The oculomotor system of Daphnia magna

Summary The highly mobile cyclopic compound eye of Daphnia magna is rotated by six muscles arranged as three bilateral pairs. The three muscles on each side of the head share a common origin on the carapace and insert dorsally, laterally and ventrally on the eye. The dorsal and ventral muscles are each composed of two muscle fibers and the lateral muscle is composed of from two to five fibers, with three the most common number. Individual muscle fibers are spindle-shaped mononucleated cells with organized bundles of myofilaments. Lateral eye-muscle fibers are thinner than those of the other muscles but are otherwise similar in ultrastructure. Two motor neurons innervate each dorsal and each ventral muscle and one motor neuron innervates each lateral muscle. The cell bodies of the motor neurons are situated dorsally in the supraesophageal ganglion (SEG) and are ipsilateral to the muscles they innervate. The dendritic fields of the dorsal-muscle motor neurons are ipsilateral to their cell bodies; those of the ventral-muscle motor neurons are bilateral though predominantly contralateral. The central projections of the lateral-muscle motor neurons are unknown. In the dorsal and ventral muscles one motor axon synapses principally with one muscle fiber; in each lateral muscle the single motor axon branches to, and forms synapses with, all the fibers. The neuromuscular junctions, characterized by pre- and postsynaptic densities and clear vesicles, are similar in all the eye muscles.     

Sexual differentiation of identified motor terminals in Drosophila larvae

In Drosophila, we have found that some of the motor terminals in wandering third-instar larvae are sexually differentiated. In three out of the four body-wall muscle fibers that we examined, we found female terminals that produced a larger synaptic response than their male counterparts. The single motor terminal that innervates muscle fiber 5 produces an EPSP that is 69% larger in females than in males. This is due to greater release of transmitter from female than male synaptic terminals because the amplitude of spontaneous miniature EPSPs was similar in male and female muscle fibers. This sexual difference exists throughout the third-instar: it is seen in both early (foraging) and late (wandering) third-instar larvae. The sexual differentiation appears to be neuron specific and not muscle specific because the same axon produces Is terminals on muscle fibers 2 and 4, and both terminals produce larger EPSCs in females than males. Whereas, the Ib terminals innervating muscle fibers 2 and 4 are not sexually differentiated. The differences in transmitter release are not due to differences in the size of the motor terminals. For the terminal on muscle fiber 5 and the Is terminal on muscle fiber 4, there were no differences in terminal length, the number of branches, or the number of synaptic boutons in males compared to females. These sexual differences in neuromuscular synaptic physiology may be related to male-female differences in locomotion.     

Scanning and freeze-fracture study of larval nerves and neuromuscular junctions in Manduca sexta

The nerves and nerve terminals to tonic larval muscle fibers in third and fifth instar caterpillars were studied to compare them with those formed by the same motor neurons on phasic flight muscles in adult moths. Scanning micrographs showed a primary nerve branch running the length of each fiber, with secondary nerve branches extending from it at intervals. There was a great deal of variability in both the length of the branches and the distance from the nerve at which the neuromuscular junctions were formed. The rapid increase in muscle fiber size during larval development may be responsible for this variability. The nerves and junctions were often found to be obscure by overlying fibroblasts and tracheoblasts or entering the deep muscle clefts. Those examined were similar in appearance to the adult junctions formed by the same neurons, although some may have formed single branches instead of y-shapes. The membrane specializations of the synapse seen in freeze-fractured specimens were similar to those of the adult junction. However, the overall shape of the nerve terminal within the junction differed. The larval nerve terminals appeared varicose instead of having a uniform diameter. The spacing of the nerve plaques varied, in contrast with the relatively straight alignment and even spacing of plaques found in adult junctions. Such differences could result from an interaction between the motor neuron and the two different types of muscle fiber that it innervates, an intrinsic change in the motor neurons themselves that occurs with metamorphosis, or a plastic functional response that occurs as a result of the different types of motor patterns that are used in the two stages.     

Fine structure of a spider joint receptor and associated synapses.

