 1) are there any marked effects of ATP applied by iontophoresis. On the other hand, a number of cells in the cuneate nucleus to that of are strongly excited by ATP (471), whose potency is often comparable glutamate, although its action tends to be much more prolonged. This is likely to be related to the chelating power of ATP, since ADP is very ineffective, whereas citrate is also quite a strong excitant. 2. ,4t periphery a) Inhibition. A very different inhibitory transmitter role of ATP in the gut has been proposed more recently by Burnstock et al. (190). An inhibitory action of adenosine and its derivatives on smooth muscle has long been known (70, 355, 356, 12 18). Its electrical characteristics have been examined in detail by Imai and Takeda (6 18) and Axelsson and Holmberg (50), who found a direct hyperpolarizing action on the smooth muscle membrane. Burnstock et al. (190) obtained clear evidence that ATP and several derivatives of adenosine are released by transmural electrical stimulation that is believed to have a nonadrenergic inhibitory that ATP could be released from the isolated effect. They showed, moreover, Auerbach’s plexus and that its action was not prevented by blocking the intestinal Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 A. Adenosine Derivatives TRANSMITTERS 490 IS. KRNJEVIC Volume 54 significant neurotransmitter mechanism, even though the term “purinergic” might not be quite appropriate. Summary. Although it may seem wasteful to use a high-energy compound like ATP as a neurotransmitter, several features would make it very suitable for such a function. Its leakage from active cells seems to be a very widespread phenomenon -seen even in plants (806). Its strong anionic charge can make it react with divalent cations attached to the cell membrane, causing the membrane to contract and thus possibly change significantly its permeability characteristics (cf. 22, 722). As suggested by Ambrose (22), ATP could in this way transfer information between small groups of cells. It should not be surprising if the same mechanism was utilized for junctional transmission. Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 nerve fibers with tetrodotoxin; they therefore concluded that ATP was probably the transmitter released by the intestinal nonadrenergic inhibitory nerve fibers. This hyperpolarizing action is thought to be due to a specific increase in GH (189; cf. also 1183) ; it is presumably unrelated to a chelating mechanism since ATP and ADP are equipotent in this respect. The substantial case for ATP (or a close derivative) as the general nonadrenergic inhibitory transmitter in the gut is somewhat weakened by the rather mixed excitatory and inhibitory effects of ATP seen with most gut preparations other than the guinea pig taenia coli, which make it necessary to postulate the presence of at least two kinds of ATP receptors (190). b) Excitation. Burnstock et al. (191) have confirmed Ambache and Zar’s (20) observation that applications of ATP reproduce the effects of nerve stimulation and therefore propose that the atropine-resistant synaptic excitation of the urinary bladder is mediated by ATP or some close derivative. As further supporting evidence they report a block of both types of excitation by quinidine. Their observations differ from those of Ambache and Zar’s (20) in one very significant respect : they could not produce a desensitization to ATP without also blocking the neural effect. The possibility of peripheral junctional transmission by the release of ATP or related purines from “purinergic” nerves has been fully discussed by Burnstock (189). This review lists an extensive series of findings showing that ATP is released from a variety of tissues and has a marked excitatory or inhibitory action on several kinds of smooth muscle and places much emphasis on the presence of adenosine and its derivativesas well as enzymes concerned in the metabolism degradation of ATP-in nerves that appear to be neither cholinergic nor adrenergic. Although this accumulation of data is quite impressive, more conclusive evidence is needed that the postulated “purinergic” nerves do not in fact release NE or ACh, according to the classical schemes. For example, questions have been raised about the supposed absence of adrenergic neurons from Auerbach’s plexus (99). As pointed out above, it is not reasonable to expect junctions with narrow gaps to be as readily blocked by antagonists as the corresponding effects of topical applications of NE or ACh: of course, ATP might be released in conjunction with other transmitters [cf. its release together with catecholamines in the adrenal medulla (347)], but if ATP has a marked postsynaptic action, this would be a A&d VERTEBRATE 1974 SYNAPTIC TRANSMISSION 491 B. Ergothioneine An excitatory action of cerebellar extracts (255) has been ascribed by Crossland et al. (256) to the presence of ergothioneine (897). However, it is unlikely that ergothioneine can be an important excitatory transmitter in the cerebellum, since cerebellar neurons are very little affected by direct applications of this comin the brain does not conform to that of the putapound (756) and its distribution tive cerebellar excitatory factor ( 165). 1. Substance P This name covers a group of agents that cause strong contractions of intestinal smooth muscle, are vasodilators and sialogogues, and were first extracted from the gut ( 12 18). These polypeptides (2 13, 12 10) are found in many parts of the CNS (24, 1284) and in especially high concentration in the substantia nigra (1284). Their possible function as neurotransmitters-especially at the first synapse in the afferent pathway (813)-has been much discussed (24, 1132, 1284), but there is only limited evidence so far (957) that they have a significant action on neurons excited by primary afferent fibers (471). According to more recent observations by KrnjevX and Morris (747a) pure substance P sometimes has a powerful action on cells in the cuneate nucleus; this is probably a slow depolarization, which can lead to inactivation of cell discharges rather than overt excitation. The comparatively slow time course and great variability of this effect make it unlikely that substance P could be the quickly acting excitatory transmitter released by primary afferent fibers (813), but it may well have some other significant action. 2. Other polypeptides Excitatory polypeptides can be obtained from cerebrospinal fluid (439) and the frog’s skin (408, 957). Whether these are of significance for synaptic function remains to be established. The possible role of various polypeptides in neurotransmission has been discussed at some length recently (129). 