GABAergic neurons in the embryonic olfactory pit/vomeronasal organ: maintenance of functional GABAergic synapses in olfactory explants

In previous work, we showed a robust gamma-aminobutyric acid (GABAergic) synaptic input onto embryonic luteinizing hormone-releasing hormone (LHRH) neurons maintained in olfactory explants. In this study, we identify GABAergic neurons in olfactory pit (OP) of embryonic mice in vivo and study, using patch-pipet whole-cell current and voltage clamp techniques, synaptic interactions of these neurons in explant cultures. In vivo, glutamate decarboxylase (GAD, the enzyme which synthesizes GABA) mRNA was first detected in nasal regions on Embryonic Day (E) 11.5. From E12.5 to E13.5, robust GAD expression was localized to cells primarily in the ventral aspect of the OP. GAD mRNA was not detected over dorsally located cells in olfactory sensory or respiratory epithelium. In addition, GAD mRNA was not observed in cells along olfactory axons. GAD mRNA was dramatically reduced in the OP/vomeronasal organ by E16.5. Using antibodies against both GABA and GAD, immunopositive axonal-like tracts were detected in the nasal septum on E12.5. GABAergic staining decreased by E13.5. To examine synaptic interactions of these GABAergic cells, embryonic olfactory explants were generated and maintained in serum-free media. As explants spread, neuron-like cells migrated into the periphery, sometimes forming ganglion-like clusters. Cells were recorded, marked intracellularly with Lucifer Yellow and post-fixation, immunocytochemically examined. Forty-six cells, typically multipolar, were GABAergic, had resting potentials around -50 mV, and exhibited spontaneous action potentials which were generated by spontaneous depolarizing GABAergic (GABAA) synaptic activity. OP neurons depolarized in response to GABA by increasing Cl- conductance. The biophysical properties of OP-derived GABAergic neurons were distinct from those reported for olfactory receptor neurons but similar to embryonic LHRH neurons. However, unlike LHRH neurons, GABAergic neurons did not migrate large distances in olfactory explants or appear to leave the olfactory pit in vivo.     

Biphasic response of spinal GABAergic neurons after a lumbar rhizotomy in the adult rat

The expression of gamma-aminobutyric acid (GABA) and of the isoforms of the enzyme involved in its synthesis, glutamic acid decarboxylase (GAD), is modified in several rat brain structures in different injury models. The aim of the present work was to determine whether such plasticity of the GABAergic system also occurred in the deafferented adult rat spinal cord, a model where a major reorganization of neural circuits takes place. GABAergic expression following unilateral dorsal rhizotomy was studied by means of non-radioactive in situ hybridization to detect GAD67 mRNA and by immunohistochemistry to detect GAD67 protein and GABA. Three days following rhizotomy the number of GAD67 mRNA-expressing neurons was decreased in the superficial layers of the deafferented horn, while GABA immunostaining of axonal fibres located in this region was highly increased. Seven days after lesion, on the other hand, many GAD67 mRNA-expression neurons were bilaterally detected in deep dorsal and ventral layers, this expression being correlated with the increased detection of GAD67 immunostained somata and with the reduction of GABA immunostaining of axons. GABA immunostaining was frequently found to be associated with reactive astrocytes that exhibited intense immunostaining for glial fibrillary acidic protein (GFAP) but remained GAD67 negative. These results indicate that degeneration of afferent terminals induces a biphasic response of GABAergic spinal neurons located in the dorsal horn and show that many spinal neurons located in deeper regions re-express GAD67, suggesting a possible participation of the local GABAergic system in the reorganization of disturbed spinal networks.     

