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AM. ZOOLOGIST, 2:79-96 (1962). ORGANIZATION OF NEUROPIL DONALD M. MAYNARD Department of Zoology, The University of Michigan Most complex nervous systems consist of central ganglia connected with outlying sensory organs and effectors by peripheral nerves. Information moves to and from the ganglia in the nerves in coded form, as a spatial-temporal pattern of all-or-none impulses. Decoding of the incoming message and recoding of the appropriate outgoing response occur somewhere in the ganglia. Many of the fundamental problems of neurophysiology revolve around the related questions of the functional significance of impulse patterns in the central ganglia (codes), and of the mechanisms of central neural integration (coding and decoding). Information about impulse patterns and the properties of integrative systems can be derived from input-output analyses (Wiersraa, 1962), but problems regarding detailed mechanism eventually require penetration into the ganglion and dissection of the integrating system itself. Let us look, then, at the organization of a central invertebrate ganglion. The emphasis will be on arthropod material, for I am most familiar with this group; most of the comments should apply with little modification to many of the annelids, molluscs, and lower vertebrates. NEUROPIL In the central ganglia of most higher invertebrates four histological divisions are evident (Fig. 1): 1) An outermost sheath of non-neural connective tissue cells or glial elements separates the central neural tissue from general body cavities. 2) Just within the sheath tissue are the cell bodies of the monopolar neurons. 3) Tracts of fibers form the third division. These run from Original work on Panulirus reported in this paper was carried out at the Bermuda Biological Station. It was supported by Rackham Grants from the University of Michigan, an ONR grant to the Biological Station, and U.S.P.H.S. grant B 3271. Miss C. Goodrich collaborated on the experiments with the stomatogastric ganglion. cell bodies into the neuropil, from the neuropil to the origin of peripheral nerves, and from neuropil to neuropil as connectives or commissures. The fibers of the tracts are either axons or extended, unbranched portions of the dendritic arborizations. 4) The fourth and final division is the central neuropil, the neuron feltwork. In many cases it represents the major portion of the ganglion. The term neuropil, however, has beef, used in different ways by a number of authors, and is not a precisely defined concept (see Herrick, 1948; Dempsey and Luse, 1958; Friede, 1960). Classically it is an anatomical term referring to regions of the central nervous system composed of tangles or meshworks of nerve processes and non-neural elements such as glia and blood vessels. By extension it also refers to such tangles of collaterals, dendrites, and axon terminations in peripheral ganglia. Neuropil has also been considered synonymous with synaptic field, however, and thus has acquired functional connotations as the primary site of integrative neuron activity (Herrick, 1948). The structural limits of neuropil are apparent in forms such as the crayfish or lobster where the majority of the centra' neurons are monopolar with cell bodies located in the periphery of the ganglion. As one moves either to such non-vertebrates as the Coelenterata or Platyhelminthes, however, or to the higher vertebrates, bipolar or multipolar neurons in the central nervous system increase and the cell body becomes increasingly mixed with dendritic fields. Finally the original distinction between cell body regions and fibrous regions is lost; there is a continual gradation from the classical neuropil, a synaptic field which included processes only, to the synaptic field which places the ce.ll body in the center and surrounds it with dendrites and terminal arborizations, as for example in the vertebrate olivary nucleus (Fig. 2). In organisms with the latter kind of synaptic field the (79) 80 DONALD M. MAYNARD BIG. 1. Brain of craylish, Astacus fliwiatilis, as seen from above and behind. Methylene blue stain showing peripheral cell bodies, fiber tracts, and neuropil. Peripheral nerves from top to bottom: anterior me- dian nerve, optic tract, tegumentary nerve, antennal nerve, and circumesophageal connectives. (From Retzius, 1890, Biol. Untersuch. N.F.) cell body seems more intimately involved with electrical activity of the neuron, and it is reasonable to consider the entire integrating portion of the nervous system as functional neuropil. Many authors have been impressed with the apparent structural disorder of neuropil (Dempsey and Luse, 1958; Horridge, 1961). It is indeed heterogeneous and complex, and because of this has resisted most attempts at a systematic analysis. It has been called the terra incognita of neuroanatomy and neurophysiology. Nevertheless, neuropil often seems to conform to some underlying structural pattern, even when showing random connectivity. We shall begin this discussion, therefore, with a preliminary structural classification of neuropil. The recurrence of similar patterns in regions subserving similar functions in widely divergent organisms suggests that much of the structure may have unique functional significance. The direct measurement of neural activity within neuropil has just begun, but electrical recordings have revealed several functional properties that in some cases correlate closely with neuropil structure. l i d . 2. (ells of inferior olivary nucleus of new born human, a, axon: b, collateral. (From Cajal, 1904, Textura del sistcma nervioso del hombre y rle los vertebrados. II. Part 1.) ORGANIZATION OF NEUROPIL FIG. 3. Plexiform neuropil. Nerve net of bipolar cells in mesentary of Metridium. Whole mount, Holmes' silver. (From Pantin, 1952, Proc. Roy. Soc. London, B.) STRUCTURE OF NEUROPIL Microscopic Morphology On the basis of gross fiber configurations, all neuropil can be placed in either of two general classes, structured or unstructured neuropil. These in turn can be subdivided: the unstructured into plexiform and diffuse neuropil, the structured into glomerular and stratified neuropil. The groupings are not sharply bounded or exhaustive, but they do emphasize significant differences in organization and, without undue stretching, seem to include most kinds of neuropil. M tic junctions seem to be lacking, and the nerve net has been considered syncytial (Mackie, 1960). Plexiform neuropil may form the major portion of the nervous system in primitive, sessile, or burrowing organisms. In the Coelenterata, for example, the entire nervous system is a two-dimensional