3. Antidiuretic hormone There is some supraoptic nucleus If confirmed, this action mediated by Summary. The has been shown to evidence that the hormone secreted by the neurons of the is also released by their inhibitory recurrent branches (935). would be the first example of a neurotransmitter inhibitory a polypeptide. most promising other transmitter candidate is ATP, which be released (as ATP or adenosine) by stimulation of the gut, Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 C. Polyfeptides 492 K. KRNJEVIC Volume 54 VII. SOME A. Chemical SPECIAL ASPECTS Transmission OF CHEMICAL TRANSMISSION in Retina Because of its comparatively simple organization and the fact that it is thin enough to allow in vitro studies, the retina lends itself to systematic investigations such as have not been feasible, at least so far, on any other central synapses in vertebrates. Over the last few years, they have revealed some very unexpected features of the mechanisms of operation of central synapses. The predominant response of both photoreceptors and horizontal cells to illumination is a hyperpolarization associated with an increase in membrane resistance (78, 79, 656, I 145, 1186, 1248). It appears that, in the dark, photoreceptors are depolarized and therefore release from their terminals a depolarizing transmitter that keeps horizontal cells and bipolar cells also depolarized. On illumination, the high membrane permeability (presumably P,,) of the photoreceptor is reduced. This allows them to repolarize, reduces the release of depolarizing transmitter from their nerve terminals, and therefore leads to a hyperpolarization of the horizontal and some bipolar cells (78, 1186, 1190). The second point of great interest is that only graded potentials are recorded from all the cells of the outer layers, including the bipolar cells. Clear spikes appear to be generated only by cells in the inner layer-the amacrine and the ganglion cells (79, 348, 656, 875, 1248, 1270). This is the first convincing demonstration of the effectiveness of local graded potentials for the transmission of information not only within a given neuron, but across one or even two synapses. What evidence do we have about the identity of the chemical transmitters in the retina? Like most central neurons, the ganglion cells are excited by L-glutamate and inhibited by GABA (942). Glycine also has a clear blocking action. The presence of complex inhibitory mechanisms of both strychnine and picrotoxin (23, 185). is suggested by the excitatory Catecholamines are also quite effects strong depressants of ganglion cell activity ( 1140), whereas cholinomimetics appear to have a mainly facilitatory action, which has been interpreted as consistent with the presence of nicotinic synapses (23). The site of these postulated cholinergic Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 peripheral nerves, and the brain. Although by no means widely active in the CNS, it does excite strongly some relay cells in primary afferent nuclei and therefore could be a significant transmitter if released by primary afferent terminals. Since ATP and derivatives depress intestinal smooth muscle, there is reason to believe that ATP may be the transmitter for nonadrenergic inhibition in the gut. Some evidence suggests that ATP may also be involved in excitatory transmission in the gut and the bladder So far none of the physiologically active polypeptides, such as substance P, have shown properties consistent with a simple function as neurotransmitter, but there is a possibility that antidiuretic hormone may act as an inhibitor in the supraoptic nucleus. April 1974 VERTEBRATE SYNAPTIC TRANSMISSION 493 Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 synapses is not clear; it is evidently neither on photoreceptors nor on horizontal cells (920). Unlike the horizontal cells, which are readily depolarized by both L-glutamate and L-aspartate (209, 920) and hyperpolarized by glycine and GABA (920), photoreceptor cells appear to be singularly insensitive to the usual chemical transmitters (920). Because the hyperpolarizing responses of horizontal and bipolar cells cannot be easily reversed, Nelson (929a) has suggested that they are not due to movements of a single ion species and, further, that they may be caused by changes in the amount of K+ released by the photoreceptors. The possible involvement of transmitters in retinal function is supported by various neurochemical findings. Like other parts of the CNS, the retina contains the usual large amounts of glutamate, aspartate, and GABA, as well as glycine (728, 969, 1157). All the enzymes of the GABA system are present in various layers of the retina (785). According to Graham (51 l), GABA is localized particularly in the amacrine cells and horizontal cells. A number of recent autoradiographic studies have attempted to localize sites of uptake of labeled amino acids (particularly [3H]GABA) in the retina of several vertebrate species (382, 384, 793, 927). Th ere is some disagreement about the distribution of radioactivity. For example, according to Ehinger and Falck (384) and Ehinger (382), labeled GABA and glycine are taken up mainly by amacrine cells, whereas Lam and Steinman (793) find that changes in [3H]GABA uptake caused by exposure to light are greatest in horizontal cells. Neal and Iversen (927) believe that most of the labeled GABA goes into Mtiller cells (glia); Ehinger and Falck (384), on the other hand, find that labeled excitatory amino acids are absorbed particularly by the Mtiller cells. The presence of neurons containing catecholamines, particularly dopamine, has been demonstrated by several authors (383, 858) ; they appear to be mainly in the nuclear layer. Although the exceptionally high choline acetyltransferase content of the retina (546) is strong indication of a cholinergic mechanism, it has been difficult to obtain consistent evidence for the location of the presumed cholinergic synapses, especially in view of the widely contradictory results of histochemical studies of the distribution of acetylcholinesterase (cf. 555, 818, 932). One can conclude that there is strong evidence for the participation of the conventional neurotransmitters in synaptic processes in the retina. The absence of spike activity at several synapses in the retina of course would minimize any significant contribution of electrical transmission. The high sensitivity of most horizontal cells and ganglion cells to L-glutamate and L-aspartate makes it quite likely that one or the other of the excitatory transmitters is released by photoreceptors and some other cells. At least some horizontal cells and amacrine cells probably have an inhibitory function, but the evidence that they act by releasing GABA or glycine