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AMER. ZOOL., 35:566-577 (1995) Specific Associations of Neurosecretory or Neuromodulatory Axons with Insect Skeletal Muscles1 MARY B. RHEUBEN Department of Anatomy, Michigan State University, East Lansing, Michigan 48824-1316 SYNOPSIS. The general process of neuromodulation in the skeletal muscles of various insects is accomplished via several different structural forms. In addition to motor axons, which may contain a modulatory substance as a co-transmitter, a second class of axons is found in close association with insect skeletal muscles. These axons typically contain dense cored vesicles. Some come directly into contact with the sarcolemma, but do not form a typical neuromuscular junction. Others have finely distributed branches within the muscle but only directly contact glial cells of the motor nerve branches. Immunocytochemistry has shown that these nerve types contain one or more of several potential transmitters: glutamate, octopamine, serotonin, leucokinin, proctolin, or insulin. While some of these substances are known to modulate muscle fiber contractile abilities directly, or to affect the mechanisms of neuromuscular transmission, others can be hypothesized to be involved in development, respiration, and other undiscovered functions. Nerve terminals containing large numbers of dense cored vesicles, or a mixture of dense and clear cored vesicles, are also seen in a variety of locations, including adjacent to muscles. The dense cored vesicles vary in appearance in different terminal types, implying that their enclosed substances might differ. For example, five distinct types of dense cored vesicles were described in the peripheral neurosecretory system in stick insect (Fifield and Finlayson, 1978), and Jia et al. (1993) report three morphologically distinct types of terminals that contain dense cored vesicles in Drosophila skeletal muscles. Sometimes the terminals containing dense cored vesicles differ from typical central, motor or sensory synapses in that their postsynaptic target is either not evident at all morphologically, being a distant target reached by diffusion (neurohormonal association), or their structural associations with the target cell are less well defined. A contact region may partly resemble a synapse, but often lacks obvious structural postsynaptic specializations. Evidence for a specific region 1 From the Symposium Invertebrate Neuromuscular of the terminal dedicated to release of neuOrganization: Peripheral Contributions to Behavioral rosecretory material and reuptake of memVariability presented at the Annual Meeting of the brane can consist of a distinct cluster of clear American Society of Zoologists, 27-30 December 1993, cored vesicles resembling those found at at Los Angeles, California. INTRODUCTION Skeletal muscles in most insect species receive excitatory motor innervation from one or more nerve terminals containing large numbers of electron lucent vesicles, averaging 20-60 nm in diameter, with an occasional larger dense cored vesicle among them (Osborne, 1975). These nerve terminals typically produce excitatory junction potentials (EJPs) of two general types: either "slow," in which the EJP is small, of longer timecourse, and facilitates readily, or "fast" in which the EJP is larger, usually above threshold for an active membrane response, and may depress on repeated stimulation. Generally speaking, the structural differences between "slow" and "fast" nerve terminals are quantitative rather than qualitative (Rheuben, 1985;Titmus, 1981). Some muscles are also innervated by inhibitory motor neurones, and the vesicles of these are also electron lucent or "clear cored" (Titmus, 1981; Aizu, 1982). 566 NEUROMODULATORY AXONS IN INSECT MUSCLE normal synapses ("synaptoid vesicles"), an electron dense tuft or bar, resembling the presynaptic active zone, and large dense cored vesicles nearby. These regions of the terminal or axon are often termed "synaptoid contacts" (Raabe, 1989). They may occur in apposition to nothing more substantial than a layer of basal lamina. The nature of the functional relationships between the several types of nerve terminals and their targets is something of a continuum. The distinction between neurotransmission, neuromodulation, and hormonal effects is one of degree, and the same substance may function in all of the three ways. In this paper the term neurotransmission is used to mean the action of a substance which is limited to a synaptic cleft; in the case of insect muscle this refers either to excitatory or inhibitory neuromuscular junctions which produce an immediate electrical effect on the muscle