Regeneration studies on a crayfish neuromuscular system. I. Connectivity changes after intersegmental nerve transplants

The superficial flexor muscles of the crayfish are a neuromuscular system of a few muscle cells innervated by six neurons in a precise position-dependent pattern. The neurons are capable of regenerating their normal connectivity patterns within a short span of time when conditions are favorable. The superficial flexor muscles of the second and third segments, despite their similarities in neuronal and muscle cell size and number, have distinctive connectivity patterns; some homologous neurons form similar patterns but other homologous neurons form patterns that are reversed between segments. We transplanted each segment's nerve into each other's muscle in order to observe regeneration of the nerves into a target area that differed in connectivity patterns from their original muscle. During the first weeks of regeneration all neurons formed a connectivity pattern with more connections medially and declining connections laterally, a pattern determined by the medial location of the nerve transplant. This pattern is maintained for most of the neurons, but for some there is an eventual reduction in medial connections as maximum synapse formation shifts to the lateral muscle fibers. Three of the eight neurons studied were able to regenerate connectivity patterns that corresponded to their segment of origin and not to their host muscle. This suggests that intersegmental muscle differences are not influencing the formation of these connectivity patterns, so the neurons will follow their inherent synaptogenesis program.     

Synaptic repression at crayfish neuromuscular junctions. I. Generation after partial target area removal

Synaptic repression, the inability of synaptic junctions to generate normal-sized postsynaptic potentials under normal physiological conditions, is reported here for crayfish neuromuscular synapses. The synapses in the superficial flexor muscle system of the crayfish change their efficiency in generating a postsynaptic response as a result of a specific alteration in their immediate environment. When the superficial flexor nerve is cut halfway into the target muscle field and the lateral muscle fibers are removed, the intact medial synapses do not generate normal-sized junction potentials (JP) at the 17° –19°C temperature of the Ringers solution. JPs cannot be recorded in 83% of the muscle fibers at 2 weeks after the operation and of the few JPs that can be detected, 80% are smaller than 1 mV in size. By 8 weeks after the operation, JPs were detected in 55% of the muscle fibers, and now only 46% of these are smaller than 1 mV. When the lateral muscle fibers are left in place during the original operation, providing a target area for the cut nerve to grow into, JPs were then detected in 60%–80% of all medial fibers at all time periods after the operation; their size profile, with 10%–25% of the muscle fibers having JP's less than 1 mV, was similar to control values. These results suggest that the efficiency of these synaptic contacts become affected as a result of partial axotomy and removal of the target area of the cut branches of the axons. © 1993 John Wiley & Sons, Inc.     

Regeneration of an identifiable motoneuron in the crayfish. II. Patterns of reconnection and synaptic strength established in the presence of an extra nerve

The regeneration of neuromuscular connections to the superficial flexor muscle system in the crayfish has been studied under a variety of experimental manipulations. These have provided insight into the factors that can influence the regeneration program of neurons. In this work the regeneration of the largest excitor motoneuron was studied under two different conditions: (1) when the original neuron and a transplanted neuron were growing simultaneously into a denervated target, and (2) when a transplanted neuron was growing into a target that had its original nerve supply intact. In condition 1 both the transplanted and the original neuron formed normal patterns of connectivity and synaptic strength in comparable periods of time. In condition 2 the rate of growth of the transplanted neuron is significantly reduced and does not extend into the lateral fibers of the muscle. It is concluded that the regeneration program of this neuron is not affected by the presence of other neurons growing at the same time into a denervated muscle. Since regeneration is seriously affected if growth occurs into a fully innervated target area, it is suggested that lack of growth stimuli from the target or competitive interactions between established and growing synaptic terminals could influence the regeneration program of this neuron.     

Regeneration studies on a crayfish neuromuscular system. II. Effect of changing the nerve entry point into the muscle field on the gradient of innervation

The superficial flexor muscle of the crayfish is a neuromuscular system in which the neurons form position-dependent connectivity patterns with the muscle fibers. This system could be formed with the help of a single medial-to-lateral gradient during development that embodies positional information. To test this gradient hypothesis we changed the nerve's normal medial entry point into the muscle by transplanting it to the middle of the muscle sheet. When all the muscle fibers were present in the target area, most of the neurons studied passed through a stage during regeneration in which they showed preference for either medial or lateral synapse formation. Those neurons that in normal animals innervated preferentially the medial fibers showed a medial preference for new contacts; the neuron that normally innervated the lateral fibers showed a lateral preference for new contacts; the neuron that normally innervated everywhere regenerated equally well into both medial and lateral fibers. Therefore, these neurons are able to detect information regarding their position within the muscle mass and respond to it by preferential synapse formation. The effect of a positional gradient could not be detected when half of the target field was removed prior to regeneration. In this instance, the neuron that innervated the missing target area now regenerated to almost all the available fibers. It is suggested that the interplay of positional cues with other factors at different points in time could determine the final connectivity patterns formed by these cells.     