is still inferential. The possible function of dopaminergic neurons is quite obscure. Summary. With the exception of the photoreceptors, most retinal neurons are excited by L-glutamate and aspartate and inhibited by GABA and glycine. Catecholamines also depress excitability, whereas ACh has a facilitatory action. There is also plentiful evidence of the presence in the retina and particularly of the 494 K. active uptake of these agents, various synapses. B. Chemical Transmission KRNJEVIC some or all of which Volume may well be transmitters 54 at at Sensory Endings 1. Carotid body chemoreceptors The high sensitivity of chemoreceptor afferents to acetylcholine and their close association with large glomus cells led to several early proposals that a cholinergic synapse may be interposed between the chemoreceptor elements and the afferent nerve fibers (3 10, 108 1, 12 19). This idea has been strongly advocated more recently by Eyzaguirre and his collaborators, who have shown that chemoreceptor activity can be depressed, at least to some extent, by magnesium or by nicotinic-blocking agents (4 12, 4 15, 936) and that acetylcholine is released from the carotid body (412, 416). This evidence, though very suggestive, is not conclusive and doubts have been raised about the interpretation (114, 962, 963). The essential counterargument of Biscoe (114) is that the fibers with which glomus cells appear to form synapses are not sensory but efferent fibers, which degenerate when the glossopharyngeal nerve is cut intracraniallyintracranial sections are proximal to the sensory ganglion and therefore should cause a degeneration of only efferent fibers (“decentralization”), whereas extracranial sections are distal to the sensory ganglion, Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 Many sensory nerve endings are relatively easily excited by a variety of neurotransmitter substances or related compounds injected into their blood supply or applied directly (5 14, 96 1, 962). It is not yet certain whether this is a pharmacological peculiarity or whether this indicates, as some have thought (306, 521, 7 1 1), that at least some nerve endings are normally excited by chemical transmitters released from specialized cells acting as transducers. It seems unlikely, however, that a mechanism of chemical transmission is operative at most sensory endings since the chemical sensitivity of certain receptors can be blocked by specific antagonists without blocking the responses evoked by the natural mechanical stimulus. Thus the carotid sinus baroreceptors go on responding to pressure changes even after their sensitivity to acetylcholine has been totally blocked by curarelike agents (5 14). Similarly, the chemical responsiveness may be enhanced specificallyfor example by eserinewithout changing the threshold of the mechanical receptor. Although there is only a very remote possibility that chemical transmission is a general feature of sensory endings (for recent critical reviews see 962, 963), there are at least two somewhat specialized types of receptors for which there is quite strong evidence indicating a mechanism of synaptic chemical excitation. [A further possible site of chemical transmission, at the sensory terminals of the eighth nerve (306), is not considered in any detail here as there is insufficient evidence for any useful conclusion.] April VERTEBRATE 1974 SYNAPTIC TRANSMISSION 495 2. Electrical receptors Strong evidence has been obtained by Bennett and his collaborators that electrical receptors in fish utilize a chemical transmitter mechanism (93, 943). Electrically sensitive receptor cells appear to be connected synaptically to afferent fibers. In the ampullae of Lorenzini of the skate, the receptor cells appear to be electrically excitable on one side- the luminal face that is exposed to applied currents -and they probably release a chemical transmitter from the other (serosal) face. There is no direct evidence about the identity of this substance, but, judging by the high sensitivity of the nerve terminal to L-glutamate, this could be the natural transmitter (1127). Summary. Chemical transmission is not likely to be a regular feature of sensory transduction in all types of sensory receptors, but certain specialized receptor cells may excite the peripheral terminals of afferent fibers via a chemical synapse: for example, in the carotid body and the ampullae of Lorenzini. C. Presynaptic Actions of Transmitters There is now some conclusive evidence that transmitters cant action on nerve terminals, but it is still not clear whether quence for normal synaptic transmission. can have a signinthis is of any conse- 1. Acetylcholine The first and most extensively studied presynaptic actions are those of acetylcholine, in muscle and in ganglia. a) Muscle. The presynaptic action of ACh was first suggested by the discovery that anticholinesterases promote a repetitive discharge of motor terminals that can be recorded antidromically in ventral root fibers (441, 874, 1258). The evident possibility that nerve terminals might be excited by postsynaptic electrical Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 and so make all fibers degenerate. Biscoe’s findings are certainly incompatible with the hypothesis of a sensory synapse; but they have themselves been seriously questioned. Hess and Zapata (569) have repeated the experiments and found that after decentralization the relevant nerve fibers and synapses in the carotid body remained intact, and there was no change in chemosensory discharges. They concluded that the synapses formed by the glomus cells must be nearly all sensory. The presence of choline acetyltransferase activity in the cells rather than the nerve endings (59) agrees with this interpretation. However, Biscoe’s proposal that the chemoreceptor nerve terminals are directly depolarized by changes in Paz, Pco2, or pH is partly supported by Eyzaguirre’s (414) suggestion that such direct effects -operating in parallel with a cholinergic junction between the chemoceptive glomus cell and the nerve endingsmay explain why ACh antagonists do not produce a complete block of transmission= 496 K. KRNJEVIC? Volume 54 2. Presynaptic action Ofneurotransmitter amino acids These have already been discussed in detail in the section dealing with the excitatory and inhibitory amino acids (see sect. III). Although depolarizing effects, or an increase in terminal excitability, have been described by several authors, the significance of these phenomena for norm.al transmission is by no means established. This is also true for the specific hypothesis that GABA is released at axoaxonal synapses and is responsible for presynaptic inhibition. It appears that presynaptic