fiber. "Neuromodulation" refers to the process by which a second nerve can chemically modify synaptic transmission by affecting either pre- or postsynaptic mechanisms. The term neurohormone refers to substances which act after diffusing some distance or after being transported to broadly distributed sites of effect via the bloodstream. A neuromodulatory substance could conceivably arrive at a muscle or neuromuscular junction either as a neurohormone or via a more closely applied nerve process. See Raabe (1989) for a complete description of the ways that these general terms are applied to insects. The most obvious function in skeletal muscle to be modulated is contraction—its amplitude and time course—and the mechanisms can range from direct and indirect effects on muscle fiber membrane properties to influences on cAMP levels. One of the best known neuromodulatory substances to be used in this fashion in insects is octopamine, which enhances the capabilities of flight muscle via both pre- and postsynaptic effects (reviewed by Orchard et al, 1993). For example, octopamine increases miniature excitatory junction potential (MEJP) frequency as well as the amplitude, the contraction rate, and the rate of relaxation of the nerve-evoked twitch in the dorsal longitudinal flight muscles of cricket and locust 567 (Whim and Evans, 1988; O'Gara and Drewes, 1990). In Manduca, octopamine can decrease input resistance and hyperpolarize the muscle fiber membrane, increase the amplitude and timecourse of the excitatory junction potential, and increase the amplitude and frequency of the MEJP. The precise effects are dependent upon the stage of development of the animal (Klaassen and Kammer, 1985; Klaassen et al, 1986; Fitch and Kammer, 1986). However other functions besides contraction, such as those involving the metabolism and respiration of the muscle fiber, its development, or the development of its innervation, should also be considered. The diversity of structural types of neurosecretory axons in close association with skeletal muscles support the likelihood of diverse transmitters and diverse functions, and, as will be described below, the structural arrangements appear to be characteristic of the particular insect group. In 1971 Osborne (Osborne et al, 1971) described neurosecretory type endings in association with striated muscles from three insect species and from frog. At the end of the discussion, those authors commented "Thus somatic muscles in general are innervated by a variety of axons, some of which appear to be neurosecretory. The physiological significance of these endings clearly remains to be discovered." In 1994, while the number of structural examples have increased, and while the potential identities of their transmitters have been deduced in some cases using immunocytochemistry, little is known about their normal uses. The objective of this article is to summarize what is known about the subset of neurosecretory or neuromodulatory axons that specifically associate with skeletal muscles, to add some recent work from our laboratory, and to suggest some future directions for study. PERIPHERAL NEUROSECRETORY STRUCTURES IN INSECT SKELETAL MUSCLE The neurosecretory or neuromodulatory endings found in close association with skeletal muscle fibers may be categorized into three broad structural types for the purposes of discussion: 568 MARY B. RHEUBEN 1. The neuromodulatory function is combined directly with that of synaptic transmission, so that the neuromodulatory substance is actually a co-transmitter in a "normal" motor neuron. 2. The neuromodulatory ending is a separate structure from the motor neuron. Its terminal contains numerous dense cored vesicles, and is in direct contact with the muscle fiber membrane. In some there may be, in addition to the dense cored vesicles, a "synaptoid" or synapse-like structure, with its cluster of clear vesicles surrounding an electron dense bar. A distinct postsynaptic specialization may be lacking. 3. The neuromodulatory ending accompanies the fine motor nerve branches within the muscle, but does not directly contact the muscle fiber. Apparent synaptoid release sites may occur in juxtaposition with the glial cells ensheathing the neurosecretory axon or only with basal lamina. There is no significant structural barrier between a released substance and the hemolymph. Each of these structural designs may be used in different parts of an insect's nervous system, depending on the species. /. The neuromodulatory substance is a co-transmitter with the excitatory transmitter in the motor neurones The best investigated example of an insect skeletal muscle receiving innervation from a motor neuron having two