Uncoupling store-operated Ca2+ entry and altered Ca2+ release from sarcoplasmic reticulum through silencing of junctophilin genes

Junctophilin (JP) mediates the close contact between cell surface and intracellular membranes in muscle cells ensuring efficient excitation-contraction coupling. Here we demonstrate that disruption of triad junction structure formed by the transverse tubular (TT) invagination of plasma membrane and terminal cisternae of sarcoplasmic reticulum (SR) by reduction of JP expression leads to defective Ca2+ homeostasis in muscle cells. Using adenovirus with small hairpin interference RNA (shRNA) against both JP1 and JP2 genes, we could achieve acute suppression of JPs in skeletal muscle fibers. The shRNA-treated muscles exhibit deformed triad junctions and reduced store-operated Ca2+ entry (SOCE), which is likely due to uncoupled retrograde signaling from SR to TT. Knockdown of JP also causes a reduction in SR Ca2+ storage and altered caffeine-induced Ca2+ release, suggesting an orthograde regulation of the TT membrane on the SR Ca2+ release machinery. Our data demonstrate that JPs play an important role in controlling overall intracellular Ca2+ homeostasis in muscle cells. We speculate that altered expression of JPs may underlie some of the phenotypic changes associated with certain muscle diseases and aging.     

Transplantation of Xenopus laevis Tissues to Determine the Ability of Motor Neurons to Acquire a Novel Target

The evolutionary origin of novelties is a central problem in biology. At a cellular level this requires, for example, molecularly resolving how brainstem motor neurons change their innervation target from muscle fibers (branchial motor neurons) to neural crest-derived ganglia (visceral motor neurons) or ear-derived hair cells (inner ear and lateral line efferent neurons). Transplantation of various tissues into the path of motor neuron axons could determine the ability of any motor neuron to innervate a novel target. Several tissues that receive direct, indirect, or no motor innervation were transplanted into the path of different motor neuron populations in Xenopus laevis embryos. Ears, somites, hearts, and lungs were transplanted to the orbit, replacing the eye. Jaw and eye muscle were transplanted to the trunk, replacing a somite. Applications of lipophilic dyes and immunohistochemistry to reveal motor neuron axon terminals were used. The ear, but not somite-derived muscle, heart, or liver, received motor neuron axons via the oculomotor or trochlear nerves. Somite-derived muscle tissue was innervated, likely by the hypoglossal nerve, when replacing the ear. In contrast to our previous report on ear innervation by spinal motor neurons, none of the tissues (eye or jaw muscle) was innervated when transplanted to the trunk. Taken together, these results suggest that there is some plasticity inherent to motor innervation, but not every motor neuron can become an efferent to any target that normally receives motor input. The only tissue among our samples that can be innervated by all motor neurons tested is the ear. We suggest some possible, testable molecular suggestions for this apparent uniqueness.     

Regeneration of an identifiable motoneuron in the crayfish. I. Patterns of reconnection and synaptic strength established in normal and altered target areas

The superficial flexor muscles of the crayfish are innervated in a position-dependent connectivity pattern, which can be reestablished when the nerve to the muscle is cut. This article deals with the regeneration of the largest excitor motoneuron under three different target scenarios: (1) a normal target with all the muscle fibers present, (2) a reduced target lacking the medial or the lateral muscle fiber population, and (3) when the nerve enters the target in the middle of the muscle field. In scenario 1 the neuron is able to regenerate the normal connectivity pattern within 10 weeks after surgery: all the lateral fibers become innervated, with a linear decline in the probability of connections over the medial fibers. The medial fibers become transiently hyperinnervated before the normal pattern of connections is established. In scenario 2 the normal pattern of connections is established only when the lateral fibers were present; with only medial cells as a target, the transient hyperinnervation stage is stable and no decline in connections was observed. Analysis of regenerated junction potential sizes during the stable hyperinnervation stage show abnormal patterns, suggesting that some aspects of the regeneration program of this neuron can be affected when signals from its prime target cells are missing. In scenario 3 growth begins in both directions until the entire muscle becomes innervated. The normal pattern of connectivity finally emerges after continued lateral growth and diminished medial growth, suggesting that the position of the muscle fibers influences connectivity patterns during the final stages of regeneration.     