depolarizing actions of GABA may operate through a quite different Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 activity or by excitatory agents, such as potassium, released by the postsynaptic elements was practically eliminated in studies in which the muscle fibers were largely cut away, so that the presynaptic spikes were abolished and any other activity reduced to a minimum (72, 1025). In a different kind of experiment, Ciani and Edwards (219) and Hubbard et al. (613), by making measurements of the quanta1 content of the end-plate potential, found that extrinsic acetylcholine significantly reduces ACh release from the motor terminals. Hubbard et al. further showed that this effect is associated with an increase in terminal excitability. These presynaptic actions of ACh are evidently in the opposite direction from the potentiation of transmitter release postulated by Koelle (7 11). The presynaptic receptors for ACh cannot be very different from the postsynaptic ones since they are also blocked by curare. The question of a possible presynaptic component of the blocking action of curare has not yet been resolved. The most recent studies still lead to contradictory conclusions. Auerbach and Betz (41) could not detect a significant change in quanta1 release, whereas Hubbard and Wilson (614) found that curare reduces the quanta1 content and further accelerates the decline in quanta1 content seen during repetitive activity; however, Galindo (470) b e 1ieves that curare reduces transmitter release and raises the incidence of presynaptic failures of nerve conduction. This long-standing controversy is therefore still not closed. 6) Autonomic ganglia. The studies of Koketsu and Nishi (715), Nishi (937), and Gingsborg (491) h ave clearly shown that acetylcholine can cause a presynaptic depolarization and even a transient block of conduction in nerve terminals in sympathetic ganglia; as in muscle, these presynaptic acetylcholine receptors are nicotinic and therefore blocked by nicotine and tubocurarine. The most conclusive observations are those of Pilar (995, 996), who applied ACh while recording directly from inside the presynaptic nerve terminals in avian ciliary ganglia. He thus obtained incontrovertible evidence that ACh can depolarize the nerve terminal and lower its membrane resistance, and at the same time reduce the quanta1 content of ACh release. Although these results are interesting, it is unlikely that they indicate a very significant physiological presynaptic action of ACh (228). G) Central neruous system. Apart from some evidence that dorsal root terminals may be depolarized by ACh (702, 7 14) and that ACh may have a presynaptic action in the hippocampal dentate gyrus (1280), there is very little information available. April 1974 VERTEBRATE SYNAPTIC TRANSMISSION mechanism from that responsible for central inhibition permeability) and the possibility has not been excluded GABA may be mediated indirectly. 497 (an increase in chloride that the observed effect of 3. 0 ther agents acting presyna@‘cally D. Denervation Supersensitivity The phenomenon of postdenervation receive a good deal of attention. supersensitivity (199) has continued to 1. Muscle jibers The principal aim has been to try and decide whether the spread of the AChsensitive area is caused by the loss of a trophic factor, normally released from the Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 The unmyelinated terminal portion of nerve fibers appears to be particularly susceptible to various kinds of agents, ‘both pharmacological and natural agents associated with activity. For example, there is strong suggestive evidence that even relatively small increase in tissue PCO~ may cause a block of nerve conduction in activity a block of conthe region of the terminals (915, 916). D uring repetitive duction on the motor nerve terminals is frequently observed (741, 744) and there is reason to think that this is caused by the accumulation of extracellular potassium (1025). The large potentials that are recorded in afferent fibers close to their terminals (69, 378, 7 13) may well be caused at least partly by the release and accumulation of potassium as a result of nerve activity. Until recently any evidence that potassium was indeed accumulating around unmyelinated nerve fibers during activity was obtained by intracellular recording from glial cells (772) or by studies on isolated unmyelinated nerves (698); but with the use of K+-specific microelectrodes it has now been possible to show directly that there are very significant increases in extracellular potassium activity in the region of afferent nerve terminals, both in the spinal cord (747, 1224) and the cuneate nucleus (747), whose time course is very similar to that of the slow potential changes recorded in the same region and along afferent fibers. Although the precise significance of extracellular changes in potassium concentration for synaptic transmission is not yet clear, it is unlikely that their effects could be totally negligible; whether they are mainly presynaptic (239, 830, 1156) or postsynaptic (cf. 929a) remains to be determined. Summary. That some presynaptic terminals have receptors sensitive to the transmitters they release has now been incontrovertibly established, especially for cholinergic endings in the ciliary ganglion. It is less certain that t-glutamate and GABA have significant direct effects on central presynaptic terminals. On the other hand, the recent demonstration of an accumulation of K+ around active terminals may explain previous observations of slow presynaptic potential changes and perhaps corresponding alterations in synaptic efficacy. 498 K. KIWJEVIt? Volume 54 2. Denervation supersensitivity of nerve cells There is much less precise evidence about supersensitivity of peripheral or central neurons after denervation. Although KufHer et al. (773) were able to show a spread of ACh sensitivity in postganglionic parasympathetic cells in the frog heart after denervation, in view of the relatively short distance between points of innervation on the surface of the cell, it is not certain that this effect would make a very large difference to the chemical sensitivity of the cell as a whole. There is reason to think that other factors, particularly the large reduction in acetylcholinesterase activity after denervation, play a much greater role in making ganglia more sensitive to ACh ( 17 1). Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 motor nerve terminals (523, 900, 1223), or whether it is the result of a loss of mechanical activity. The second possibility is strongly indicated by two kinds of experiments. Lomo and Rosenthal (838) found that chronic paralysis caused by blocking the motor nerve with a long-lasting local anesthetic was associated with a clear spread of ACh sensitivity, even though neuromuscular transmission was not impaired by the prolonged inactivity; moreover, the supersensitivity could be prevented by stimulating the nerve at a point distal to the site of block. In complementary experiments, Vrbova (1223) and Drachman and Witzke (349) were able to reduce greatly supersensitivity by stimulating the denervated muscle directly with suitable electrical pulses. These findings strongly suggest that mechanical activity of the muscle is an important (perhaps the most important) stimulus for the normal suppression of gene activity controlling the synthesis of new ACh receptors [the widespread sensitivity after denervation is not just due to the uncovering of occluded receptors; Fambrough (419) has shown that the development of ACh supersensitivity in muscle in vitro is prevented by inhibitors of RNA and protein synthesis]. A significant role of a trophic factor released by nerve fibers, however, is indicated by some other observations. Trophic factors may be expected to be manufactured in the motor nerve cell bodies and carried to nerve endings by axoplasmic transport (cf. 353, 509, 845, 949, 1245). It therefore seems significant that colchitine, one of the agents known to block axoplasmic transport when applied locally to nerve fibers (60, 290, 729), causes a spread of ACh sensitivity in the corresponding muscles (58 1). According to these authors, colchicine has no effect on nerve conduction of neuromuscular transmission, and therefore the supersensitivity can only be explained by the interruption of a flow of some trophic factor to the muscle. This claim is not wholly consistent with observations of several other authors, who have found that colchicine reduces significantly the efficacy of synaptic transmission when applied either to the presynaptic nerve (684, 978) or the postsynaptic cell or nerve (998, 1080). A more direct influence of the nerve terminals than the induction of muscular activity is also suggested by the observation that the junctional region at the end plate has a higher sensitivity to ACh and a greater density of receptors than any other part of the muscle, even after denervation (5 1, 433, 538, 649, 898). April VERTEBRATE 1974 SYNAPTIC TRANSMISSION 499 E. Role of Glial Cells In his discussion of the functions of neuroglia, Lugaro (843) considered among other possibilities that neuroglia may provide a form of chemical insulation,. especially in the region of the nerve endings, where he postulated that unwanted products of neuronal activity were especially likely to be released. The idea that neuroglia may form a useful diffusion barrier around synaptic sites has been emphasized more recently by some authors. For example, De Robertis (332) points out that neuroglia do appear to form a continuous system of cellular membranes around nerve endings and synapses and therefore could prevent the diffusion of transmitters away from sites of release and so prevent an unwanted action on other cells. A more active role was proposed by KrnjevZ and Schwartz (763) : that glia may be involved in the removal of transmitter substances released during neuronal activity. This suggestion was based on the observation that cortical unresponsive cells-which are probably glia, as was subsequently demonstrated by intracellular staining (5 18, 692, 123 1) -were depolarized by applications of GABA and ACh, an effect that was not associated with an increase in membrane conductance and therefore might indicate an electrogenic process of active uptake [an alternative explanation (804), that the glial cell was depolarized by potassium released from adjacent cells, does not take into account the fact that GABA inhibits by increasing chloride permeability and that ACh excites cortical cells by reducing potassium permeability]. There is now rapidly increasing biochemical and auto- Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 Very precise tests have not been possible in the CNS, but measurements of mean neuronal sensitivity in neurally isolated islands of cortical tissue in cats have shown no evidence of increased responses to glutamate, GABA, or ACh applied by microiontophoresis (734, 760). With a similar technique, Spehlmann et al. (1117, 1119) also failed to see any sign of increased sensitivity to acetylcholine in partially deafferented cortex. Although the density of synapses may not be much greater on mammalian cortical neurons than on the frog’s parasympathetic neurons [probably less than 10 % of the surface area is actually covered by synapses (cf. 232, 536)], when synapses are separated by distances of 5 pm or less, the responses of‘ the cell may be determined primarily by the high-sensitivity spots. It seems very likely that much of the apparent increase in ACh sensitivity of isolated cortical tissue can also be ascribed to a marked loss of tissue cholinesterase (760, 1033). Another factor that may cause the apparent sensitivity of central nervous tissue to, increase is a disturbance of the normal neurotransmitter uptake mechanisms; such an effect is largely responsible for the denervation supersensitivity of tissues innervated by adrenergic nerves (476, 1189). Summary. Several lines of evidence indicate that the loss of muscular activity promotes the development of new ACh receptors, but a significant trophic action of the nerve fibers has not been eliminated. In denervated ganglia or isolated portions of the CNS, a reduced efficiency of transmitter removal may be more significant than the formation of new receptors in causing supersensitivity. 500 K. KRNJEVIC Volume 54 VIII. GENERAL A. Origin CONSIDERATIONS and Nature of Chemical ABOUT SYNAPTIC TRANSMISSION Transmitters One might suppose that in the course of evolution special substances were developed for the sole purpose of intercellular communication and that, as a result of their release at certain points, specific receptors were induced by a process of adaptation comparable to a postulated mechanism of antibody formation (386, 539, 972). Such a scheme might seem consistent with the fact that in muscle the region of innervation is much more sensitive to the transmitter released by the nerve fibers (ACh) than is the rest of the muscle fiber (173, 770, 798, 899). This view of chemical transmission has been taken sufficiently seriously that the widespread actions of certain amino acids in many areas of the vertebrate CNS have been considered as evidence that these compounds cannot be natural transmitters (129, 522, 548). However, it is becoming more and more evident that a chemical sensitivity of the cell membrane is a quite fundamental property