transmitters with differing functions is found in the cockroach. The peptide proctolin has been identified in a slow motor neuron (Ds) that innervates the coxal depressor muscle (O'Shea and Bishop, 1982; Adams and O'Shea, 1983). Both the cell body and its fine branches within the depressor muscle have been shown to contain a proctolin-like substance using both single cell neurochemical methods and immunocytochemistry (O'Shea et al, 1982). Application of proctolin to the muscle directly produces a slow rise in tension, without membrane depolarization. Tonic high frequency stimulation of the nerve also causes a slow increase in tension superimposed upon the nerve evoked twitches. The single evoked excitatory junction potential was similar to that seen from non-proctolin containing motor neurones, so it has been suggested that proctolin is present in conjunction with another excitatory transmitter, presumably glutamate (Adams and O'Shea, 1983). Only particular divisions of muscle 177 in the metathoracic segment received innervation from Ds. The tonic bundle of the locust extensor tibia muscle exhibits great sensitivity to proctolin, which enhances the force of the spontaneous myogenic contractions at 5 x 10-" M (May et al, 1979) and proctolin has been identified in its slow motor neuron (Worden et al, 1985). Unspecified body wall muscles in the cockroach abdomen (O'Shea et al, 1982) also receive branches from a small number of proctolin containing neurones (lateral white cells) that can be identified within the abdominal ganglia. Proctolin-containing beaded endings are seen at the light microscope level on the mandibular closer muscle in locusts, and proctolin enhances the neurally evoked contractions as well as elevating inositol trisphosphate (IP3) (Baines et al, 1990a). From a survey of skeletal muscles for proctolin-like immunoreactivity it has been suggested that proctolin may be contained in some but not all of the slow motor neurones associated with tonic (slow) muscles (Witten and O'Shea, 1985). In the skeletal muscles of insects and other arthropods that have been studied physiologically, proctolin enhances contraction or generates a slow rise in tension similar to a "catch" state (O'Shea and Shaffer, 1985), so it may often have a role in maintaining resting tension. Octopamine and serotonin have also been associated with sustained tension or postural muscle control in insects and Crustacea (Hoyle, 1984; review by Kravitz, 1988). Besides forming endings in close association with skeletal muscles, neurones containing proctolin or a proctolin-like substance also extensively innervate the heart, digestive tract, oviduct musculature, and neurohaemal regions (Nassel and O'Shea, 1987, blowfly; Witten and O'Shea, 1985, cockroach; Veenstra et al, 1985, Colorado potato beetle; Davis et al, 1989, Lepidoptera) so that proctolin may commonly operate widely as a neurohormone as well as a neuromodulator, perhaps with general NEUROMODULATORY AXONS rN INSECT MUSCLE functions in feeding and digestion. In the visceral organs the structural specializations associated with the proctolin-containing terminals include both direct synaptoid contacts with muscle fibers and neurohemal type release sites (Klemm et al, 1986). 569 fibers directly. At least four types of axon have been observed so far to innervate particular muscles of this group. These axons differ characteristically in size of bouton and type of vesicle. One category, "Type I," corresponds best to a motor nerve function. It has been divided into two subtypes, Is and Ib (Atwood et al., 1993) or CV and CVo (Jia et ai, 1993) for muscle fibers 6 and 7, and is characterized by relatively large boutons, clear cored vesicles, and the presence of a subsynaptic reticulum formed by the muscle fiber at the synaptic sites. Occasional dense cored vesicles have been seen in Type Is terminals. The Ib and Is terminals appear to give rise to slow and fast synaptic potentials respectively, (Atwood et al., 1993; Kurdyak et al., 1993) and are described more fully elsewhere in this volume (Atwood and Cooper, 1995). The second axon type, "Type II" (Johansen et al., 19896; Budnik and Gorczyka, 1992; Monastirioti et al., 1995) is characterized at the light microscope level by substantially smaller (less than 2 pm) rounded boutons and a wider, more irregular distribution of its terminals over the muscle fibers. Type II terminals are immunoreactive for the Small Synaptic Bouton antigen "SSB," whereas the Type I motor nerve terminals are not (Budnik and Gorczyka, 1992). An ultrastructural comparison of muscle fibers 6 and 7 (which do not usually have type II endings) and muscle fibers 12 and 13 (which do) suggests that at least some of the type II endings have elliptical dense cored vesicles as well as clear cored vesicles (Jia et al., 1993). These are referred to as "mixed vesicle", MV, type axons. In subsequent studies, Monastirioti et al. (1995) have found that two octopamine immunoreactive neurones supply nearly all of the body wall musculature via these characteristic type II endings. 