Sequence and expression of glutamic acid decarboxylase isoforms in the developing zebrafish

We describe the isolation two glutamic acid decarboxylase (GAD) cDNAs from zebrafish with over 84% identity to human GAD65 and GAD67. In situ hybridization studies revealed that both GAD65 and GAD67 were expressed in the early zebrafish embryo during the period of axonogenesis, suggesting a role for GABA prior to synapse formation. Both GAD genes were detected in the telencephalon, in the nucleus of the medial longitudinal fasciculus in the midbrain, and at the border regions of the rhombomeres in the rostral hindbrain. In the caudal hindbrain, only GAD67 was detected (in neurons with large-caliber axons). In the spinal cord, both GAD genes were detected in dorsal longitudinal neurons, commissural secondary ascending neurons, ventral longitudinal neurons, and Kolmer-Agduhr neurons. Immunohistochemistry for gamma-aminobutyric acid (GABA) revealed that GABA is produced at all sites of GAD expression, including the novel cells in the caudal hindbrain. These results are discussed in the context of the hindbrain circuitry that supports the escape response. We conclude that fish, like mammals, have two GAD genes. The zebrafish GAD65 and GAD67 are present in identified neurons in the forebrain, midbrain, hindbrain, and spinal cord, and they catalyze the production of GABA in the developing embryo.     

The activation of cannabinoid receptors in striatonigral GABAergic neurons inhibited GABA uptake

Cannabinoid receptors (CNRs) in basal ganglia are located on striatal efferent neurons which are gamma-aminobutiric acid (GABA)-containing neurons. Recently, we have demonstrated that CN-induced motor inhibition is reversed by GABA-B, but not GABA-A, receptor antagonists, presumably indicating that the activation of CNRs in striatal outflow nuclei, mainly in the substantia nigra, should be followed by an increase of GABA concentrations into the synaptic cleft of GABA-B receptor synapses. The present study was designed to examine whether this was originated by increasing GABA synthesis and/or release or by decreasing GABA uptake. We analyzed: (i) GABA synthesis, by measuring the activity of glutamic acid decarboxylase (GAD) and GABA contents in brain regions that contain striatonigral GABAergic neurons, after in vivo administration of CNs and/or the CNR antagonist SR141716; (ii) [3H]GABA release in vitro in the presence or the absence of a synthetic CN agonist, HU-210, by using perifusion of small fragments of substantia nigra; and (iii) [3H]GABA uptake in vitro in the presence or the absence of WIN-55,212-2, by using synaptosomes obtained from either globus pallidus or substantia nigra. Results were as follows. Delta9-tetrahydrocannabinol (delta9-THC) and HU-210, did not alter neither GAD activity nor GABA contents in both the striatum and the ventral midbrain at any of the two times tested, thus suggesting that CNs apparently failed to change GABA synthesis in striatonigral GABAergic neurons. A similar lack of effect of HU-210 on in vitro [3H]GABA release, both basal and K+-evoked, was seen when this CN was added to perifused substantia nigra fragments, also suggesting no changes at the level of GABA release. However, when synaptosome preparations obtained from the substantia nigra were incubated in the presence of WIN-55,212-2, a decrease in [3H]GABA uptake could be measured. This lowering effect was specific of striatonigral GABAergic neurons since it was not observed in synaptosome preparations obtained from the globus pallidus. In summary, the activation of CNRs located on striatonigral GABAergic neurons, which primarily access to GABA-B receptor synapses, was accompanied by a reduction in neurotransmitter uptake, thus prolonging the presence of GABA into the synaptic cleft. This mechanism might underly the CN-induced motor inhibition through the potentiation of the inhibitory effect of GABA on neuronal activity, in particular of nigrostriatal dopaminergic neurons.     

Origin, course, and laterality of spinocerebellar axons in the North American opossum, Didelphis virginiana