that is largely unrelated to Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 radiographic evidence that glial cells are indeed sites of uptake of various neurotransmitters (382, 563, 584, 608, 864, 927, 955). It may well be a major function of neuroglia to keep the extracellular spaces of the CNS free of neurotransmitters and perhaps other potent agents that might otherwise interfere with efficient neuronal activity. If neuroglia are capable of accumulating substantial amounts of various neurotransmittersand perhaps even participate in their synthesis-one evidently must consider the possibility that this uptake process may, under some conditions, be reversed, so that a significant release of transmitter perhaps takes place from neuroglia. This could be an abnormal phenomenon, resulting from a tissue disturbance. Of greater interest would be a possible release triggered by adjacent neuronal activity. There are various ways in which such interaction could take place. An obvious one would be through potassium leakage from active neurons. Excess potassium readily accumulates in the minute space between neurons and glia (cf. 206, 518, 519, 747, 761, 775, 1224). A n even more direct form of coupling between neurons and glia in tissue culture has been reported by Walker and Hild (1227). Depolarization of the glial cells may conceivably lead to a discharge of stored transmitters. There is evidence that, in denervated muscle, ACh is released from Schwann cells when they are depolarized by an electrical current (328). Another mechanism of coupling between neurons and glial cells through the release of catecholamines has been recently proposed by Gilman and Nirenberg (487). Summary. It is increasingly likely that glial cells are very much involved in mechanisms of chemical transmission in the CNS, particularly in the active uptake and perhaps metabolism of excitatory and inhibitory transmitters, and they may even be sites of transmitter release, which could play a significant role in the control of neuronal discharge. April 1974 VERTEBRATE SYNAPTIC TRANSMISSION 501 Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 innervation although innervation usually has a significant modifying action. Chemical sensitivity is thus often seen best and most widely at an early stage of development and later tends to be repressed to some degree when the cells become innervated. A good illustration of this is provided by skeletal muscle. The sensitivity of muscle fibers to ACh is particularly high and widely distributed in the fetus or in very young animals (341, 488); in fact, as recent work has shown, it appears at a very primitive stage of myogenesis, long before innervation (42 1, 944; cf. also 362, 964). When contact is made by a nerve fiber, the ACh-sensitive area becomes largely restricted to the immediate junctional zone. But removal of the nerve (5 1, 488, 898) or even prolonged nerve block (838) rapidly leads to a reversal to the more primitive condition of widespread sensitivity. Even such noninnervated cells grown in tissue culture as the mouse neuroblastoma cells (535, 929) or the fibroblastic L cells (928) have been shown to be sensitive to ACh. These facts demonstrate that sensitivity to ACh is an intrinsic property of many (but not necessarily all) cells. On the other hand, ACh or macromolecules related to the metabolism of ACh appear to be involved in very basic cellular processes. As pointed out in section IIJ, there is some reason to think that acetylcholinesterase may provide sites for cation movements through cell membranes. Another possibility is that ACh and its related macromolecules may have a function in the transport or accumulation of raw materials essential for the synthesis of some membrane component, such as lecithin or other choline-containing phospholipids. A function of this kind is perhaps indicated by the fact that ACh markedly accelerates the incorporation of inorganic phosphate in to phosphatidic acid and phosphatidylinositol (587, 588, 801), although the full significance of this effect is still obscure (540). The presence of very large amounts of enzymes of the ACh system in the placenta (553) and the cornea (1267) may be also related to a comparable mechanism of selective transport or of synthesis of new membranes. In this context, it is perhaps significant that regions where new membranes are being formed at an exceptionally high rate because of local injury or some other factors also tend to be sites of relatively high ACh sensitivity: for example, the musculotendinous junction (432, 672), injured muscle fibers (673), the growing tips of regenerating sensory nerve fibers (338), and perhaps some peripheral sensory receptors (5 14, 96 1). An interesting observation is that degenerating nerve fibers stimulate the formation of ACh receptors (1223), though it is not clear whether this effect is mediated by the release of a specific chemical factor or by a physicochemical interaction. An essential sensitivity to excitatory and inhibitory amino acids in the vertebrate brain is indicated by the high sensitivity to glutamate and GABA shown by cerebral cortical neurons in the newborn kitten (755) and by cerebellar neurons in the newborn rat before synapses are formed (1272). E ven some cells that lie outside the CNS and therefore are probably never innervated by GABA-releasing nerve fibers such as sympathetic ganglion cells (5, 3 15, 944) and dorsal root ganglion cells (3 18, 944)-which presumably have no synapses on them-nevertheless show an appreciable sensitivity to this inhibitory amino acid. Different synaptic inputs are known to be segregated on different regions of 502 IS. KRNJEVIC Volume 54 Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 the somadendritic complex (377, 841, 1020, 1201, 1202). This is particularly evident for some inhibitory inputs that appear to be concentrated on the soma of certain cells (232, 373). It is not known at present whether there is a corresponding differential sensitivity of various parts of the cell membrane, so that for example the region of innervation by inhibitory fibers is particularly sensitive to GABA-as might be suggested by the observation of spots of maximal ACh sensitivity corresponding to the synaptic sites on the surface of parasympathetic ganglion cells (536). A sharp localization of GABA sensitivity on certain portions of the Mauthner cell was apparent in Diamond’s (339) initial experiments, but more precise tests revealed no significant difference in GABA sensitivity over a large area of the cell body and dendrite (340). In any case it is clear that various cells are not uniquely sensitive to particular transmitters. Most central neurons have proved to be sensitive