2. A separate neurosecretory type axon forms a direct contact with skeletal muscle fibers In Dipterans many muscles receive direct innervation from axons containing large numbers of dense cored vesicles as well as from more conventionally structured motor axons. Because their peripheral axons and nerve terminals are less well covered by glial cells than in other types of insects, they are amenable subjects for immunocytochemical studies. The larval abdominal musculature has several neurosecretory type axons which have been shown by electron microscopy to make direct contact with the sarcolemma: For example, muscle fiber " 8 " (the abdominal muscles in Dipterans are described anatomically by number) in larval Drosophila, Calliphora, and Phormia is innervated by three axons, two of which have terminals containing dense cored vesicles and which occupy shallow depressions in the muscle fiber membrane. One of the terminals having dense cored vesicles is immunoreactive to antiserum to leucokinin I (LKIR). A single LKIR neuron innervates each of 7 abdominal hemisegments, makes intimate contact only with muscle fiber 8 in each of these segments, has an extension to a trachea, and has superficially placed varicosities along part of its axon which could release material into the hemolymph. In the adult the LKIR reactive axons innervate abdominal spiracles. Consequently LKIR axons could have not only a specific and direct effect on muscle 8, but also be involved in respiration and the function of adjacent muscles which are influenced by A third structural type of axon, "Type LKIR indirectly through the hemolymph III," was first identified from nerve termi(Cantera and Nassel, 1992). nals immunoreactive for an insulin-like Muscle fibers 6, 7, 12, and 13 in Dro- peptide. These terminals exclusively innersophila have been studied extensively. Some vate muscle fiber 12 in segments 2-5, and of these fibers have, in addition to motor have boutons intermediate in size between innervation, two or more kinds of neurose- those of typical type I and type II endings, cretory type axons which contact the muscle and which are more elongated or oval in 570 MARY B. RHEUBEN shape (Gorczyca et al, 1993). They were immunologically distinct from the type of terminal stained by the SSB (small synaptic bouton) antibody, associated with type II terminals (Budnik and Gorczyca, 1992). Jia et al, (1993), after comparing terminal types at the electron microscopic level on muscles 6, 7, 12, and 13, suggest that a set of terminals containing spherical dense cored vesicles may correspond to the type III insulin-containing terminals. These axons also contained clusters of clear cored vesicles in association with presynaptic densities, forming a synaptoid; each of the latter structures was found in apposition to the muscle fiber in depressions of the sarcolemma near to junctions made by type I terminals (Atwood et al, 1993; Jia et al., 1993). Even though the insulin-containing terminals are restricted to a particular muscle, virtually all body wall neuromuscular junctions exhibit localized immunoreactivity for insulin receptors, which appeared to be in the postsynaptic muscle membrane. This suggests both immediate and long distance effects for an insulin-like peptide. Immunoreactivity for proctolin (or proctolin-like substances) has also been seen in terminals supplying muscle fibers 12 and 13 and selected other fibers in the abdominal musculature (Anderson et al., 1988; Keshishian et al, 1993). It is not yet certain which type of terminal houses the proctolin, or whether it is co-localized to motor nerve type I terminals containing glutamate, or to neurosecretory type terminals also containing insulin or octopamine. Furthermore, glutamate immunoreactivity has been observed in most if not all type I and type II fibers in Drosophila (Johansen et al, 1989a, b), with anti-HRP and anti-glutamate antibodies identifying the same terminals in double label experiments done on muscle fibers 2, 4, 6, 7, 12, and 13. Therefore, in the case of the specialized, type II, dense cored vesicle-containing terminals, glutamate may be a co-transmitter to the octopamine housed in the dense cored vesicles! When all these points are considered, it seems likely that some dense cored vesiclecontaining axons of either Type II