Inhibition of neurons containing gamma-aminobutyric acid (GABA) may underlie some of the excitatory effects of opioids in the central nervous system (CNS). In the present study, we examined the relationship of the cloned mu- and delta-opioid receptors (MOR1 and DOR1, respectively) to GABAergic neurons in brain and spinal cord. This was done by combining immunofluorescent staining for MOR1 or DOR1 with that for GABA or glutamic acid decarboxylase (GAD); fluorescent retrograde tract-tracing was used in some cases to identify neurons with particular projections. In rats, cells double labeled for GABA and MOR1 were observed in layers II-VI of the parietal cortex and in layers II-IV of the piriform cortex. In the hippocampus, double labeling was observed in the dentate gyrus and in regions CA1 and CA3. Double labeling was very prominent in the striatum and in the reticular nucleus of the thalamus; it was also observed in other portions of the diencephalon. However, double labeling for GABA and MOR1 was never observed in the cerebellar cortex. Cells double labeled for GABA and MOR1 were common in the periaqueductal gray (PAG) and the medial rostral ventral medulla (RVM) of both rats and monkeys, suggesting that involvement of GABAergic neurons with supraspinal opioid antinociception may extend to primates. In the RVM of rats, many of those double-labeled neurons were retrogradely labeled from the dorsal spinal cord. In contrast, double-labeled neurons in the PAG were almost never retrogradely labeled from the RVM. No unequivocal examples of double labeling for DOR1 and GAD were found in any region of the CNS that we examined in either rats or monkeys. However, GABAergic neurons were often apposed by DOR1 immunoreactive varicosities. Our findings suggest that activation of mu-opioid receptors directly modulates the activity of GABAergic neurons throughout the CNS, including neurons involved in the supraspinal component of opioid analgesia. In contrast, delta-opioid receptors appear to be positioned to modulate the activity of GABAergic neurons indirectly.     

The vesicular GABA transporter, VGAT, localizes to synaptic vesicles in sets of glycinergic as well as GABAergic neurons

A transporter thought to mediate accumulation of GABA into synaptic vesicles has recently been cloned (McIntire et al., 1997). This vesicular GABA transporter (VGAT), the first vesicular amino acid transporter to be molecularly identified, differs in structure from previously cloned vesicular neurotransmitter transporters and defines a novel gene family. Here we use antibodies specific for N- and C-terminal epitopes of VGAT to localize the protein in the rat CNS. VGAT is highly concentrated in the nerve endings of GABAergic neurons in the brain and spinal cord but also in glycinergic nerve endings. In contrast, hippocampal mossy fiber boutons, which although glutamatergic are known to contain GABA, lack VGAT immunoreactivity. Post-embedding immunogold quantification shows that the protein specifically associates with synaptic vesicles. Triple labeling for VGAT, GABA, and glycine in the lateral oliva superior revealed a higher expression of VGAT in nerve endings rich in GABA, with or without glycine, than in others rich in glycine only. Although the great majority of nerve terminals containing GABA or glycine are immunopositive for VGAT, subpopulations of nerve endings rich in GABA or glycine appear to lack the protein. Additional vesicular transporters or alternative modes of release may therefore contribute to the inhibitory neurotransmission mediated by these two amino acids.     

lin-14 regulates the timing of synaptic remodelling in Caenorhabditis elegans

In the nematode Caenorhabditis elegans six GABAergic motor neurons, known as DDs, remodel their patterns of synaptic connectivity during larval development. DD remodelling involves a complete reversal of the direction of information flow within nerve processes without marked changes in process morphology. We used a marker localized in vivo to DD presynaptic zones to analyse how the timing of DD remodelling is controlled. In wild-type animals, DDs remodel their synaptic outputs within a 3-5-hour period at the end of the first larval stage. We show that the heterochronic gene lin-14, which controls the timing of stage-specific cell lineages, regulates the timing of DD synaptic output remodelling. In lin-14 loss-of-function mutants, DDs remodel precociously. The degree of precocious remodelling is correlated with the level of lin-14 activity. Expression of lin-14(+) in the DDs of lin-14-null mutants rescues the precocious remodelling, indicating that lin-14 can act cell-autonomously. Consistent with this hypothesis, LIN-14 protein levels decrease in the DDs before remodelling. Our observations reveal a role of heterochronic genes in non-dividing cells, and provide an example of cell-autonomous respecification of neuronal connectivity.     