to several chemical agents : for example, notwithstanding their rather specialized function, Renshaw cells are sensitive to a variety of transmitters : ACh (both as a nicotinic and as a muscarinic agent), glutamic acid, GABA, glycine, and probably some monoamines. They must have several distinct chemical receptors appropriate for the different transmitters, though it is by no means certain that each Renshaw cell is innervated by fibers of all the corresponding types [cf. neurons in the medulla, which are very sensitive to both glycine and GABA, but appear to receive only a GABA-releasing innervation (696, 1172)]. In conclusion, it seems that the chemical sensitivity of a given cell is an intrinsic property, probably coded genetically like the specific transmitter synthesizing capacity (cf. 17). Why and how these different sensitivities originally developed are not fully evident, but one can readily understand that the charged surface of the cell membrane must be affected by its ionic environment, particularly by multivalent ions that tend to interact strongly with all charged surfaces. Practically all cells appear to have a surface negative charge (398a), so that any cation (especially if multivalent) would significantly alter the charge density and therefore the conformation of membrane macromolecules, possibly inducing in this way various specific changes in membrane permeability and potential through which excitability can be either raised or lowered (cf. 489, 737). The membranes of even unicellular organisms show some marked effects of charged molecules introduced into their environment (22). Since small charged molecules leak out of cells and the rate of such leakage is likely to be a function of activity, the interaction of such small molecules with adjacent cells could, from a very early stage of evolution, have effectively provided a means of intercellular communication. Processes of synaptic transmission could readily develop from such relatively distant interactions, and it may well be that the synaptic transmitters used by even the highest organisms had their origin in this way, not as special molecules specifically developed for purposes of communication, but simply as elements of basic metabolic or synthetic processes common to large families of cells. Clearly it is advantageous to utilize for transmission molecules that are readily available from metabolic processes widely distributed in living organisms. All that is needed for effective transmission is that these molecules should interact with certain mem- April 1974 VERTEBRATE SYNAPTIC TRANSMISSION 503 B. Electrical Transmission Embryonic tissues may show a high degree of intercellular coupling-to an as well as extent quite unsuspected until recently -so that quite large molecules electric currents may readily pass between adjacent cells (94, 630, 833, 834). This situation is ideal for the efficient distribution of essential chemicals during development, but it disappears as the cells differentiate; most nerve and other cells in the fully developed animal are independent units, separated by largely impermeable membranes, even in areas of functional contact. Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 brane groups acting as receptors. As Watkins (1233) pointed out, it is perhaps significant that several of the neurotransmitter molecules are indeed strikingly closely related to very common constituents of cell membranes. All the rapidly acting putative neurotransmitters have proved to be relatively small molecules (molecular weights less than 200) of relatively simple structure, having only a few (but well-defined) charged groups, typically with pK’s well away from seven, so that they are fully ionized at a physiological pH. Such molecules probably do not carry a great deal of biological information. This is fully consistent with the very limited amount of information necessary for synaptic transmission. The neurotransmitters as a rule do not travel more than a minute distance, because the synaptic spaces are small, and therefore they need specify neither their site of origin nor site of action. For practical purposes all that they have to embody is a simple yes-or-no statement. Much more complex information, of course, is required for the establishment of synaptic connections, and no doubt more complex molecules are involved in that process. It is now clear that the genetic information needed for protein synthesis-and indeed specifying the whole organismis transcribed in terms of 20 simple amino acids. These building blocks are thus also units of information, which are combined to form polypeptides and proteins containing correspondingly greater amounts of information (387, 629, 1234). It therefore is appropriate that the elementary messages of synaptic transmission should be mediated by such amino acids or closely related molecules of comparable simplicity. The huge information-handling capacity of the brain presumably resides in the vast number of cells and synapses available and the enormously varied possibilities of interconnections between units that each contain relatively little information. This system offers a marked contrast to the storage of genetic information in single cells in the form of giant macromolecules. In general the complexity of molecules used for the transfer of information seems to be related to the complexity of the message: at one extreme, synaptic transmitters that carry minimal information are probably the simplest molecules longer chains of polypeptides make up the releasing of this type; increasingly factors or hormones, which circulate over relatively large areas and therefore must convey more elaborate information to identify the specific target cells or to initiate the appropriate changes in enzyme activity, metabolism, and ultimately behavior. 504 K. KRNJEVIt? Vohme 54 relatively few junctions, where speed of transmission and are of particular importance (92), although even at most mechanisms also seem to operate (cf. 46 1, 72 1, 796, 87 1). transmission is most advantageous where sharp electrical sharp synchronization of these sites chemical Furthermore, electrical signals are generated; since action potentials are only necessary for conduction over relatively large distances, whereas primitive nerve cells must have had very limited dimensions compatible with efficient signaling by electrotonic conduction, it is very unlikely that electrical transmission could have been of much use for junctional transmission until a relatively late stage in the evolution of larger and more complex organisms. Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 Only in a small proportion of cells does one find a type of contact apparently specialized to facilitate cellular coupling. At these junctions the usual synaptic space is absent: at first this was believed to be due to partial fusion of the membranes (966), but subsequent investigations have revealed a narrow gap (which can be filled with lanthanum and horseradish peroxidase) as well as a polygonal system of connections across the gap, possibly providing channels for intercellular currents and diffusion ( 166, 974, 1035). The first evidence of functionally significant electrical’ transmi .ssion in the vertebrate CNS-probably at synapses of this type-was obtained in the medulla of the goldfish by Furukawa and Furshpan (462): this was seen as an inhibitory synaptic potential of very brief latency, relatively insensitive to the membrane potential of the postsynaptic cell, which could be reproduced with an anodal current flowing from an extracellular micropipette. Shortly after Furshpan (461) described an excitatory PSP with comparable properties, also recorded in the Mauthner cell of the goldfish. It therefore appeared that, in addition to conventional chemical EPSP’s and IPSP’s, these cells normally receive significant electrotonic signals. Other electrotonic junctions have been described in the brain and the retina of fish (92, 95-98, 656, 721), in the spinal cord of the frog (5 17), and in the ciliary ganglion of birds (87 1). In the latter case, where electrical coupling is facilitated by the presence of exceptionally large presynaptic terminals as well as effective electrotonic transmission develops relatively late during gap junctions, embryogenesis or only after hatching (568, 796, 997). According to morphological evidence of the presence of gap junctions, electrotonic transmission may be expected at several sites in the mammalian CNSparticularly in the vestibular nuclear complex, the retina, and the mesencephalic nucleus of the trigeminal nerve (166) and perhaps even in the neocortex (1102) and the cerebellum (1 1 1 1)-but so far there has been only rather limited support from electrophysiological evidence (55, 600). As pointed out by Bennett (92), electrotonic transmission in theory could produce just about all the transmitter effects needed to operate the.CNS-though it is difficult to see how any electrical mechanism could efficiently generate the very long inhibitions so characteristically seen in the brain (durations of > 100 ms). role in the functional organization Since inhibition in fact has such a predominant of the CNS (cf. 226, 374, 377) chemical transmission may well have had an evolutionary advantage, which explains the relegation of electrotonic mechanisms to April 1974 VERTEBRATE C. Chemical Ihferentiation SYNAPTIC TRANSMISSION 505 in Nervous System Downloaded from http://physrev.physiology.org/ by 10.220.32.246 on June 17, 2017 Why do different synapses seem to operate by distinct chemical means? It would have been simpler (especially for the investigator) if only one excitatory and possibly one inhibitory transmitter were utilized for all the junctions throughout the organism. Yet it is beyond question that certain types of cells are consistently particularly sensitive to certain agents or tend to release a given transmitter. Such differences are probably genetically determined (cf. 17). The simplest explanation is that various aspects of function and behavior may be executed by neurons of a certain genotype, each type having its own chemical properties, which may be related to the function of the cells or may have arisen fortuitously. The chemical differentiation of the peripheral nervous system is well known (cf. the various divisions of the autonomic system). Even in the CNS it is evident that different populations of cells have a distinct chemistry: not only are there regional variations in the distribution of monoamines and other substances, but the effects produced at various sites by these agents differ quantitatively and even qualitatively, sometimes in a systematic manner. For example, in the spinal cord glycine plays a very important inhibitory role that is rarely seen in some of the upper regions of the CNS, whereas monoamines appear to have excitatory effects in the brainstem that are not evident in other parts of the brain. It seems likely that the excitatory and inhibitory amino acids are the transmitters released by the large fibers of the rapidly conducting pathways in the most recently developed part of the CNS, the outermost portion that contains the long tracts of white matter, well-defined nuclei, and large cells concerned in the interaction between the organism and environment (cf. 1278). The core of the CNS, on the other hand, is made up of a diffuse network of small fibers and cells and has only poorly differentiated nuclei and tracts. Its function may well be to control the internal state of the organism (cf. 1278). Cholinergic and monoaminergic mechanisms seem to be particularly prominent in this core and the surrounding intermediate system that, according to Yakovlev (1278), is essentially responsible for the outward expression of the internal state- what in the higher animals would correspond to psychic states. As suggested by many authors, the monoamines and ACh may be heavily involved in basic drives such as hunger, thirst, aggression, mating, and the related emotional states, as well as in other mechanisms that maintain the internal economy, presumably including sleep. There is a vast literature already referred to which discusses the role of possible derangements of this system and its chemistry, in relation to psychic disorders, and much use is being made of drugs that are believed to exert their effects on the metabolism of the corresponding transmitters. Of course it could be argued that the more recent, faster conducting pathways have largely taken over the function of the core-and this might seem to be indicated by the surprisingly insignificant behavioral effects of 6-OH-dopaminebut this would be a very rash conclusion without much more evidence than is yet available. An important technical problem arises from the fact that a diffuse network of small fibers and cells is not readily amenable to systematic unit analysis by electrophysiological means. Inevitably the electro- 506 K. KRNJEVIC Volume 54 The author is grateful to Mrs. L. Simon for her great assistance review, particularly for typing the manuscript, and to the Canadian for its financial support. This review was essentially completed in May 1973. in the Medical preparation Research of this Council REFERENCES 1. ABDERHALDEN, E., AND A. WEIL. Vergleichende Untersuchungen iiber den Gehalt der verschiedenen Bestandteile des Nervensystems an AminosPuren. 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