or Type III morphologies may well contain different and possibly multiple transmitter types. If this is the case then one must define motor axons and neuromodulatory axons very carefully, since their functions will be dependent on the presence and responses of receptors to their transmitters in the postsynaptic membranes and in adjacent regions. Further physiological and ultrastructural double labelling studies will be necessary to clarify the situation. In summary, in Drosophila and other Dipterans, there are at least four or five types of axon which make direct contact with the skeletal muscle fibers. Two or three of these may be excitatory or inhibitory motor axons, but the other endings could be responsible for supplying leucokinin I, octopamine, proctolin, insulin, or other modulatory substances to specific locations on specific muscle fibers. While these latter endings often closely parallel the motor endings, their separate nature would allow an independent, more versatile action on the muscle fiber when compared to the arrangement where the modulatory substance is a co-transmitter in the motor nerve terminal, and where release is still presumably linked to motor axon activity. In addition, the finding of an insulin-like peptide and insulin receptors that increase in distribution during development, and by analogy with the vertebrate system, has suggested that growth and development of muscle and neuromuscular junctions are phenomena in insects that could be regulated by the neurosecretory type axons (Gorczyca et al, 1993). 3. The neuromodulatory or neurosecretory axons are in close association with the motor nerve branches within the muscle but do not directly contact the muscle fibers Insect neurohormones are released from a variety of specialized organs such as the corpora cardiaca, which is a glandular structure near the brain, the perisympathetic organs which are segmentally arranged in association with the median and transverse nerves, and various neurohemal release sites along peripheral nerves (Raabe, 1989). The single neurosecretory type axons which have release sites along the fine motor nerve NEUROMODULATORY AXONS IN INSECT MUSCLE branches might be viewed as a special case of neurohemal organs or might represent a distinctly different mechanism depending upon the actual target. For example, serotonin immunoreactive neurites have been found to form a meshwork in the outer layers of the glial sheath surrounding motor nerves. In Periplaneta, a small number of serotonergic cells sends processes by a very circuitous route from the subesophageal ganglion to the nerves supplying the muscles of mastication. When the serotonergic axons reach the regions of the nerve adjacent to the muscles, they divide to form many fine branches along the surface of the motor nerve trunk (Davis, 1987). Fine beaded branches are also seen to run in close proximity to muscle fibers of the mandibular closer muscle (locust, Baines et al., 19906). In Calliphora, the homologous nerves contain serotonergic profiles in the outer neural sheath that are seen at the electron microscope level to be separated from the hemolymph only by a layer of basal lamina. These 5-HT immunoreactive axons contain both clear and dense cored vesicles (Nassel and Elekes, 1984). Their precise function relative to the skeletal muscles is unknown; in visceral muscles of several species exogenously applied 5-HT results in enhancement of contraction or increase in frequency of contraction (Nassel, 1988). Another neuromodulatory cell type whose terminals form a distant association with muscle fibers is the octopamine-containing DUM (dorsal unpaired median) neurones of the thoracic and abdominal ganglia in orthopterans, lepidopterans, and hemipterans. DUM cells, unlike most other central neurones, are capable of generating an action potential in their somata. Each sends out a single primary axon which then bifurcates to provide mirror image branches to the right and left halves of the thorax (reviewed by Evans, 1980). Two DUM neurones have been investigated in particular, the DUMETi (dorsal unpaired median cell to the extensor tibia muscle) and DUMDL (dorsal unpaired median cell to the dorsal longitudinal muscle). Hoyle et al. (1980) traced the axon of DUMETi to the periphery using a combi- 571 nation of electrophysiological and ultrastructural methods. It was observed to accompany the fast motor neuron, initially within the common glial sheath. In branches of the nerve within the muscle itself, the presumed DUMETi axon lay in or on the outermost glial layer, paralleled the fast motor axon when it branched, formed "swellings," but was not seen to come into direct contact