nerve net (or nets) and cell bodies are continuous with processes. In Balanoglossids, on the other hand, the cell bodies are separated from a thicker plexiform fiber mat (Bullock, 1945a). Plexiform neuropil also forms limited regions of more complex nervous systems or serves as a component of structured neuropil. It is the common pattern of innervation for hollow organs such as the digestive tract. Diffuse neuropil presents the classical picture of "a tangled confusion" (Horridge, 1961) (Fig. 4). Neurons contributing to it in most invertebrates are either monopolar elements of average or large size, or peripheral bipolar sense cells whose axon terminations ramify centrally. Many of the monopolar cells are motor neurons or interneurons connecting several ganglia or segments of the organism (Fig. 5). Processes are characteristically tortuous, extensively Unstructured neuropil Unstructured neuropil is characterized by ill-defined fiber configurations. Areas of fiber interaction are not sharply differentiated within the general fiber network, and the domain of a single neuron may be extensive. The neuropil may take either of two forms. Plexiform neuropil is homogeneous and net-like (Fig. 3). The neurons forming it are usually simple mono-, bi- or multipolar structures. The processes are often unbranched, straight, and of constant diameter. Synaptic junctions appear simple, of few varieties, and may occur at almost any point on the process. Many appear to be unpolarized, and transmit impulses in either direction. In some forms, the synap- FIG. 4. Diffuse neuropil. Central region of brain of spiny lobster, Panulirus argus. Thin section, Masson's trichrome stain. Ramifying dendrites and fiber tracts form a "haystack" with regions of varying texture. Width of field, about 1.5 millimeters. 82 DONALD M. MAYNARD FIG. 5. Diffuse neuropil. Last thoracic and abdominal ganglia of Carcinus maenas. Whole mount, methylene blue stain. Note extensive branching of motor unit in right lobe of thoracic ganglion. (From Jiethe, 1895, Arch. Mikroscop. Anat. u. Entwitklungsinech.) branched, and of widely varying diameters. They spread to varying degrees throughout the neuropil so that it seldom presents a homogeneous appearance. Each neuron, however, tends to have its own domain of arborizations. Synaptic junctions are not easily recognized with light microscopy, but are probably more heterogeneous than in plexiform neuropil. There is no good anatomical information concerning the existence of unpolarized synapses or syncytial interneuron bridges. Diffuse neuropil is characteristic of the more posterior central ganglia of most higher invertebrates, and usually contributes to the cerebral ganglia. It is also found in the brainstem of lower vertebrates and in many peripheral invertebrate ganglia; the crustacean stomatogastric ganglion is an example. tions. Areas of fiber interaction are usually clearly distinguished from surrounding fiber networks. The domain of a single neuron may be quite limited, but it often makes contact with other elements whose domain is extensive. The rigidity and preciseness of the fiber patterns is impressive. They usually seem to represent a reduplicating system; a complex structure built up of large numbers of relatively few kinds of units which in turn are grouped into repeating sub-structures. Such precise but redundant structural fabric probably occurs where stability, accuracy, and temporal or spatial complexity are functional prerequisites. It seems unlikely that functional lability and the potentiality for "learning" predominate in the more excessively structured neuropil. Structured neuropil is most evident in anterior ganglia of complex organisms. It may be directly associated with complex sensory organs. There are many geometrical patterns of structured neuropil, but Structured neuropil Structured neuropil is characterized by regular, often repeating fiber configura- ORGANIZATION OF NEUROPIL riG. 6. Glomerular neuropil. Nucleus rolundus of sea robin, l'rionolus evolans. Thin section, upper part drawn as it appears after silver impregnation according to Bodian. Lower part, reconstruction from Golgi preparations. Ch., commissura horizontalis of Kritsch; Gl., glomeruli; Tr.r.-l., tractus rotundo-lohaiis. (I'rom Scharrer, 1945, J. Coinp. Neurol.) most seem to fall into one or the other of two groups, glomerular and stratified. Glomerular neuropil is characterized by tight, profuse ramifications of pre- and postsynaptic elements at the site of junction (Fig. 6). These ramifications form the discrete, knot-like clusters of fibers that give this kind of neuropil its name. The glomeruli themselves rarely occur in precise relationship with each other; they may be in layers or apparently unstructured clumps. The size and form of the glomerulus and the number and pattern of elements contributing to it may vary greatly. For example, an afferent fiber in the vertebrate olfactory bulb or the insect antennal lobe terminates in but one glomerulus, while neurons of the insect corpora pedunculata or mitral cells of bird olfactory lobes may have branching processes which enter a number of glomeruli. The minimal number of neurons necessary to form a glomerulus is not clear, but the olfactory glomeruli of the rabbit must represent a near maximum. Each glomerulus is claimed to receive as many as 26,000 incoming fibers and has about 90 outgoing fibers (Allison, 1953). This is a particularly clear example of the convergence that often seems to occur in glomeruli. 83 The internal structure of glomeruli is largely unknown (see Bodian, 1952). Silver and methylene blue stains suggest that differences exist between the arborization patterns of pre- and post-units. The neurons whose processes form glomeruli range widely in function and anatomy. They may be motor, but more commonly are sensory or internuncial elements. Among the latter, the dendrites of small globular neurons of the invertebrates (Hanstrom, 1928) and granule cells of the vertebrate cerebellum characteristically form glomerular junctions. It is evident that glomerular neuropil does not represent a homogeneous class, but further subdivision is undesirable until more information on structure and function is available. Stratified neuropil (Fig. 7) is a regular, three-dimensional fiber lattice formed from precisely oriented neuron processes. The anatomy presumably reflects the functional connectivity. The basic pattern consists of vertically or radially oriented fibers that pass at right angles through horizontal or tangential layers of ramifying processes. Synaptic junctions occur between the vertical and horizontal fibers. The arthropod optic ganglia represent a typical, highly structured example of stratified neuropil (Fig. 7). The vertical elements are quite different in form from the horizontal fibers. Some of the vertical elements have collaterals and junctions in only one layer, others send collaterals into several layers. Often the collateral structure differs according to the layer or the particular vertical neuron; presumably it reflects differences in the functional properties of the junction. The lateral extent of the synaptic field of a vertical unit is usually limited. Consequently most vertical units probably synapse with relatively few horizontal fibers in any one layer. In contrast, the horizontal fibers branch diffusely. They often travel in preferred directions, but apparently form junctions with a large number of vertical units in their level. Within a layer they are probably the major link between vertical units, just as the vertical units appear to be the major link between layers. 