The fine structure of a joint receptor (R10) in a spider leg (Zygiella x-notata) was examined with light and electron microscopy. The R10 receptor consists of a compact ganglion which is situated near the dorsal joint membrane of the femur/patella joint. Each of the ten sensory cells comprising the ganglion sends one branching dendrite into the hypodermis underlying the joint membrane. All dendritic branches together form a sheet-like meshwork 50 microns wide and 1 microns thick, which is traversed obliquely by hypodermis cells. When the joint is stretched shearing forces are apparently transmitted to the receptive dendritic branches via microtubular bundles inside the hypodermis cells. The soma and dendrites of the sensory cells receive numerous synaptic input from presumably efferent fibres. The fine structure of these synapses is described and compared with other peripheral and central spider synapses. All R10 synapses contain small synaptic vesicles (32 nm diameter), whereas motor endplates possess large vesicles (38 nm). Central synapses have two significantly different vesicle populations which are either of the small or large variety. Since synapses with small vesicles are supposedly inhibitory, receptor cells in spiders might be under efferent control. Such a system is unknown in insects or crustaceans, but may be typical for arachnids.     

Identified motor terminals in Drosophila larvae show distinct differences in morphology and physiology

In Drosophila, the type I motor terminals innervating the larval ventral longitudinal muscle fibers 6 and 7 have been the most popular preparation for combining synaptic studies with genetics. We have further characterized the normal morphological and physiological properties of these motor terminals and the influence of muscle size on terminal morphology. Using dye-injection and physiological techniques, we show that the two axons supplying these terminals have different innervation patterns: axon 1 innervates only muscle fibers 6 and 7, whereas axon 2 innervates all of the ventral longitudinal muscle fibers. This difference in innervation pattern allows the two axons to be reliably identified. The terminals formed by axons 1 and 2 on muscle fibers 6 and 7 have the same number of branches; however, axon 2 terminals are approximately 30% longer than axon 1 terminals, resulting in a corresponding greater number of boutons for axon 2. The axon 1 boutons are approximately 30% wider than the axon 2 boutons. The excitatory postsynaptic potential (EPSP) produced by axon 1 is generally smaller than that produced by axon 2, although the size distributions show considerable overlap. Consistent with vertebrate studies, there is a correlation between muscle fiber size and terminal size. For a single axon, terminal area and length, the number of terminal branches, and the number of boutons are all correlated with muscle fiber size, but bouton size is not. During prolonged repetitive stimulation, axon 2 motor terminals show synaptic depression, whereas axon 1 EPSPs facilitate. The response to repetitive stimulation appears to be similar at all motor terminals of an axon.     

Scanning electron microscopy of nerve-muscle contacts in embryonic cell culture.

Scanning electron microscopy (SEM) of cell cultures of dissociated nerve and muscle from chick embryos has shown that developing muscle fibers can be contacted at many sites by one or more than one neuron, and that a single nerve can send branches to several myofibers. At these contact regions of nerve with muscle, the neurons send out terminal or lateral sprouts with fine tips which initially lack terminal swellings, but later acquire small “bouton”-like structures in contact with the sarcolemma, which resemble embryonic synapses. At these points, the sarcolemma does not appear to differ in ultrastructure from other surface regions of the myofiber. Transmission electron microscopy (TEM) has revealed the presence of both electron lucent and dense-cored vesicles at some nerve terminals. However, fluorescence histochemistry (Falck-Hillarp technique) failed to detect the presence of catecholamines in these cultures. The SEM pictures at substantially higher resolutions than the light microscope, and the enhanced three dimensional perspective of this technique, provide additional information about the developmental morphology of the nerve-muscle cell culture system. The results are correlated with previous findings by light microscopy, TEM and electrophysiology, and discussed in relationship to proposed innervation processes of skeletal muscle fibers in vivo.     