Junctophilins emerge as novel therapeutic targets

Junctophilins (JPs) emerge to play key role in human pathophysiology. This family includes four subtypes (JP1–4), which are differentially detected in excitable cells. Previous work demonstrated the knockout of JPs that seriously damage physiological functions in skeletal muscle, cardiac, and neurons. Here, we summarize latest papers on the essential function of JPs in some Ca²⁺-related diseases and neurological diseases, such as primary muscle disease, cardiomyopathies, Type 2 diabetes, gastrointestinal cancer, Huntington’s disease-like 2, and Charcot-Marie-Tooth disease. Growing evidence suggests that targeting JPs might be a promising therapeutic approach to achieve better clinical efficacy in Ca ²⁺-related diseases and neurological diseases.     

Junctophilin 1 and 2 proteins interact with the L-type Ca2+ channel dihydropyridine receptors (DHPRs) in skeletal muscle

Junctophilins (JPs) anchor the endo/sarcoplasmic reticulum to the plasma membrane, thus contributing to the assembly of junctional membrane complexes in striated muscles and neurons. Recent studies have shown that JPs may be also involved in regulating Ca2+ homeostasis. Here, we report that in skeletal muscle, JP1 and JP2 are part of a complex that, in addition to ryanodine receptor 1 (RyR1), includes caveolin 3 and the dihydropyridine receptor (DHPR). The interaction between JPs and DHPR was mediated by a region encompassing amino acids 230-369 and amino acids 216-399 in JP1 and JP2, respectively. Immunofluorescence studies revealed that the pattern of DHPR and RyR signals in C2C12 cells knocked down for JP1 and JP2 was rather diffused and characterized by smaller puncta in contrast to that observed in control cells. Functional experiments revealed that down-regulation of JPs in differentiated C2C12 cells resulted in a reduction of intramembrane charge movement and the L-type Ca2+ current accompanied by a reduced number of DHPRs at the plasma membrane, whereas there was no substantial alteration in Ca2+ release from the sterol regulatory element-binding protein. Altogether, these results suggest that JP1 and JP2 can facilitate the assembly of DHPR with other proteins of the excitation-contraction coupling machinery.     

Identification of sensory neurons supplying receptors in lingual muscles of the rat: histochemical and retrograde labeling study with horseradish peroxidase.

Sensory innervation of lingual musculature was studied in young adult Wistar rats using retrograde labeling by horseradish peroxidase (HRP) and combined silver impregnation and acetylcholinesterase (AchE) methods. Intra-lingual injection of HRP resulted in labeling of neuronal somata in the trigeminal, superior vagal, and second cervical spinal (C2) ganglia. When HRP was directly applied to the proximal stump of severed hypoglossal nerve, labeling occurred only in the cervical and superior vagal ganglia. Morphometric analysis revealed that the labeled neurons were of the small-sized category in all ganglia. However, in the trigeminal and C2 ganglia, labeling occurred also among the medium-sized neurons. Combined silver and AchE preparations from lingual muscles revealed the absence of typical muscle spindles. Instead, there were free and spiral nerve terminals in the interstitium, and epilemmal knob-like or bouton-like endings surrounding non-encapsulated muscle fibers. These terminals showed AchE -ve reaction in contrast to the motor ones. Few ganglionic cells were scattered along the hypoglossal nerve with uniform AchE +ve reaction in their perikarya. This indicates that medium-sized neurons in the trigeminal and C2 ganglia, and probably sensory neurons along the hypoglossal nerve mediate lingual muscle sensibility perceived by atypical sensory terminals.     