84 DONALD M. MAYNARD areas of cerebral ganglia. The cortex of the vertebrate or possibly the central body of the arthropod are examples. These regions, however, often lend to merge in form with diffuse, unstructured neuropil, and the fiber domain of a single neuron appears less rigidly defined than in the optic systems described above. Hislochemistry and Sub-microscopic Anatomy The above discussion gives a general view of fiber patterns in neuropil, but it is necessarily superficial. In addition to the obvious limitations resulting from this superficiality, there are at least two serious drawbacks in the methods themselves which reduce the functional significance of the findings. On the one hand, the usual preparative techniques of classical micro-anatomy do not reveal chemical differences among fibers of the neuropil, and on the other, light microscopy does not have sufficient resolution to show the detailed intracellular structure of fibers and synapses. Both of these aspects are particularly relevant in a consideration of the functional anatomy of neuropil. Histochemislry FIG. 7. Stratified neuropil. Diagram of retina and optic ganglia of honey bee, Apis melifica L. I, retina; II, lamina ganglionaris; III, medulla externa; IV, medulla interna. (From Cajal and Sanchez, 1915, Investig. Biol.) Both vertical and horizontal fiber groups contain afferent and efferent elements. In the case of optic ganglia, however, the primary input fibers from the visual receptors are vertical rather than horizontal. In contrast, only horizontal fibers seem to carry information from the optic ganglion complex into more central areas of the brain. The retina and superior colliculus in the vertebrate and the retina profundus of the octopus are formed from stratified neuropil essentially like that of the arthropod. Such a configuration may be important in dealing with complex spatial patterning, and will be considered in more detail below. Less precise layering is evident in other There is considerable evidence for several neurotransmitters in the nervous system (Florey, 1962). A detailed map showing the localization of these, or substances involved in their production or break-down could prove of aid in determining the functional properties of a given neuropil. Many of the transmitters seem to be specific for particular kinds of neurons. We have been interested in the localization of cholinesterases in crustacean ganglia (Fig. 8, Maynard and Maynard, 1960a). There are high concentrations of the enzyme in peripheral glial tissue (see also, Wigglesworth, 1958), but it also occurs in some neuron somata and particularly in certain regions of the neuropil. Experiments with more peripheral structures in the lobster (Maynard and Maynard, 1960b) suggest that concentrations of the enzyme may correlate with physiological character- ORGANIZATION OF NEUROIML dendrite and axon in the neuropil is lacking. The peripheral cell bodies are devoid of terminal contacts, and fiber junctions are confined to the neuropil. Three kinds of junction are recognized: 1) cross contacts, 2) longitudinal contacts, and 3) endknobs (Fig. 9). In all, neuron membranes come into direct contact with each other, but there is no fusion of neuroplasm. Although membranes are often flattened or indented at the point of contact, invaginations as found in the synapse between giant fibers in the crayfish (Robertson, 1953) are apparently absent. Only the end-knob junction contains the accumulations of microvesicles and mitochondria often considered characteristic of synapses (De Robertis, 1958; Palay, 1958). Microvesicles, however, also occur along fibers in non-junctional areas. ol FIG. 8. Distribution of cholinestcrase in brain of lobster, Homarus americmuis. Thin section, acelylthiocholine substrate. Al, accessory lobe, a glomerular neuropil; gc, globular cells, two different groups; ol, olfactory lobe, another glomerular neuropil. Enzyme concentration to right of accessory lobe represents a fiber tract; light areas surrounded by a ring of enzyme are large neuron somata; other areas of high enzyme concentration are in diffuse neuropil. (Courtesy of E. A. Maynaid) istics, but this remains to be established in the central ganglia. Perhaps the final interpretation o£ such chemoarchitectonics must await clarification of the role of acetylcholine esterase in neural activity. Sub-microscopic anatomy A number of scattered observations on the fine structure of invertebrate nervous systems exists, but a systematic, comprehensive investigation of neuropil has not been published. One of the closer approximations to such a needed work is a preliminary investigation of caterpillar ganglia (Trujillo-Cenoz, 1959). Most fibers in the (diffuse?) neuropil of a caterpillar ganglion are naked and lack glial sheaths. In this they differ from the sheathed fibers in interganglionic connectives. Fine structural distinction between FIG. 9. End-knob in neuropil of caterpillar, Pholus labruscoe L. Electron micrograph. E.K., end-knob; M, mitochondria; S.M., synaptic membranes; T.F., thin fibers (post-synaptic?). Microvesicles accumulate to left of mitochondria. Width of field, about 2.3 microns. (From Trujillo-Cenoz, 1959, Zeit. f. Zellforsch.) 86 DONALD M. MAYNARD Several important conclusions can be drawn from these observations. 1) Neuron interaction in caterpillar ganglia, if dependent upon close anatomical contact, seems to be confined to the neuropil. 2) Some junctions in the neuropil are similar to the classical synapse; one may therefore anticipate similar properties of synaptic transmission. 