Two types of neuromuscular junctions in the pharyngeal retractor muscle ofHelix lucorum

The pharyngeal retractor muscle of the snailHelix lucorum is innervated by a pair of nerves containing axons of two types, for which there are two corresponding types of myoneural junctions with the muscle cells. The junctions of type I correspond to the thick axons. The terminals of these axons, which contain numerous spherical transparent vesicles (41±5 nm) and fewer vesicles of the dense-core type (67±3 nm), make contact mainly with noncontracting sarcoplasmic projections of the muscle cells. Junctions of type II correspond to thin axons, containing many granules. The terminals of these axons make contact with contractile parts of the muscle cells and they contain a heterogeneous population of vesicles: small spherical clear vesicles (44±2 nm), granules with fine-grained contents (135±5 nm), and a few spherical dense-core vesicles. The distance between the muscle cells is usually great — over 50 nm, but in the region of the sarcoplasmic processes the surface membranes come together to form a gap which in some areas does not exceed 10 nm.N. K. Kol'tsov Institute of Developmental Biology, Academy of Sciences of the USSR, Kiev. Translated from Neirofiziologiya, Vol. 9, No. 5, pp. 539–542, September–October, 1977.     

Immunocytochemical and electron-microscopic study of the elasmobranch nucleus sacci vasculosi

Summary The elasmobranch nucleus sacci vasculosi was studied by means of electron microscopy (in the dogfish) and immunocytochemistry (in the dogfish and the skate) by using antibodies against tyrosine hydroxylase, alpha-melanocyte-stimulating hormone, somatostatin, serotonin, and substance P. Ultrastructural study of the dogfish nucleus sacci vasculosi shows the presence of medium-sized cells that possess numerous mitochondria but that have no dense-core vesicles in the cytoplasm or in cell processes. Fibres of the conspicuous tractus sacci vasculosi have a beaded appearance and form conventional synapses with dendrites and cell perikarya of the nucleus sacci vasculosi. The perikarya of this hypothalamic nucleus were not immunoreactive to any of the antibodies tested, and fibres immunopositive to tyrosine hydroxylase, alpha-melanocyte-stimulating hormone, somatostatin, serotonin, and substance P were scarce within this nucleus, in both the dogfish and the skate. Dorsal to the nucleus sacci vasculosi, there are numerous positive neuronal processes in addition to many small neurons that show immunoreactivity to alpha-melanocyte-stimulating hormone, somatostatin and tyrosine hydroxylase. Two types of neuron occur in this dorsal region, displaying dense-core vesicles of either 100–160 nm or 60–100 nm diameter in their cytoplasm; they were identified as peptide-containing and monoamine-containing neurons, respectively. The neuropil of this region has a significantly different ultrastructure from that of the nucleus sacci vasculosi, with many processes containing dense-core vesicles. This group of neurons, located dorsal to the nucleus sacci vasculosi and showing (a) immunoreactivity to neuropeptides or to monoamine-synthesizing enzyme, and (b) cytoplasm with dense-core vesicles, was considered not to be a part of the nucleus sacci vasculosi but rather part of the nucleus tuberculi posterioris. These results support the non-peptidergic and non-aminergic character of the nucleus sacci vasculosi.     

Light and electron microscopic studies of slowly adapting abdominal stretch receptors of the American river crayfish Orconectes limosus (RAF)]