with the muscle membrane. The DUMDL, although not continuously traced from ganglion to muscle, seemed to have a similar structure. These DUM cell axons branch to associate with specific muscles, but yet appear not to form a structural specialization with the fibers directly. This suggests a mechanism of modulation that might differ from those used by the previous two types of neurosecretory innervation. In the case of the extensor tibia, one known effect of octopamine and/or stimulation of the DUMETi neuron is slowing of a myogenic rhythm present in one of the muscle bundles. For the dorsal longitudinal flight muscles both octopamine and activation of the DUM neuron is associated with a complex enhancement of muscle function and stretch receptor function at the initiation of flight (Orchard et al, 1993), although distinguishing the effects of circulating octopamine from those supplied by the DUM cells directly to the muscles is difficult. In Manduca, we have investigated a similar type of neurosecretory axon in the larval and adult mesothoracic muscles. The association of neurosecretory type axons with individual muscle fibers was examined in detail using extensive serial sectioning (Rheuben and Autio, in preparation). It was found for larval dorsal longitudinal muscles "A," "B," and "C" that contribute to the adult dorsal longitudinal flight muscles, that a single neurosecretory axon followed and branched with the single motor axon along the surface of the muscle fiber. The neurosecretory twig was not ensheathed completely by glial cell processes as was the motor axon, but rather lay loosely wrapped in the outermost layer of the glial sheath. In several long sets of serial sections, the neurosecretory twig was found to enlarge to form varicosities at intervals. These varicosities contained both clear and dense cored 572 MARY B. RHEUBEN vesicles. Occasionally clusters of clear cored vesicles were found in conjuction to a structure comparable to a presynaptic density, forming a "synaptoid" contact. This contact was often in direct apposition to a glial cell membrane or occasionally to the thick layer of basal lamina that surrounded the outside of the nerve as a whole (Figs. 1, 2). Even though the motor axon branched off to form neuromuscular junctions at intervals, the neurosecretory twig did not. No direct contacts were seen between the neurosecretory axon and the muscle fiber, either within or adjacent to the neuromuscular junctions. Virtually all larval terminal motor nerve branches were found to have an associated neurosecretory twig in the outer part of the glial sheath. This type of synaptoid contact with glial processes also occurs in the dorsal longitudinal nerve in flightless grasshoppers even though the muscle is greatly reduced or absent (Arbas and Tolbert, 1986). In contrast, in the adult dorsal longitudinal muscles innervated by some of the same larval motor axons (Rheuben and Kammer, 1980) the neurosecretory type axon has been seen within the muscle in affiliation with the terminal branches of the motor axon only very rarely. Axons containing similar types of dense cored vesicles have been seen in the outer sheaths of main nerve branches close to the third axillary muscles (Rheuben and Kammer, 1983) in adults, and are frequent at the bases of the nerves supplying the dorsal longitudinal muscles, near the neurohemal organs at each segmental ganglion (Wasserman, 1985). The reasons for this developmental change in apparent distribution for one particular nerve type are not known, nor is the transmitter. The functions and fiber types of larval and adult muscles are quite different, as are some of the adult muscles from each other, so the presence or absence of a modulatory neurosecretory axon could relate either to developmental changes or to not yet understood differences in function. FUNCTIONS OF THE NEUROSECRETORY TYPE AXONS SUGGESTED BY THEIR STRUCTURE In both the structural type in which a modulatory substance is carried as a co- transmitter, and that in which a separate neurosecretory type axon makes direct contact with the muscle sarcolemma, it would seem that the most immediate target is the postsynaptic muscle fiber and the most immediate function is modulation of contraction. However, the morphology does not exclude secondary targets such as adjacent glial cells, tracheoblasts, or adjacent muscles and their innervation. The existence of the third structural type, in which the neurosecretory axon only comes as close as the tertiary branches of the motor axon, further suggests that other targets and other functions may be important. It might be useful to speculate on some of these. 