3) Several forms of fiber junction occur, suggesting the possibility of more than one mechanism of neuron interaction. It should be clear, however, that full benefit of the increased resolution of electron microscopy will be apparent in neuropil analysis only when correlations between fine structure and specific microscopic structures are possible. Glia and Capillaries Thus far we have spoken of neuropil as though it were composed solely of neuron processes. This is misleading, for both glial cells and vascular elements are also present. As mentioned earlier, glial elements usually form an outer sheath enveloping the entire nervous system. They may also surround cell bodies and individual fibers in tracts and connectives (Wigglesworth, 1959). Just how far glial processes interpenetrate the denser fiber tangles of invertebrate neuropil is not known (Trujillo-Cenoz, 1959). In the mammalian grey matter, of course, glia and neuron processes intermingle, and glia-neuron junctions have been described (Scheibel and Scheibel, 1958). The physiology of glia is of current interest. In the past glia have been considered supporting or nutritive elements (Wigglesworth, 1960), but they also have been implicated in the control of extracellular environment in the neuropil (Hoyle, 1953; Twarog and Roeder, 1956; Treherne, 1961). In addition, there are reports of electrical activity in vertebrate glia (Tasaki and Chang, 1958), and current speculations suggest that they may be involved in integration in the nervous system (Galambos, 1961). This topic is beyond the scope of the present paper, but obviously glia cannot be completely dismissed from a final functional analysis of the nervous system. Capillaries. Capillary beds are usually dense in neuropil areas. In fish, for example, the glomeruli of the olfactory lobe and nucleus rotundus are heavily vascularized. This often correlates with concentrations of mitochondria or oxidative enzymes (Scharrer, 1945; Friede, I960) suggesting that neuropil is a region of unusually high physiological activity. Even in the lobster and crayfish, which are generally considered to have an open circulatory system, the central nervous system, particularly areas of structured neuropil, is laced with a capillary network. In both vertebrates and lobsters, the higher correlative functions of the nervous system are very sensitive to oxygen deprivation. FUNCTIONAL PROPERTIES OF NEUROPIL Forms of Electrical Activity Electrical changes provide one of the most sensitive measures o£ neural activity. For the past two years, in collaboration with J. Clarridge, V. Shoemaker, R. Stephens, and M. J. Cohen, we have been using intracellular microelectrodes to probe into parts of neurons in the perfused spiny lobster brain. One of our purposes was to answer the simple question: What kind of electrical activity normally occurs in complex central ganglia? Are there propagated impulses? Are there slow potentials? Do all parts of the neuron act alike? General remarks First, we find that in fibers in tracts only full-sized action potentials occur. These are indistinguishable from those found in any peripheral nerve. Second, electrodes placed in peripheral cell bodies record only attenuated, slow depolarizations in our preparations. The soma membrane seems to be inactive, and the recorded potentials presumably represent passive, electrotonic spread from distant sites of activity in the neuropil (Hagiwara and Bullock, 1957; Preston and Kennedy, 1960). Such a finding perhaps is not surprising in the light of observations made by Bethe (1897) and Hardy (1929) that reflex activity can continue for short periods after surgical removal of cell bodies. 87 ORCAN'IZATIO.N OF NF.UROPIL FIG. 10. Synaptic noise recorded with intracellular electrode from a nerve process in brain of spiny lobster. Read from right to left. One spike potential rises from unusually large synaptic potential. Both epsp and ipsp probably contribute to subthreshold activity. Calibrations, 0.1 second; 4 millivolts. Thirdly, ramifying processes in the unstructured neuropil are the site of much complex electrical activity. This is gratifying, for neither the all-or-none spikes of the tracts nor the small, relatively simple slow potentials of the neuron soma are the kinds of activity expected in a center of complex integration. Some of the neuropil elements are silent, but many are either spontaneously active or subject to continuous subthreshold, presynaptic bombardment (Fig. 10). There are at least four types of activity: 1. Excitatory post-synaptic potentials (epsp) are the most prominent. These are depolarizing potentials lasting several milliseconds. They are non-propagated, and consequently of greatest amplitude at the site of the synaptic junction. Since those recorded in a single cell often vary considerably in amplitude, they must represent different junctions either at unequal distances from the recording site, or possibly, of unequal effectiveness. Epsp initiated by different presynaptic elements may sum algebraically, and upon reaching critical depolarization, initiate propagated potentials that travel down the axon (Fig. 11). 2. Action potentials are recorded from most neuropil processes as small, all-ornone spikes lasting 1-2 milliseconds. These may propagate distally, for they correlate perfectly with impulses recorded in peripheral nerves. They usually rise from generator or synaptic potentials. In the latter case, they tend to be smallest in those units with the largest epsp. This suggests that they may originate some distance from the site of the synaptic junction and do not invade the junctional regions. Elements may have more than one size spike potential, implying that one neuron may have several different impulse sites, each capable of maintaining an independent action potential (Bullock and Terzuolo, 1957; Preston and Kennedy, 1960; Spencer and Kandel, 1961). 3. Generator potentials are slowly growing depolarizations that usually give rise to propagated action potentials. (They are not to be confused with the generator potentials of receptors.) They presumably originate spontaneously in the normal unit, but their genesis is obscure and they also occur in injured axons. It is not clear how much of the spike activity of neuropil elements results from generator potentials and how much from synaptic noise. 4. Inhibitory post-synaptic potentials (ip- * • I « • * * t • FIC. 11. Epsp. Intracellular recording from unit process in diffuse neuropil of spiny lobster brain (tracing). Input via homolateral antennular nerve with increasing stimulus strengths from bottom trace up. Strongest stimulus evokes an action potential whose peak is not shown. Time mark, 2 millisecond intervals; voltage calibration, 10 millivolts. 