The slowly adapting abdominal stretch receptors of Orconectes limosus (RAF) have been investigated morphologically; 1. Despite their variety of size and shape all slowly adapting receptor neurons show common characteristic features which in addition distinguish them clearly from the fast adapting receptor neuron type SN2. The slightly globular cells have always several dendrites (often 4-6). They originate apical or lateral to the neuron, are oriented mainly longitudinal to the muscle fibres and are brodly ramified. The fine dendrites form a 3-dimensional fibrilar network. 2. The structure and distribution of the connective tissue in the "intertendon" of the muscle receptor organ correspond to the dendrite ramification; In this area, some muscle fibres end direktly at tendon-like connective tissue structures, but a number of different fibres run uninerruptedly through the whole muscular fascicle. 3. The perikaryon of every sensory neuron shows 2 "cytoplasm types" which are clearly distinguishable one against the other. A characteristic feature of the granular-lamellar neuroplasm that closely surrounds the nucleus are many flat vesicles of the granular endoplasmatic reticulum, accumulations of free ribosomes, numerous mitochondria and Golgi fields. The fibril-rich neuroplasm on the contrary contains only few mitochondria, but very many neurofilaments, here and there also neurotubuli. It projects directly into the dendrites and neuritek. Cell bodies, axon and dendrites are surrounded alternatingly by sheath cells and connective tissue of collagenous nature. The innermost layer of the coat cells borders directly on the neuron membrane. Finer dendrites are enclosed by nothing more but a thin layer of sheath cell plasm and intercellular substance. The dendrite terminals are either stored directly in connective tissue ground substance or border immediately on the sarcoplasm. 5. The axo-dendritic or axo-somatic synapses, respectively, contain numerous ellipsoidal (250-350 X 400-500 A), but also many spherical, vesicles. Some vesicles have a slightly larger diameter (700-900 A) and contain an electron-dense core. The synaptic gap measures 150 to 200 A. The neuromuscular (supposedly excitatory) synapses are filled much lighter with vesicles as compared with those just mentioned, which show a relatively unique form and size (nearly all spherical, phi 400-500 A). There are less vesicles with an electron-dense centre. On the average, the synaptic gap is broader (200-250 A) and the contact zone is larger. Apart from these, terminals could be observed in the dendritic ramification area, too, resembling the axo-dendritic and axo-somatic ones, respectively. 6. Finer dendrite branches contain vesicles differing slightly from those mentioned above as far as shape and size are concerned. Their diameters vary between 500 and 1 000 A. "Dense bodies" could be observed sporadically in these vesicles.     

Embryonic development of the innervation of the locust extensor tibiae muscle by identified neurons: formation and elimination of inappropriate axon branches

Intracellular dye fills have been used to reveal the pattern of embryonic growth of each of the four neurons which innervate the extensor tibiae muscle (ETi) of the hind leg of the locust. The growth cone of the slow extensor tibiae motoneuron (SETi), the first of the four neurons to leave the central nervous system, pioneers nerve 3 (N3). The fast extensor motoneuron (FETi), the next neuron to grow out, follows earlier outgrowing motoneurons into the periphery in nerve 5 (N5) and then rejoins SETi in N3. As it transfers from N5 to N3, it is transiently dye-coupled to the Tr1 pioneer neuron which spans the gap between the two nerves. It then follows SETi onto the ETi muscle in the femur. The common inhibitory neuron and the dorsal unpaired median neuron (DUMETi) follow SETi and FETi in nerves 3B2 and 5B1, respectively. SETi's growth cone requires almost twice as long to reach ETi as those of the three later motoneurons, all of which follow preexisting neural pathways. At least three of the four developing motoneurons form one or more axon branches not found in the adult. These branches may occur (1) at segmental boundaries; (2) where the nerve, which the growth cone is following, itself branches or the growth cone encounters another nerve; or (3) when the axon continues to grow beyond its target muscle. These findings contrast with the apparent absence of inappropriate axon branches in another developing locust neuromuscular system and during the innervation of zebrafish myotomes, but resemble in some ways the transient production of inappropriate axonal branches reported for embryonic leech motoneurons.     

Observations on the ultrastructure of ganglion cells in the circumvallate papilla of rat and mouse

Ganglion cells in the circumvallate papilla of adult rodents are described as typical autonomic neurons. Some neurons are aggregated to form a discrete structure in the base of the papilla; others are scattered through the core, along the nerve bundles, and particularly near the dome. The term "circumvallate ganglion" is applied to the entire population. Satellite cells completely ensheathe each neuron. Preganglionic fibers, containing clear vesicles, synapse on the soma and stumpy dendrites of the neurons. Axons, containing dense-cored vesicles, are observed in close proximity to the neurons. However, these fibers do not establish true morphological synaptic contacts with the neurons. We have not observed serial or reciprocal synapses on or in the vicinity of the ganglion cells. The hypothesis that the axons of the circumvallate ganglion neurons act as parasympathetic vasodilators is indicated by the proximity of the two structures and by nerve terminations on the arteriole muscle cells. Direct modulation of taste transduction by these neurons is ruled out.     