Genes that guide growth cones along the C. elegans ventral nerve cord

During nervous system development, growth cone pioneering and fasciculation contribute to nerve bundle structure. Pioneer growth cones initially navigate along neuroglia to establish an axon scaffold that guides later extending growth cones. In C. elegans, the growth cone of the PVPR neuron pioneers the left ventral nerve cord bundle, providing a path for the embryonic extensions of the PVQL and AVKR growth cones. Later during larval development, the HSNL growth cone follows cues in the left ventral nerve cord bundle provided by the PVPR and PVQL axons. Here we show that mutations in the genes enu-1, fax-1, unc-3, unc-30, unc-42 and unc-115 disrupt pathfinding of growth cones along the left ventral nerve cord bundle. Our results indicate that unc-3 and unc-30 function in ventral nerve cord pioneering and that enu-1, fax-1, unc-42 and unc-115 function in recognition of the PVPR and PVQL axons by the AVKR and HSNL growth cones.     

Novel synthesis and release of GABA in cerebellar granule cell cultures after infection with defective herpes simplex virus vectors expressing glutamic acid decarboxylase

The inhibitory amino acid neurotransmitter gamma-aminobutyric acid (GABA) is synthesized from glutamate in a single step by the enzyme glutamatic acid decarboxylase (GAD). We sought to determine whether viral vectors containing GAD cDNA could be used to enhance synthesis and stimulation-evoked release of GABA in cultures of CNS neurons. For this purpose, we generated double-cassette defective herpes simplex virus (HSV) vectors that expressed one of the two GAD isoforms (GAD65 or GAD67), and Escherichia coli LacZ. Infection of cerebellar granule cell (CGC) cultures with vectors containing GAD cDNA resulted in a significant increase in isoform-specific expression of GAD, synthesis of GABA, and stimulation-evoked GABA release. GAD65 and GAD67 vector-infected neurons exhibited a comparable profile of GABA levels, synthesis and release, as well as GAD protein distribution. In CGCs cultured for 6 days in vitro (DIV), GABA synthesized after vector-derived GAD expression was released by treatment with glutamate or veratridine, but only in a Ca2+-independent fashion. In more mature (10 DIV) cultures, both Ca2+-dependent, K+ depolarization-induced, as well as Ca2+-independent, veratridine-induced, GABA release was significantly enhanced by GAD vector infection. Treatment of CGCs with kainic acid, which destroys most of the GABAergic neurons (<1% remaining), did not prevent vector-derived expression of GAD nor synthesis of GABA. This suggests that defective HSV vector-derived GAD expression can be used to increase GABA synthesis and release in CNS tissue, even in the relative absence of GABAergic neurons. The use of such GAD vectors in the CNS has potential therapeutic value in neurologic disorders such as epilepsy, chronic pain, Parkinson's and Huntington's disease.     

SVOP, an evolutionarily conserved synaptic vesicle protein, suggests novel transport functions of synaptic vesicles

We describe a novel synaptic vesicle protein called SVOP that is distantly related to the synaptic vesicle proteins SV2A, SV2B, and SV2C (20-22% sequence identity). Both SVOP and SV2 contain 12 transmembrane regions. However, SV2 is highly glycosylated, whereas SVOP is not. Databank searches revealed that closely related homologs of SVOP are present in Caenorhabditis elegans and Drosophila (48% sequence identity), suggesting that SVOP is evolutionarily ancient. In contrast, no invertebrate orthologs of SV2 were detected. The sequences of SVOP and SV2 exhibit homology with transport proteins, in particular with mammalian organic cation and anion transporters. SVOP and SV2 are more distantly related to eukaryotic and bacterial phosphate, sugar, and organic acid transporters. SVOP is expressed at detectable levels only in brain and endocrine cells where it is primarily localized to synaptic vesicles and microvesicles. SVOP is present in all brain regions, with particularly high levels in large pyramidal neurons of the cerebral cortex. Immunocytochemical staining of adjacent rat brain sections for SVOP and SV2 demonstrated that SVOP and SV2 are probably coexpressed in most neurons. Although the functions of SV2 and SVOP remain obscure, the evolutionary conservation of SVOP, its hydrophobic nature, and its homology to transporters strongly support a role in the uptake of a novel, as yet unidentified component of synaptic vesicles. Thus synaptic vesicles contain two classes of abundant proteins with 12 transmembrane regions that are related to transporters, nonglycosylated SVOP and highly glycosylated SV2, suggesting that the transport functions of synaptic vesicles may be more complex than currently envisioned.     