88 DONALD M. MAYNARD n FIG. 12. Ipsp. Iniracellular recording as in Fig. 11. Read from right to left. Input at 50 stimuli per second via medial bundle of heterolateral antennular nerve. Calibrations, 0.1 second; 10 milivolts. Upper trace of this and following figures taken from monitoring electrodes on antennular nerve; lower trace from microelectrode within ganglion. sp) are similar to the epsp but often hyperpolarize rather than depolarize (see Furshpan and Potter, 1959b). On occasion these reduce the excitability of the unit and either block spike initiation or reduce spontaneity (Fig. 12). Like the epsp, the ipsp originate from activity in presynaptic elements, and like them, help comprise the continuous subthreshold synaptic noise found in most units of the neuropil. T h e activity described above is well known from other preparations, but it is important that it be demonstrated in neuropil itself. Other kinds of electrical activity are probably to be found in the ganglia (see below), but the above are the most obvious, and from them we may begin to build a tentative picture of functional neuropil as a coding center of the nervous system. 1. Integrative activity is largely confined to the neuropil. Tracts only conduct impulses, and cell bodies seem to be electrically inactive. 2. Although both input and output from the ganglion, and communication between neuropils within, involves propagated action potentials and a pulsed code, the electrical events concerned with actual integration may be more similar to a non-pulsed, analog system. 3. Portions of the neuron concerned with reception and integration of information from other neural elements are distinct from, and have much more variable activity than portions concerned solely with impulse propagation. T h e entire neuron, therefore, does not a d as an all-or-none unit, and the eventual propagated impulse leaving the neuropil depends upon the preceding summed, integrated, non-propagated activity of the dendritic arborizations. 4. At least some neuropil integration involves typical synaptic transmission, both excitatory and inhibitory. 5. Many, if not most, of the neuropil association or motor elements receive continuous, subthreshold presynaptic bombardment. This is not immediately apparent in the output of the system, but is probably the basis of a variable central excitatory state. It seems unlikely that normal neuropil can ever be considered truly at rest or in a static state. Specific remarks on single units One of the remarkable things about many neuropil elements is the extent of their arborizations. We have suggested that functional differences exist between arborizations and axons, but one may also ask: Are all excitatory synapses alike? How does one branch of a neuron affect another if it is several millimeters distant? Although we cannot fully answer these questions, certain observations on units in the lobster brain are suggestive. First: Many of the synaptic potentials caused by primary afferent fibers from the antennular nerve in the spiny lobster do not show temporal facilitation. In fact, they begin to diminish in amplitude at low frequencies of repetitive stimulation and soon fall below the spike-initiating threshold. In contrast, other epsp with longer latencies do not fatigue and usually require temporal summation to reach maximum effectiveness in spike initiation. There are, therefore, at least two kinds of epsp in central as well as peripheral crustacean ganglia (see Terzuolo and Bullock, 1958; Furshpan and Potter, 1959b). These give the integrating neuron the potential of responding in very different manners to similar temporal patterns from different inputs (Fig. 13). Second: Several observations suggest that a neuron can maintain independent spike potentials in more than one of its processes. Although evidence is not conclusive, it is possible that these potentials represent propagated impulses rather than synaptic 89 ORGANIZATION OF NEUROPIL RMB I I I 1 I M 1 11 T T I r LMB 0.2 sec FIG. 13. Patterned responses from different inputs. Intracellular recording from one unit as in Fig. 11. Read from right to left. Input at 10 per second via medial bundle of homolateral antennular nerve (upper pair of traces) and via medial bundle of het- erolateral antennular nerve (lower pair of traces). Note different patterns of response to first stimulus of train and differences in adaptation or fatigue. Synaptic potentials were not present in this record. potentials (Furshpan and Potter, 1959a). Jf so, they provide a mechanism of intracellular interaction in extensively branched neurons (Spencer and Kandel, 1961; Preston and Kennedy, 1960). The propagated impulse would occur in stretches of the dendrite connecting separate arborizations, and not appear as the final output of the neuron unless capable of exciting the efferent axon itself. Our records indicate that this does not always occur (Fig. 14), and, if our hypothesis is correct, that synaptic potentials initiating a propagated impulse in one portion of a single neuron's arborizing processes may ultimately fail because of failure of the impulse at some dendrite-axon junction. This implies that the single neuron may be analogous to a two-neuron chain, and the point of potential spike failure or lowest safety factor is analogous to a synaptic junction. Possibly the extensively branched neurons often found in the arthropod neuropil (see Fig. 5) should not be regarded as functional units. They may embody in one morphological structure two or more functional elements and may be, in fact, similar to a synaptic chain of several less ramifying neurons. One may recall that many invertebrates have remarkably few neurons in their nervous system and yet are capable of much complex, adaptive behavior. FIG. 14. Various spike potentials in one unit. Recording as in Fig. 11. Unit responds in different manner to each of four different inputs: LLB, lateral bundle of left antennular nerve: LMB, medial bundle of left antennular nerve; RLB, lateral bundle of right anlciinular nerve; RMB, medial bundle of right antennular nerve. Note synaplic potential following RMB stimulation and smaller spikes following RLB. Under other conditions, these smaller spikes were capable of initialing the larger spike. Small spikes never appeared upon stimulation of bundles of the left antennular nerve. AsyitapUc in term: I ion The observations reported above indicate that at least some of the integrative activity of the neuropil involves synaptic transmis- 90 DONALD M. MAYNARD sion. There is evidence from other preparations, however, that synapses may not be the only means of significant interaction between neurons; non-synaptic, electronic