Elicitation and abrupt termination of behaviorally significant catchlike tension in a primitive insect

Sustained steady contractural or catchlike tension (CT) occurs in the metathoracic extensor tibiae muscle of the primitive insect the weta (Orthoptera: Stenopelmatidae) during its characteristic leg-extension defense behavior or following leg-position conditioning. Similar action occurs occasionally in semi-intact preparations and is abruptly turned off by a single peripheral inhibitory impulse. These phenomena were reproduced routinely by first infusing saline containing 10^?8M (or stronger) octopamine into the muscle for 12 min, and then stimulating the slow excitatory motor neuron SETi with a brief burst. Direct stimulation of the dorsal unpaired median neuron, innervating the extensor tibiae (DUMETi) prior to SETi stimulation, also led to CT. Both octopamine and DUMETi markedly enhanced the tension developed in response to a burst of impulses in SETi.     

Ultrastructure of the larval firefly light organ as related to control of light emission

The firefly larva has a pair of light organs consisting of a layer of interdigitating, light emitting cells, covered dorsally with a layer of opaque, white cells. Each light organ is ventilated by one large and several smaller tracheal branches and is innervated by a branch of the segmental nerve containing two axons. These axons branch profusely in the photocyte layer so that several nerve profiles are seen around any photocyte. Nerve terminals contain large dense-core vesicles and small light-core vesicles. Clusters of light-core vesicles surrounding irregularly shaped membrane densifications, presumably the synapses between nerve and photocyte, are common in nerve terminals. Light emitting cells in insects characteristically contain photocyte vesicles. In the larva there are both full and empty photocyte vesicles; the full vesicles contain a matrix with tubular membrane invaginations in contrast to the empty vesicles which contain amorphous membrane invaginations.     

Electrophysiology of Supramedullary Neurons in Spheroides maculatus : I. Orthodromic and antidromic responses

This series of three papers presents data on a system of neurons, the large supramedullary cells (SMC) of the puffer, Spheroides maculatus, in terms of the physiological properties of the individual cells, of their afferent and efferent connections, and of their interconnections. Some of these findings are verified by available anatomical data, but others suggest structures that must be sought for in the light of the demonstration that these cells are not sensory neurons. Analysis on so broad a scale was made possible by the accessibility of the cells in a compact cluster on the dorsal surface of the spinal cord. Simultaneous recordings were made intracellularly and extracellularly from individual cells or from several, frequently with registration of the afferent or efferent activity as well. The passive and active electrical properties of the SMC are essentially similar to those of other neurons, but various response characteristics have been observed which are related to different excitabilities of different parts of the neuron, and to specific anatomical features. The SMC produce spikes to direct stimuli by intracellular depolarization, or by indirect synaptic excitation from many afferent paths, including tactile stimulation of the skin. Responses that were evoked by intracellular stimulation of a single cell cause an efferent discharge bilaterally in many dorsal roots, but not in the ventral. Sometimes several distinct spikes occurred in the same root, and behaved independently. Thus, a number of axons are efferent from each neuron. They are large unmyelinated fibers which give rise to the elevation of slowest conduction in the compound action potential of the dorsal root. A similar component is absent in the ventral root action potential. Antidromic stimulation of the axons causes small potentials in the cell body, indicating that the antidromic spikes are blocked distantly to the soma, probably in the axon branches. The failure of antidromic invasion is correlated with differences in excitability of the axons and the neurite from which they arise. As recorded in the cell body, the postsynaptic potentials associated with stimulation of afferent fibers in the dorsal roots or cranial nerves are too small to discharge the soma spike. The indirect spike has two components, the first of which is due to the synaptically initiated activity of the neurite and which invades the cell body. The second component is then produced when the soma is fired. The neurite impulse arises at some distance from the cell body and propagates centrifugally as well as centripetally. An indirect stimulus frequently produces repetitive spikes which are observed to occur synchronously in all the cells examined at one time. Each discharge gives rise to a large efferent volley in each of the dorsal roots and cranial nerves examined. The synchronized responses of all the SMC to indirect stimulation occur with slightly different latencies. They are due to a combination of excitation by synaptic bombardment from the afferent pathways and by excitatory interconnections among the SMC. Direct stimulation of a cell may also excite all the others. This spread of activity is facilitated by repetitive direct excitation of the cell as well as by indirect stimulation.     