Strength of synaptic transmission at neuromuscular junctions of crustaceans and insects in relation to calcium entry

Crustacean and insect neuromuscular junctions typically include numerous small synapses, each of which usually contains one or more active zones, which possess voltage-sensitive calcium channels and are specialized for release of synaptic vesicles. Strength of transmission (the number of quantal units released per synapse by a nerve impulse) varies greatly among different endings of individual neurons, and from one neuron to another. Ultrastructural features of synapses account for some of the physiological differences at endings of individual neurons. The nerve terminals that release more neurotransmitter per impulse have a higher incidence of synapses with more than one active zone, and this is correlated with more calcium build-up during stimulation. However, comparison of synaptic structure in neurons with different physiological phenotypes indicates no major differences in structure that could account for their different levels of neurotransmitter release per impulse, and release per synapse differs among neurons despite similar calcium build-up in their terminals during stimulation. The evidence indicates differences in calcium sensitivity of the release process among neurons as an aspect of physiological specialization.     

A powerful GABA(B) receptor-mediated inhibition of GABAergic neurons in the arcuate nucleus

We combined histofluorescence with in situ hybridization to identify GABAergic neurons in the arcuate nucleus (ARC) following electrophysiological recordings, using GAD65 as a marker. Intracellular recordings were made in hypothalamic slices prepared from ovariectomized guinea pigs. Over 90% of ARC neurons tested with the GABA(B) receptor agonist baclofen responded with a membrane hyperpolarization or an outward current. The hyperpolarization was dose-dependent, and the GABA(B) receptor antagonist CGP 35,348 produced a rightward shift in the agonist dose-response curve. Agonist potency was lower, and the efficacy greater, in GAD-positive neurons. The use of this novel technique for identifying GABAergic neurons thus reveals differences in the pharmacodynamics of GABA(B) receptor activation GABAergic and non-GABAergic ARC neurons.     

Neurotrophic factors increase neuregulin expression in embryonic ventral spinal cord neurons

Neuregulins (NRGs) are expressed in spinal cord motor neurons and accumulate at the neuromuscular junction where they may increase the synthesis of postsynaptic acetylcholine receptors and voltage-gated sodium channels. We demonstrate here that NRG expression is selectively increased in rat ventral spinal cord neurons at approximately the time that nerve-muscle synapses first form. A rapid increase in NRG mRNA and protein expression was induced in vitro in cultured rat spinal motor neurons by brain-derived neurotrophic factor, neurotrophin-3, neurotrophin-4, or glial-cell-line-derived neurotrophic factor. Agrin expression was not affected by these factors over the same time course. Brain-derived neurotrophic factor, but not neurotrophin-3, selectively regulated immunoglobulin domain-containing splice variants of NRG, which are likely to be important for binding to the synaptic basal lamina. Regulation of NRG expression in motor neurons by muscle-derived neurotrophic factors may represent one portion of a reciprocal, regulatory loop that promotes neuromuscular synapse development.     

Retinal ganglion cell depletion alters the phenotypic expression of GABA and GAD in the rat retina

We have looked at the phenotypic expression of gamma-aminobutyric acid (GABA) and the two isoforms of its synthetic enzyme [glutamic acid decarboxylase (GAD)-65 and -67] in adult rat retinas that had the superior colliculus, pretectum and optic tract lesioned unilaterally at birth. It has been shown previously that this type of manipulation induces retrograde degeneration of retinal ganglion cells presumably without affecting other intraretinal neurons. We present evidence that GABAergic amacrine cells are affected by such manipulation. The number of cells immunoreactive for GABA, GAD-65 and GAD-67 decreased in the inner nuclear layer. In the retinal ganglion cell layer, however, the number of GABA- and GAD-65-labelled cells increased, while the number of GAD-67-labelled cells did not change. Biochemical assay showed that overall GAD activity was not altered in retinas of lesioned animals. Our results support the notion that, while neonatal lesion reorganizes the expression of GABA and GAD in the retina, enzyme activity is maintained within normal levels.     