influences may exist (Watanabe, 1958; Bennett, I960). Unlike synaptic potentials, the electrotonic influence does not occur as discrete events, but appears in the second unit as an attenuated and distorted reflection of prolonged potential changes originating in the first unit. This kind of interaction should tend to insure that a group of neighboring neurons remain at the same excitability state. In two preparations where they have been analyzed, electrotonic interactions are associated with synchronized discharge of elements of the system. Synchronized discharges of neighboring neurons as found in the cockroach corpora pedunculata (Maynard, 1956) may also result from such mechanisms of interaction. Summary T o return to our first question, we may say that the electrical activity in neuropil is complex, and that a large portion of the complexity appears as a property of individual units rather than as the result of complex connectivity patterns. The relatively simple model of synaptic action as we know it from the neuro-muscular junction, the vertebrate motor horn cell, or the squid giant axon synapse therefore seems less applicable to an analysis of central integration than the more complex patterns found in such peripheral autonomic ganglia as the crustacean cardiac ganglion. Tt may be misleading, when analyzing central integration, to concentrate too exclusively on wiring diagrams, functional or anatomical, and to ignore the properties of the switches and oscillators of the system. Functional Connectivity Functional connectivity patterns are important, however, and much of our structural analysis of neuropil is based on the anatomical substrate of such patterns. I should like to turn about and review several observations which seem to demonstrate this importance in the determination of physiological characteristic* of total neu- ropil. They also suggest correlations between the morphology and the physiology of specific neuropils. Connectivity in diffuse neuropil From anatomy one might presume that in diffuse neuropil each element connects with almost every other, in an unorganized fashion. Preliminary electrical recordings from single elements in the lobster brain do not entirely support such a conclusion in so far as functional connections are concerned. It is true that many neurons form junctions with a variety of presynaptic elements, but these are not disorganized. Central connections of the lobster antennular nerve can serve as an illustration. Massive stimulation produces the responses shown in Figure 15. Variation of stimulus parameters leads to the conclusion that the beginning of the initial response is produced by direct action of afferent fibers. The prolongation of the initial response seems to be the result of activity in interposed excitatory internuncials, and the following drop, the result of inhibitory internuncials. The terminal rise in excitation shows different properties and must result either from a second population of internuncials or much more slowly conducting afferent fibers. Such a patterned sequence seems less likely to result from random central connections than from some specific pattern like that illustrated. Further evidence for regular connectivity patterns in diffuse neuropil in lobsters comes from stimulating identical nerves on each side of the body while recording from one central element. Responses to the two inputs may be qualitatively different. On the other hand, stimulation of normal and heteromorphic appendages may cause similar responses, again suggesting specific connectivity within the diffuse neuropil. Although it has been implied (Horridge, 1961) that functional connectivity patterns in unstructured neuropil can be explained solely by physiological mechanisms such as neurotransmittor and neuroreceptor specificity, it seems more likely that functional patterns reflect appropriate anatomical wiring diagrams. ORGANIZATION OF NEUROPIL A. B. PHh l-'IG. 15. Connectivity in dill use neuropil. A. Intracellular recording of response to antennular stimulation from unit in spiny lobster brain (tracing). Calibrations, 10 milliseconds, 10 millivolts. B. Diagram of possible connections from antennular nerve with schematic post-synaptic potential changes which, when summed, could produce the above response. Antennular nerve fibers enter from left; dot and vertical line represent excitatory and inhibitory synapses respectively. See text for further details. Connectivity in plexiform neuropil In the above discussion we were concerned primarily with formal connectivity between units, and not with geometrical configurations. In many neuropils, however, geometrical considerations seem fully as necessary as formal connectivity diagrams for an understanding of function. This may be illustrated by two examples from a system in which spatial or geometrical patterning is unusually important, the visual system. The first example is the subretinal plexus of Liniulus as described by Hartline and his colleagues (see Ratliff, 1961). This is a plexiform neuropil interposed between the receptors of the compound eye and the 91 fibers of the optic nerve. Each ommatidium acts as a unit and serves as a node in the network of nerve branches forming the plexus. The discharge from each ommatidium depends upon both the incident light, which stimulates, and the activity in the plexus, which depresses. The plexus, therefore, is the site of the first stage of integration of the visual pattern received by the receptors. Our concern is to show how geometrical parameters play a large role in the function of this rather simple system. Non-geometrical properties of the junctions between ommatidia in the Limulus eye are as follows: 1) All interaction between elements is inhibitory. 2) The degree of inhibition is proportional to the frequency of discharge of the fiber from the presynaptic ommatidium (when above a threshold frequency). 3) The effect of one presynaptic unit may sum with the effect of others. 4) Interaction is reciprocal, but need not be symmetrical with respect to effectiveness. 5) With the possible exception of ommatidia around the borders of the eye, all seem to have similar junctions with the neuropil. 6) Under normal circumstances, some ommatidia pairs do not interact, suggesting that not all ommatidia are directly connected with each other. These properties permit fairly accurate prediction of the result of simultaneous visual stimulation of any two receptor elements, but they do not allow one to say how the message in the optic nerve, resulting from patterned illumination of the entire eye, differs from the one that would obtain if interaction in the plexus were absent. Some additional statement about geometrical connectivity is necessary. An anatomical wiring diagram of the subretinal plexus is not available, but its apparent homogeneity suggests either of the following possibilities: 1) Connectivity in the eye is truly random, and the probability of interaction between elements is completely unrelated to the position of or distance between elements. 