A radioautographic and immunocytochemical study of the GABA systems of the habenula complex in the rat

Radioautography of [³H]GABA accumulation and immunocytochemistry of glutamate decarboxylase have been used to study anatomically and morphologically the GABA system of the rat habenular (Hb) complex. Radioautographic visualisation of GABA specific neurons show a very high innervation of the complex including both stria medullaris (SM), the habenular commissure and the periventricular thalamic fibers (FPVT). A massive labeled fiber system in the SM appears to divide into two branches when it reaches the Hb nuclei: a part of fibers continue their course dorsally to the nuclei up to the habenular commissure; other fibers enter the Hb lateralis or run along the ventral Hb medialis at the level of FPVT. The staining is markedly diminished in the entire complex in response to SM lesions. In the Hb lateralis, the radioautographic-positive reaction is mainly bound to labeled fibers or axonal varicosities. However GAD immunocytochemistry reveals some GAD-positive cell bodies in the ventro-median portion of the nucleus. In the Hb medialis the radioautographic and immunocytochemical staining is observed in the neuropile between the unlabeled large cell bodies. In the subependymal layer bundles of processes are strongly labeled and form a continual strain behind the unlabeled ependymocytes. Three types of reactive terminals have been differentiated based on size and shape of vesicles. Some of them are exclusively characterized by clear round vesicles and probably have their origin in the septum. Others contain clear vesicles and some large dense-cored vesicles and disappear after mesencephalic Raphe lesions or 5,7-dihydroxytryptamine treatment. They could correspond to terminals of raphe neurons with a double potentiality GABA and 5HT. The last exhibit mainly a dense population of large dark-cored granules similar to the ones found in neurosecretory nerve endings. However numerous fibers morphologically similar to the reactive fibers are unlabeled.     

Fine structural study of interstitial cells associated with the deep muscular plexus of the rat small intestine,with special reference to the intestinal pacemaker cells

Two types of interstitial cells have been demonstrated in close association in the deep muscular plexus of rat small intestine, by electron microscopy. Cells of the first type are characterized by a fibroblastic ultrastructure, i.e. a well-developed granular endoplasmic reticulum, Golgi apparatus and absence of the basal lamina. They form a few small gap junctions with the circular muscle cells and show close contact with axon terminals containing many synaptic vesicles. They may play a role in conducting electrical signals in the muscle tissue. Cells of the second type are characterized by many large gap junctions that interconnect with each other and with the circular muscle cells. Their cytoplasm is rich in cell organells, including mitochondria, granular endoplasmic reticulum and Golgi apparatus. They show some resemblance to the smooth muscle cells and have an incomplete basal lamina, caveolae and subsurface cisterns. However, they do not contain an organized contractile apparatus, although many intermediate filaments are present in their processes. They also show close contacts with axon terminals containing synaptic vesicles. These gap-junction-rich cells may be regular components of the intestinal tract and may be involved in the pacemaking activity of intestinal movement.     

Ultrastructural immunocytochemical studies on CCK-and neurotensin-like immunoreactivity in the nucleus accumbens of the rat

Using the peroxidase-antiperoxidase technique of Sternberger and collaborators the ultrastructural localization of cholecystokinin- and neurotensin-like immunoreactivity was studied in the nucleus accumbens of the albino rat. The immunoreactive material was localized to nerve terminals making probable axodendritic or axo-axonic contacts with neighbouring nerve cells. Within the nerve terminals most immuno-reactivity was confined to so-called large dense-core vesicles.     

Serotonin-immunoreactive neurons in the antennal sensory system of the brain in the carpenter ant, Camponotus japonicus