Ultrastructural organization of GABA in the rabbit superior colliculus revealed by quantitative postembedding immunocytochemistry

We have studied the organization of gamma-aminobutyric acid (GABA)ergic profiles in the superior colliculus of the rabbit to determine whether the synaptic types found in cat and monkey also exist in a mammalian species whose visual system has a different organization. Ultrastructure of GABAergic profiles was examined by use of a polyclonal antibody to GABA and quantitative postembedding immunocytochemistry. Three distinct types of vesicle-containing profiles were labeled by the GABA antibody in the rabbit superior colliculus. One type was a putative presynaptic dendrite (PSD profile) that received synaptic input from other profiles and contained pleomorphic synaptic vesicles scattered throughout the profile. These PSD profiles frequently received retinal input and formed dendrodendritic synapses. A second type of profile was a large caliber dendrite, often horizontal in orientation (H profile), that had one or more discrete clusters of pleomorphic synaptic vesicles at sites of synaptic contact with conventional dendrites. These H profiles received few synaptic contacts. A third profile type was a putative axon terminal (F profile) with smaller, more flattened synaptic vesicles that densely and uniformly filled the profile. Quantitative analysis of gold particle density revealed that F profiles had a significantly higher gold particle density (14.3/microns 2) than did PSD or H profiles (10.4 and 10.2/microns 2), suggesting that GABAergic profile types contain different concentrations of GABA. The vesicle density of these profile types also differed, but no obvious relationship between vesicle and particle distributions was observed. We conclude that the profiles labeled by GABA in rabbit superior colliculus are similar to those in cat and monkey and must represent a phylogenetically conserved organization common to many mammals, and that particle density analysis of postembedding immunocytochemistry can distinguish different GABAergic profile types.     

Sources of GABAergic input to the inferior colliculus of the rat

We have studied the GABAergic projections to the inferior colliculus (IC) of the rat by combining the retrograde transport of horseradish peroxidase (HRP) and immunohistochemistry for gamma-amino butyric acid (GABA). Medium-sized (0.06-0.14 microliter) HRP injections were made in the ventral part of the central nucleus (CNIC), in the dorsal part of the CNIC, in the dorsal cortex (DCIC), and in the external cortex (ECIC) of the IC. Single HRP-labeled and double (HRP-GABA)-labeled neurons were systematically counted in all brainstem auditory nuclei. Our results revealed that the IC receives GABAergic afferent connections from ipsi- and contralateral brainstem auditory nuclei. Most of the contralateral GABAergic input originates in the IC and the dorsal nucleus of the lateral lemniscus (DNLL). The dorsal region of the IC (DCIC and dorsal part of the CNIC) receives connections mostly from its homonimous contralateral region, and the ventral region from the contralateral DNLL. The commissural GABAergic projections originate in a morphologically heterogeneous neuronal population that includes small to medium-sized round and fusiform neurons as well as large and giant neurons. Quantitatively, the ipsilateral ventral nucleus of the lateral lemniscus is the most important source of GABAergic input to the CNIC. In the superior olivary complex, a smaller number of neurons, which lie mainly in the periolivary nuclei, display double labeling. In the contralateral cochlear nuclei, only a few of the retrogradely labeled neurons were GABA immunoreactive. These findings give us more information about the role of GABA in the auditory system, indicating that inhibitory inputs from different ipsi- and contralateral, mono- and binaural auditory brainstem centers converge in the IC.     