Regenerated synaptic terminals on a crayfish slow muscle identify with transplanted phasic or tonic axons

Phasic or tonic nerves transplanted onto a denervated slow superficial flexor muscle in adult crayfish regenerated synaptic connections that displayed large or small excitatory postsynaptic potentials (EPSPs), respectively, suggesting that the neuron specifies the type of synapse that forms (Krause et al., J Neurophysiol 80:994-997, 1998). To test the hypothesis that such neuronal specification would extend to the synaptic structure as well, we examined the regenerated synaptic terminals with thin serial section electron microscopy. There are distinct differences in structure between regenerated phasic and tonic innervation. The phasic nerve provides more profuse innervation because innervation sites occurred more frequently and contained larger numbers of synaptic terminals than the tonic nerve. Preterminal axons of the phasic nerve also had many more sprouts than those of the tonic nerve. Phasic terminals were thinner and had a lower mitochondrial volume than their tonic counterparts. Phasic synapses were half the size of tonic ones, although their active zone-dense bars were similar in length. The density of active zones was higher in the phasic compared with the tonic innervation, based on estimates of the number of dense bars per synapse, per synaptic area, and per nerve terminal volume. Because these differences mirror those seen between phasic and tonic axons in crayfish muscle in situ, we conclude that the structure of the regenerated synaptic terminals identify with their transplanted axons rather than with their target muscle. Therefore, during neuromuscular regeneration in adult crayfish, the motoneuron appears to specify the identity of synaptic connections.     

Important role of junctophilin in nematode motor function.

Junctional complexes between the plasma membrane and endoplasmic/sarcoplasmic reticulum are shared by excitable cells and seem to be the structural ground for cross-talk between cell-surface and intracellular ionic channels. Our current studies have identified junctophilins (JPs) as members of a novel transmembrane protein family in the junctional membrane complex. Biochemical and gene-knockout studies have suggested that JPs contribute to the formation of the junctional membrane complex by spanning the intracellular store membrane and interacting with the plasma membrane. We report here invertebrate JPs in fruit fly and nematode. Three distinct JP subtype genes are found in the mammalian genome, while a single JP gene exists in either invertebrate genome. Mammalian and invertebrate JPs share characteristic structural features, although some intervening sequences are found in invertebrate JPs. A reporter assay indicated that the JP gene is predominantly activated in muscle cells in nematode. Nematodes, in which expression of JP was inhibited by RNA-mediated interference (RNAi), showed hypolocomotion. Taking account of the cell-type-specific expression and data from previous reports, the hypolocomotion is likely to be due to the deficiency of junctional membrane structures and the resulting reduction of Ca(2+) signaling during excitation-contraction coupling in muscle cells.     

Transplantation of the rat pineal organ to the brain: pinealocyte differentiation and innervation

Summary The pineal organ of neonatal rats was transplanted to the frontal part of the cerebral cortex or the cerebral interhemispheric fissure of an isogenic adult rat to determine whether pineal differentiation and pinealopetal innervation are affected by aberrant neuronal influences. Transplants were fixed for immunohistochemistry at 1, 2 and 6 months after transplantation. When treated with an anti-serotonin antibody, cells in transplants from both locations showed intense immunoreactivity and a morphology comparable to intact pinealocytes, indicating that the transplanted pinealocytes had differentiated normally. Tyrosine hydroxylase immunohistochemistry revealed that new catecholamine fibers of central nervous origin extended only into the periphery and not into the core of transplants grafted within the cortex. However, numerous catecholamine fibers were found in transplants placed in the interhemispheric fissure. These fibers were often accompanied by blood vessels, suggesting that they derived from sympathetic ganglia. Serotonin fibers, which are densely distributed in the cerebral cortex, were seldom found to enter transplants from both locations. These observations indicate that pineal cells express their characteristic properties even when transferred to a foreign milieu and that they do not receive novel innervation from the central nerves that normally do not innervate the intact pineal body; the transplant thereby retains the property of selective pinealopetal innervation.     

Junctional acetylcholine receptor channel open time is not presynaptically regulated in developing muscle

The role of motor innervation in controlling the development of acetylcholine receptor (AChR) channel open time was tested by examining synaptic current durations in transplanted muscles of Xenopus tadpoles. The presumptive lower jaw region, which gives rise to the interhyoideus muscle, was transplanted to the tail, overlying the myotomal muscle cells. The transplanted muscles became innervated, presumably by spinal nerves which normally innervate myotomal muscle. Despite development in the presence of foreign innervation, synaptic currents in the transplanted interhyoideus were predominantly long in duration and resembled those in the normally innervated interhyoideus. They did not resemble those in the myotomal muscle, where synaptic currents are brief. The apparent lack of neural influence on development of AChR function in muscle contrasts with the evidence for presynaptic control of AChR open time in frog sympathetic ganglia. This may reflect a fundamental difference between nerve and muscle in the regulation of postsynaptic function.     