Social Hymenoptera such as ants or honeybees are known for their extensive behavioral repertories and plasticity. Neurons containing biogenic amines appear to play a major role in controlling behavioral plasticity in these insects. Here we describe the morphology of prominent serotonin-immunoreactive neurons of the antennal sensory system in the brain of an ant, Camponotus japonicus. Immunoreactive fibers were distributed throughout the brain and the subesophageal ganglion (SOG). The complete profile of a calycal input neuron was identified. The soma and dendritic elements are contralaterally located in the lateral protocerebrum. The neuron supplies varicose axon terminals in the lip regions of the calyces of the mushroom body, axon collaterals in the basal ring but not in the collar region, and other axon terminals ipsilaterally in the lateral protocerebrum. A giant neuron innervating the antennal lobe has varicose axon terminals in most of 300 glomeruli in the ventral region of the antennal lobe (AL) and a thick neurite that spans the entire SOG and continues towards the thoracic ganglia. However, neither a soma nor a dendritic element of this neuron was found in the brain or the SOG. A deutocerebral projection neuron has a soma in the lateral cell-body group of the AL, neuronal branches at most of the 12 glomeruli in the dorsocentral region of the ipsilateral AL, and varicose terminal arborizations in both hemispheres of the protocerebrum. Based on the present results, tentative subdivisions in neuropils related to the antennal sensory system of the ant brain are discussed.     

Neuromuscular Junctions in Flight and Tymbal Muscles of the Cicada

The tymbal muscle fiber in the cicada closely resembles the indirect flight muscle fiber in its structural detail. We agree with other authors that the tymbal muscle is a modified indirect flight muscle. The peripheral nerve branches to the tymbal and flight muscle fibers are similar to those in the wasp leg. The axon is loosely mantled by irregular turns of the mesaxon, enclosing cytoplasm. The nerve is therefore a tunicated nerve. The neuromuscular junction in the high frequency muscle fibers shows direct apposition of plasma membranes of axon and muscle fiber, large numbers of mitochondria and synaptic vesicles in the axon, and concentrations of mitochondria, aposynaptic granules, and endoplasmic reticulum in the postsynaptic area of the muscle fiber. Of special interest is the multitude of intracellular, opposing membranes in the postsynaptic area. They form laminated stacks and whorls, vesicles, cysternae, and tubules. They occasionally show continuity with the plasma membrane, the outer nuclear envelope, and the circumfibrillar endoplasmic reticulum. The membrane system in this area is designated "rete synapticum." It is believed to add to the electrical capacity of the neuromuscular junction, to serve in transmission of potentials, and possibly is the site of the oscillating mechanism in high-frequency muscle fibers.     

Ultrastructural localization of neuropeptides and GABA in rat dorsal horn: a comparison of different immunogold labeling techniques

Several immunogold techniques were used to determine the ultrastructural localization of calcitonin gene-related peptide (CGRP), tachykinin, somatostatin, and gamma-amino-butyric acid (GABA) immunoreactivity in the dorsal horn of rat spinal cord. The immunocytochemical reactions were carried out directly on ultrathin sections from non-osmicated frozen tissue, non-osmicated low temperature-embedded (Lowicryl K4M) tissue, and osmicated epoxy-embedded material. Preservation of ultrastructural morphology and immuno-labeling efficiency were compared. Morphology of subcellular organelles was relatively good in ultra-thin frozen sections, which showed the highest immunoreactivity. However, only very small samples of tissue could be examined. Although there was relatively good immunolabeling in the Lowicryl K4M-embedded tissue, the ultrastructure of the neuropil, and particularly that of synapses, was poorly maintained. In contrast, the osmicated epoxy-embedded material offered optimal morphological preservation together with accurate subcellular localization of all antigens under study. The latter approach thus enabled clear visualization of CGRP, tachykinin, and somatostatin immunoreactivity restricted to large dense-cored vesicles (90-150 nm diameter) in many axonal and synaptic profiles in the superficial layers of the dorsal horn. CGRP- and tachykinin-positive profiles were also present in the tract of Lissauer. GABA immunoreactivity was present mainly in axons and terminals, and less frequently in somatic and dendritic profiles. In terminals, which often formed symmetrical synapses on immunonegative dendritic profiles, it was associated with small (30-60 nm diameter) clear vesicles and mitochondria. Double immunolabeling was possible on all preparations, but the osmicated, epoxy-embedded material clearly showed co-localization of peptides, especially of CGRP and tachykinins, within the same dense-cored vesicles in axonal fibers and/or terminals. On the other hand, peptide and GABA immunoreactivity were consistently seen in different nerve profiles. In a few cases, GABAnergic terminals were seen to synapse on tachykinin-positive fibers.