Distribution of GABA immunoreactivity in the central and peripheral nervous system of amphioxus (Branchiostoma lanceolatum Pallas)

On the basis of labeling with an anti-gamma-aminobutyric acid (GABA) antibody, we report for the first time the presence and distribution of GABA-immunoreactive cells in the central and peripheral nervous system of amphioxus. In the nerve cord, there is a large dorsorostral group of cerebrospinal-fluid-contacting (CSFc) cells at the caudal end of the brain vesicle that gives rise to a large ventral commissure and neuropilar region. In the middle and caudal region of the brain, numerous commissural and CSFc neurons are situated below the region of large dorsal cells. In the spinal cord, several types of GABA-immunoreactive neurons of different size, appearance, and distribution were observed. In the dorsalmost region, very small commissural cells are scattered regularly along the cord. More ventrally in the cord, GABAergic neurons, both of commissural and CSFc cell types, form segmental groups, but scattered cells are observed throughout. These cells give rise to dense longitudinal fascicles of GABAergic fibers and to scattered commissural fibers. The caudal ampulla lacks GABAergic cells and fibers. Some of the fibers of the most rostral and caudal peripheral (sensory) nerves, as well as some sensory cells of the rostral and caudal epidermis, are GABA immunoreactive. The significance of these results for the understanding of the evolution of GABAergic systems of vertebrates is discussed.     

BEN/SC1/DM-GRASP expression during neuromuscular development: a cell adhesion molecule regulated by innervation

BEN/SC1/DM-GRASP is a cell adhesion molecule belonging to the Ig superfamily that is transiently expressed during avian embryogenesis in a variety of cell types, including the motoneurons of the spinal cord. We have investigated the pattern of BEN expression during neuromuscular development of the chick. We show that both motoneurons and their target myoblasts express BEN during early embryonic development and that the protein becomes restricted at neuromuscular contacts as soon as postsynaptic acetylcholine receptor clusters are observed in muscle fibers. Muscle cells grown in vitro express and maintain BEN expression even when they fuse and give rise to mature myotubes. When embryos are deprived of innervation by neural tube ablation, BEN expression is observed in muscle fibers, whereas, in control, the protein is already restricted at neuromuscular synaptic sites. These results demonstrate that all myogenic cells intrinsically express BEN and maintain the protein in the absence of innervation. Conversely, when neurons are added to myogenic cultures, BEN is rapidly downregulated in muscle cells, demonstrating that innervation controls the restricted pattern of BEN expression seen in innervated muscles. After nerve section in postnatal muscles, BEN protein becomes again widely spread over muscle fibers. When denervated muscles are allowed to be reinnervated, the protein is reexpressed in regenerating motor axons, and reinnervation of synaptic sites leads to the concentration of BEN at neuromuscular junctions. Our results suggest that BEN cell adhesion molecule acts both in the formation of neuromuscular contacts during development and in the events leading to muscle reinnervation.     

GABAergic and pallidal terminals in the thalamic reticular nucleus of squirrel monkeys

The ultrastructure of synaptic terminals from the external segment of the globus pallidus and of other synaptic terminals positive for gamma-aminobutyric acid (GABA) was examined in the thalamic reticular nucleus (TRN) of squirrel monkeys. Two GABA-positive terminals types were commonly encountered within the TRN neuropil. The most common type of GABAergic terminals (F terminals) are filled with dispersed pleomorphic synaptic vesicles and clusters of mitochondria. These terminals establish multiple symmetric synapses upon the somata and dendrites of TRN neurons. The external pallidal terminals, labeled with WGA-HRP, arise from thinly myelinated axons and correspond to the medium to large F terminals. A less prevalent population of smaller GABAergic synaptic profiles was also identified. The synaptic profiles in this second group contain considerably fewer pleomorphic synaptic vesicles in small irregular clusters and fewer mitochondria, establish symmetric synapses, are postsynaptic to other axonal terminals, are presynaptic to dendrites and soma, and are unlabeled following pallidal injections of WGA-HRP.