2) Connectivity in the eye is biased so that the probability of interaction between elements is a function of the direction and distance between elements. If the first were true, one might ex- 92 DONALD M. MAYNARD 0.5 mm. at the eye FIG. 16. Connectivity in f.itnuhis eye. Discharge of impulses from a single receptor unit in response to a "step" pattern of illumination in various positions on the retinal mosaic (see inset). The upper (rectilinear) graph shows the frequency of discharge of the test receptor, when the illumination was occluded from the rest of the eye by a mask with a small aperture, minus the frequency of discharge elicited by a small control spot of light of constant intensity also confined to the facet of the test receptor. Scale of ordinate on the right. The lower (curvilinear) graph is the frequency of discharge from the same test receptor when the mask was removed and the entire pattern of illumination was projected on the eye in various positions, minus the frequency of discharge elicited by a small control spot of constant intensity confined to the facet of the receptor. Scale of ordinate on the left. (From Ratliff and Harlline, 1959, J. Gen. Physiol.) pect visual patterns to be transmitted with relatively little distortion, but with an increase in the uncertainty or noisiness of the message. If the second were true, and if interaction increased as distance decreased, then one might expect intensity discontinuities in the spatial or temporal visual pattern to be emphasized in the afferent message. The latter, in fact, appears to be the case in Limulus (Fig. 16). It seems of particular interest that insertion of a single, rather simple geometrical bias in an apparently random system transforms it from one which increases noise in the afferent message to one which, among other things, reduces redundancy in the message without great loss of information about the spatial pattern (Barlow, 1961). Connectivity in stratified neurofril The second example is the retina-collicular system in the frog (Maturana, et al., I960; Lettvin, et al., 1961). The pertinent anatomy is summarized first. Ganglion cells in the retina collect from rods and cones via interposed bipolars. According to our terminology the bipolars are radial or vertical elements in the structured lattice and have relatively small terminal fields, while the ramifying dendrites of the ganglion cells in the inner plexiform layer are horizontal elements. There are five kinds of ganglion cells. They differ in size, lateral extent of the dendritic tree, and the stratum or strata of the plexiform layer in which the dendrites ramify. Ganglion cell axons pass from the retina directly to the colliculus or optic tectum where they presumably separate and terminate in four horizontal strata according to ganglion cell type. There is a point-to-point representation of the retina in each layer of the colliculus, and these points are in vertical alignment. Input to the colliculus, therefore, may be likened to a stack of superimposed geographical maps, each showing a different feature of the same countryside. Output originates in dendrites of collicular cells that pass vertically through the horizontal strata. Recordings from the terminal arborizations of ganglion cells in the optic tectum show that the four horizontal layers correspond to five functional classes, one each in layers 1, 2, and 4, and two (Classes 3 and 5) in layer 3. Each class responds optimally to a different aspect of a single complex stimulus. For example, convex edge detectors (Class 2) do not respond to changes in general light intensity, but do discharge when a small object (1-3°) with a sharp edge is moved across a lighter background. All parameters are important, and if, say, the background moved with the object or were darker than the object, the response would be greatly diminished or absent. These units have also been called "fly detectors." The other classes are: Class 1, sustained edge detectors; Class 3, changing edge detectors; Class 4, dimming detectors; and Class 5, dark detectors. The functional properties of the five classes correlate rather well with the five anatomical ganglion cell types. If the suggested correlation proves correct, then the dendrites of edge detectors and of dimming detectors ramify in differ- ORGANIZATION OF NEUROPIL ent strata of the inner plexiform layer, each collecting from a different set of bipolar terminals. Only two functional classes of tectal cells have been described, "newness" detectors and "sameness" detectors. It is not necessary to go into their detailed properties at this time, but only to indicate that their receptive fields are larger than that of any ganglion cell, and that their optimal stimulus, if not more complex, at least involves new combinations of qualities not found in the single ganglion cells. Let us review: Complex integration occurs in the layered neuropil of the retina so that by the second internuncial neuron, units recognize and respond optimally to specific temporal-spatial configurations of the complex visual pattern within their receptive field. These configurations, or effective attributes of the stimulus, are limited in number and apparently correspond to the kinds of neurons involved at the pertinent level of integration. The effective attributes become potentially more complex at each stage of transmission; from receptor to bipolar, from bipolar to ganglion cell, from ganglion cell to collicular cell, and so on. The layered neuropil in this system, therefore, permits successive separation and recombination of stimulus attributes while retaining the original geometrical relations of the primary sensory layer. Physical as well as functional separation seems necessary, for each layer maps a separate attribute. Vertical elements select from these layers and apparently recombine in new configurations at later stages in the system. An analogy between the retina or colliculus and a punch card system seems rather apt. Both employ a geometric code and both are concerned with cross-reference and recombination of a limited number of attributes. .Since the attributes themselves can involve geometrical configuration of the stimulus, this may be a system which permits recognition of universals