Muscle spindle formation and differentiation in regenerating rat muscle grafts

Muscle spindle development and function are dependent upon sensory innervation. During muscle regeneration, both neural and muscular components of spindles degenerate and it is not known whether reinnervation of a regenerating muscle results in reestablishment of proper neuromuscular relationships within spindles or whether sensory neurons may exert an influence upon differentiation of these spindles. Muscle spindle regeneration was studied in bupivacaine-treated grafts of rat extensor digitorum longus (EDL) muscles. Three types of EDL graft were performed in order to manipulate the extent to which regenerating spindles might be reinnervated: (1) grafts reinnervated following severance of their nerve supply (standard grafts); (2) grafts in which intact nerve sheaths appear to facilitate reinnervation (nerveintact grafts); and (3) grafts in which reinnervation was prevented (nonreinnervated grafts). Complete degeneration of muscle fibers occurred in all grafts prior to regeneration. Initial formation of spindles in regenerating EDL grafts is independent of innervation; intrafusal muscle fibers degenerate and regenerate within spindle capsules that remain intact and viable. The extent of spindle differentiation was evaluated in each type of graft using criteria that included nucleation and ATPase activity, both of which have been shown to be regulated by sensory innervation, as well as the number of muscle fibers/spindle and morphology of spindle capsules.While most spindles contained normal numbers of muscle fibers, most of these fibers were morphologically and histochemically abnormal. Alterations of ATPase activity occurred in all spindles, but were least severe in nerve-intact grafts. While fully differentiated nuclear bag and chain fibers were not observed in regenerated spindles, large, vesicular nuclei, similar to those of normal intrafusal fibers, were present in a small number of spindles in nerve-intact grafts. Sensory nerve terminations were observed only in those spindles that also contained the distinctive nuclei. This study suggests that a specific neurotrophic influence is necessary for regeneration of normal intrafusal muscle fibers and that this influence corresponds to the properly timed sensory neuron-muscle interaction which directs muscle spindle embryogenesis. However, the infrequent occurrence of characteristics unique to intrafusal muscle fibers indicates that reinnervation of regenerating muscle grafts by sensory neurons is inadequate and/or faulty.     

Target-derived BMP signaling limits sensory neuron number and the extent of peripheral innervation in vivo

The role of target-derived BMP signaling in development of sensory ganglia and the sensory innervation of the skin was examined in transgenic animals that overexpress either the BMP inhibitor noggin or BMP4 under the control of a keratin 14 (K14) promoter. Overexpression of noggin resulted in a significant increase in the number of neurons in the trigeminal and dorsal root ganglia. Conversely, overexpression of BMP4 resulted in a significant decrease in the number of dorsal root ganglion neurons. There was no significant change in proliferation of trigeminal ganglion neurons in the noggin transgenic animals, and neuron numbers did not undergo the normal developmental decrease between E12.5 and the adult, suggesting that programmed cell death was decreased in these animals. The increase in neuron numbers in the K14-noggin animals was followed by an extraordinary increase in the density of innervation in the skin and a marked change in the pattern of innervation by different types of fibers. Conversely, the density of innervation of the skin was decreased in the BMP4 overexpressing animals. Further Merkel cells and their innervation were increased in the K14-noggin mice and decreased in the K14-BMP4 mice. The changes in neuron numbers and the density of innervation were not accompanied by a change in the levels of neurotrophins in the skin. These findings indicate that the normal developmental decrease in neuron numbers in sensory ganglia depends upon BMP signaling, and that BMPs may limit both the final neuron number in sensory ganglia as well as the extent of innervation of targets. Coupled with prior observations, this suggests that BMP signaling may regulate the acquisition of dependence of neurons on neurotrophins for survival, as well as their dependence on target-derived neurotrophins for determining the density of innervation of the target.     