1. Modulation of glial function In Manduca (Rheuben and Autio, in preparation) and in locusts (Hoyle et ah, 1980) neurosecretory type axons form varicosities and synaptoid structures with or near the glial cells which enshroud the motor axon. This could be happenstance or it could suggest that the glial cell is an immediate target. If the glial cell is the immediate target, what functions might be modulated? In locust several layers of glial cells form a "blood-brain" barrier in central and peripheral nerves, with a voltage being developed across it. Applied octopamine modulates the ability of this potential to change in response to alterations in extracellular potassium concentrations, with one possible explanation being a fall in K + permeability of the basolateral membranes (Schofield and Treherne, 1986). Octopamine also directly hyperpolarizes Schwann cell membranes in squid (Reale et al, 1986). DUM neurites form structures similar to presynaptic densities in apposition to glial cells within the ganglia of locusts (Watson, 1984). In subsequent studies, the DUM cell processes in the same regions were shown to be immunoreactive to octopamine (Stevenson et al, 1992). Taken together these findings suggest the hypothesis that octopaminergic neurosecretory type neurons, particularly the DUM neurons, may have a direct effect on glial cells that they "innervate." Similarly the observations that exogenous 5-HT affects the membrane potentials of leech glial cells (Walz and Schlue, 1982) and seroto- FIG. 1. Tertiary motor nerve branch in the vicinity of a neuromuscularjunction. An oblique section of the nerve is shown in the lower right comer of the micrograph. A varicosity of the neurosecretory axon (NS) lies in an indentation of the outermost glial process, and a small portion of the motor axon (A) is at the edge of the section. The profile of the neurosecretory axon contains a single dense cored vesicle and a cluster of clear cored vesicles. Between varicosities, the neurosecretory axons are much smaller in cross-sectional area, as little as 0.2 iim. The neuromuscularjunction and its associated glia are on the left. The neurosecretory axons were not found any closer to neuromuscular junctions or to muscle fibers than as shown here. The motor axon typically sends a terminal branch into a junction from the tertiary nerve branch, but the terminal branch is not accompanied by a process from the neurosecretory axon. G, glial process; Tr, tracheole; F, fibroblast. 51,300 x. 574 MARY B. RHEUBEN FIG. 2. Synaptoid of a neurosecretory type axon. A grazing section of a varicosity from a tertiary motor branch from a 5th instar Manduca larva is completely surrounded by a glial process in this region, with the motor axon and the rest of the glial wrappers being out of the photograph. Clear cored vesicles predominate in a synaptoid. The presynaptic electron dense specialization (arrow) is shown here in direct apposition to the glial process. Several microtubules pass through the varicosity. Between varicosities the axon contains only electron lucent cytoplasm and the continuing microtubules. Dense cored vesicles are sparsely present, with possibly one shown to the left of the synaptoid. In extensive serial sections this axon type was not found to directly contact the muscle fiber. 73,600 x. nergic neurosecretory axons are found commonly in close apposition to the glial sheath cells of the mandibular nerves evokes speculation of a possible functional relationship. The idea that glial cells might be targets of traditional neurotransmitters is not new: for example, the relationship between glial cell glycogenolysis and noradrenergic compounds has been explored extensively (Stone and Ariano, 1989). At the neuromuscular junction, Schwann cells respond to a lack of neuronal activity by upregulating glial fibrillary acidic protein. This effect appeared to be mediated via nerve terminal transmitter release (Georgiou et al., 1994). The morphology of astroglial cells is controlled by beta-adrenergic receptors (Shain et al, 1987). A trophic relationship between glia and axon in which the glial cells provide nutritive substances or transmitter precursors has long been suspected. For example, glia contain the preponderance of glycogen in the honeybee retina (Tsacopoulos et al., 1987), and use either the glycogen or glucose to make alanine which is exported to the photoreceptors for use as a mitochondrial energy source (Tsacopoulos et al., 1994). It would not be surprising if, in some systems, regulation of the rate of synthesis of these energy sources were synchronized to expected needs of the axon via a direct CNS link. NEUROMODULATORY AXONS IN INSECT MUSCLE 2. Developmental roles All insects