such as "triangle" or "circle." Further information about the mechanisms of attribute recognition or sorting is necessary, but if the above interpretations are correct, proper geometry as well as connectivity and electrical phenomena are 93 required for neural integration in visual systems. Complex Patterning in Neuropil The last section pointed out that many units in afferent neuropil may act as event detectors. Such neurons tell whether or not a pre-determined complex sensory input is present, but do not describe that input in the patterns of their own activity. One may ask whether the reverse occurs: Are there significant, complex, and relatively autonomous patterns of activity that may be elicited by simple stimuli in the appropriate neurons? To use Wiersma's phrase (1952), is there a "push-button" to initiate appropriate events as well as a signal light to indicate when events have occurred? Several analyses of animal behavior suggest that many relatively complex acts are elicited by specific stimuli and may be treated as units with all-or-nothing properties. This implies that complex, preset activity patterns exist in certain areas of the neuropil and that once initiated, these patterns are like the regenerative action potential in a single nerve fiber and are independent of the initiating stimulus. Direct recordings of responses meeting these requirements have been described in several preparations (Wiersma, 1952; Hagiwara and Watanabe, 1956; Horridge, 1961). The response of an isolated lobster stomatogastric ganglion to repeated trains of presynaptic stimuli emphasizes another aspect of such "push-button reflexes" (Fig. 17). The first train elicits no post-synaptic response; with repetition, facilitation occurs and increasing numbers of post-synaptic units become active. Eventually the pattern of Uie response alters altogether, shifting from an irregular continuous barrage to periodic bursts that continue for considerable time after the termination of the stimulus. One is reminded in some ways of the initiation of seizure activity in the vertebrate brain. Although the mechanism is not yet clear, it does appear that the ganglion shifts in a stepwise manner from one functional state into another, and that in one of these states it produces complex, maintained responses to rather brief, un- 94 DONALD M. MAYNARD 1'IG. 17. Complex response of isolaletl stomatogastric ganglion of lobster, Homarus americanus, to one second trains of presynaptic stimuli at 10 per second, repealed at five second intervals. Extracellular recording electrodes placed distal to the ganglion. Numbers indicate the number of the stimulus train; dots on records indicate individual stimuli. There is no post-synaptic response in the first record, the spikes shown come from fibers that pass without in- Structured stimuli. The potentialities for such activity must lie in the organization of the ganglion neuropil itself, for extra-ganglionic neural feed-back is absent in this preparation. If the above observations can be generalized to apply to central ganglia, then we may answer our first question as follows: Yes, there are "push-button reflexes," but activation requires two operations; first the switch must be turned on and then the button pushed. It is not sufficient for a given neural system to have the structural organization requisite for complex, semi-autonomous activity; the system must also be in the proper physiological state in order to respond to the specific, triggering stimulus. If much of an organism's behavior is organized around push-button responses, then such a priming or activating mechanism gives an added degree of freedom, and should facilitate adaptive behavior. Several functional states with corresponding differ- terruption through the ganglion, and consequently represent direct responses to the electrical stimuli. Two post-synaptic impulses occur with the 6th train of stimuli, three with the 8th, 15 with the 10th, and bursts with the 12th. Bursts continued for several seconds beyond the records shown, and the changed state of the ganglion continued still longer, as shown by test trains of stimuli. Calibration, 0.5 second. ent responses would extend still further the functional potentialities of a limited number of neurons with rigid, preset activity patterns. The anatomical and physiological properties of the hypothetical priming mechanism remain to be specified in invertebrates. Braiyi Waves A discussion of neuropil organization should not be concluded without reference to the rhythmic, often sinusoidal potential oscillations called "brain waves." These are usually recorded by surface electrodes from specific masses of nerve tissue. Although there is no complete agreement on their genesis or significance, most would concede that the waves are not envelopes of action potentials, that they reflect potential oscillations in neurons, that they appear in typical form only in masses of nerve tissue, and that they represent some synchronized activity in large numbers of units. The alpha 95 ORGANIZATION OF NEUROPIL rhythm of the vertebrate cortex is perhaps the best known example, but analogous sinusoidal oscillations have been recorded from olfactory bulb, cerebellum, optic ganglia of insects, and recently, the ocelli of some insects (see Gerard and Young, 1937; Crescitelli and Jahn, 1942; Bullock, 1945b; Bum and Catton, 1956; Ruck, 1961). The structures giving rise to the above oscillations differ rather markedly in general appearance, but they do have one factor in common. In all cases the tissue appears to be structured neuropil. Smooth oscillations apparently have not been recorded from unstructured neuropil. If the synchronization characteristic of brain waves is mediated by some mechanism not involving action potentials and synapses, possibly electrotonic potentials or chemical agents, then such geometric organization may have greater significance than originally postulated. SUMMARY Much of the factual information and many of the ideas presented in the above discussion are neither new nor original. Nevertheless, they are worth considering for they stress several points of some importance: 1. Although acceptable for general conversation, neuropil is unsatisfactory when unmodified as a technical term in that it does not refer to a commonly accepted, well-defined structure. Its original anatomical connotations have broadened to include functional aspects, and thus it logically encompasses the entire portion of the nervous system devoted to neural integration, cell bodies, processes, and all. 2. Neuropil structure is complex, but not haphazard. 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