Morphological and morphometrical characteristics of the esophageal intrinsic nervous system in the golden hamster

Very little is known about esophageal innervation in the hamster. In the present study, we used protein gene product 9.5 (PGP 9.5) to determine immunohistochemically the architectural features of the enteric nervous system in the hamster esophagus. The myenteric plexus consisted of a loose and irregular network of ganglia and interganglionic nerve bundles. The density of the neurons in the myenteric plexus was relatively low (479 +/- 75/cm(2), n = 5), with a preferentially higher density in the upper cervical portion than other parts of the esophagus. Regional differences in the number of PGP 9.5-positive neurons and ganglia were observed. PGP 9.5-immunoreactive fibers in the ganglia often branched, giving rise to expanding nerve endings of laminar morphology resembling intraganglionic laminar endings described in rats and cats. Fine varicose fibers originating from the secondary plexus were occasionally observed near the motor endplates, suggested a dual innervation of the striated muscle. The submucosal plexus was free from ganglionated plexus. A regional difference in the submucosal nervous network was observed. The number of motor endplates in the inner muscle layer was higher than that in the outer muscle layer.     

Microanatomy of the abdominal stretch receptors of the crayfish (Astacus fluviatilis L.)

Microanatomical studies on the abdominal stretch receptor organs of the crayfish Astacus fluviatilis L. have been carried out in order to establish a basis for the physiological work that has been, and is being carried out on stretch receptors of various species of crayfish. Important differences have been found between these organs and those previously described by Alexandrowicz for the lobsters Homarus vulgaris and Palinurus vulgaris. With the aid of silver-impregnated preparations the relationship of sensory endings and muscle fibers has been shown as well as the pattern of the efferent innervation. The physiological significance of the histological findings has been discussed.     

Sprouting of aberrant neuropeptide Y-immunoreactive sympathetic nerves into neonatally denervated smooth muscle.

Sympathetic nerves normally project ipsilaterally to lateral cranial targets. Following unilateral superior cervical ganglionectomy in neonatal rats, however, neurons from the contralateral superior cervical ganglion sprout into the denervated region. In the present study we examined neuropeptide Y immunoreactivity (NPY-ir) of neurons comprising ipsilateral (control) and denervation-induced contralateral pathways to the superior tarsal smooth muscle of the eyelid. Fluoro-Gold injection of the control muscle retrogradely labelled 133 +/- 18 neurons in the ipsilateral superior cervical ganglion; of these, 21 +/- 3% displayed detectable NPY-ir. Fluoro-Gold injections of the reinnervated muscle labelled 20 +/- 4 neurons in the contralateral superior cervical ganglion, of which 85 +/- 3% contained detectable NPY-ir. Examination of the control tarsal muscle revealed DBH-ir noradrenergic nerves throughout the muscle and vasculature, while NPY-ir nerves were present primarily around blood vessels. In the reinnervated preparation, NPY-ir fibers innervated both blood vessels and tarsal muscle in a pattern similar to that of DBH-ir innervation. Acute excision of the remaining superior cervical ganglion eliminated all DBH-ir fibers bilaterally; NPY-ir was reduced markedly in the reinnervated preparations, though some fibers remained. We conclude that, following neonatal denervation, the tarsal muscle is reinnervated by a subpopulation of sympathetic neurons that differs in neuropeptide phenotype from that of the normal ipsilateral innervation.     

Distribution and anatomy of GABA-like immunoreactive neurons in the central and peripheral nervous system of the snail Helix pomatia

Gamma-aminobutyric acid (GABA)-like immunoreactive neurons were studied in the central and peripheral nervous system of Helix pomatia by applying immunocytochemistry on whole-mount preparations and serial paraffin sections. GABA-immunoreactive cell bodies were found in the buccal, cerebral and pedal ganglia, but only GABA-immunoreactive fibers were found in the viscero-parietal-pleural ganglion complex. The majority of GABA-immunoreactive cell bodies were located in the pedal ganglia but a few could be found in the buccal ganglia. Varicose GABA-ir fibers could be seen in the neuropil areas and in distinct areas of the cell body layer of the ganglia. The majority of GABA-ir axonal processes run into the connectives and commissures of the ganglia, indicating an important central integrative role of GABA-immunoreactive neurons. GABA may also have a peripheral role, since GABA-immunoreactive fibers could be demonstrated in peripheral nerves and the lips. Glutamate injection did not change the number or distribution of GABA-immunoreactive neurons, but induced GABA immunoreactivity in elements of the connective tissue ensheathing the muscle cells and fibers of the buccal musculature. This shows that GABA may be present in different non-neural tissues as a product of general metabolic pathways.