undergo growth in a series of molts, and many aspects of this are under the control of circulating hormones. In addition, it is possible that some aspects of this process are regulated locally. Insulin and insulin-like growth factors may be important in synaptogenesis and maintenance of synapses, as has been shown for neurones in culture (Seecof and Dewhurst, 1974; Vanhems et al, 1990). An insulin-like compound is produced in the corpora cardiaca of locust (Hetru et al, 1991) presumably for hormonal type release. In Drosophila a single insulin containing terminal was found in certain segments at m.f. 12, but insulin receptors were found at the bases of virtually all neuromuscular junctions in the body wall musculature (Gorczyca et al., 1993). Immunoreactivity for both the insulin like compound and the insulin receptors was not detectable until late first or early second instars, with an increase thereafter. These authors speculate that it might be involved in expansion of the neuromuscular junctions as the larva grows. Glia are important to early development in some areas of the insect nervous system, forming a scaffold for transverse nerve formation in Manduca (Taghert et al., 1988), and Drosophila (Gorczyka et al, 1994), and for central nervous system tracts in Drosophila (Jacobs and Goodman, 1989). Later in development, in Manduca, the peripheral glia, both intramuscular and those surrounding main nerve branches, and which are the apparent targets for the neurosecretory type axons, undergo quite marked morphological changes during metamorphosis (Rheuben, 1992). This change in morphology of the intramuscular glial cells occurs at the same time as motor axons are withdrawing from their targets. Later, the same or similar glia are present, cradling the growing nerve tip, during the formation of the adult neuromuscular junctions (Rheuben and Kammer, 1981). Direct application of a regulatory substance by a neurosecretory axon would allow specific glial cells to respond to the changing needs of their axons in a timely fashion during such sequences of complex developmental changes. Since various muscles do not develop or degen- 575 erate in synchrony in insects, a specific control mechanism could be useful. An interesting function for a serotonin containing neurosecretory type axon has been proposed for an eclosion muscle of the tsetse fly. In this muscle a single "immunocyte" or macrophage-like cell is activated and sends out multiple processes in and amongst the muscle fibers when degeneration begins. A serotonergic axon terminates in the vicinity of the immunocyte, and cutting it stops process outgrowth of the immunocyte and concurrent muscle degeneration (Miyan and Tyrer, 1993). Consequently these authors postulate that activity of the immunocyte is controlled in some way by the neurosecretory type axon, thus indirectly regulating the timing of muscle degeneration. In summary, we have considered only a very few of the ways that neurosecretion and neuromodulation are important. However these examples suggest the need to consider diverse roles for the neurosecretory type axons in association with the skeletal muscles of insects. Although many, such as the octopaminergic axons in locust, may have primary effects on muscle contraction, others may be more important in developmental processes, in regulation of non-neuronal cells, or in respiration. Very little is concretely known about these cells at present and they offer the opportunity to add to our understanding of developmental and functional processes in insects in general. ACKNOWLEDGMENTS Support for the work in progress reported here was from NIH Grant NS 17132. We thank the Insect Physiology Laboratory, Department of Agriculture, Beltsville, MD for providing the Manduca eggs. The author is grateful to Ms. Dawn Autio for her excellent technical assistance, and to Drs. Harold Atwood, Yoshi Kidokoro, Motojiro Yoshihara, and Michael Gorczyka for their helpful discussions. REFERENCES Adams, M. E. and M. O'Shea. 1983. Peptide cotransmitter at a neuromuscular junction. Science 221: 286-289. Aizu, S. 1982. Morphological differences between 576 MARY B. RHEUBEN excitatory and inhibitory nerve terminals in cockroach coxal muscles. Tissue Cell 14:329-339. Anderson, M. S., M. E. Halpern, and H. Keshishian. 1988. Identification of the neuropeptide transmitter proctolin in Drosophila larvae: Characterization of muscle fiber-specific neuromuscular endings. J. Neurosci. 8:242-255. Arbas, E. A. and L. P. Tolbert. 1986. Presynaptic terminals persist following degeneration of "flight" muscle during development of a flightless grasshopper. J. 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