Synaptic dimorphism in Onychophoran cephalic ganglia

The taxonomic location of the Onychophora has been controversial because of their phenotypic and genotypic characteristics, related to both annelids and arthropods. We analyzed the ultrastructure of the neurons and their synapses in the cephalic ganglion of a poorly known invertebrate, the velvet worm Peripatus sedgwicki, from the mountainous region of El Valle, Mérida, Venezuela. Cephalic ganglia were dissected, fixed and processed for transmission electron microscopy. The animal has a high degree of neurobiological development, as evidenced by the presence of asymmetric (excitatory) and symmetric (inhibitory) synapses, as well as the existence of glial cell processes in a wide neuropile zone. The postsynaptic terminals were seen to contain subsynaptic cisterns formed by membranes of smooth endoplasmic reticulum beneath the postsynaptic density, whereas the presynaptic terminal showed numerous electron transparent synaptic vesicles. From the neurophylogenetic perspectives, the ultrastructural characteristics of the central nervous tissue of the Onychophora show important evolutionary acquirements, such as the presence of both excitatory and inhibitory synapses, indicating functional synaptic transmission, and the appearance of mature glial cells.     

Differential ultrastructure of synaptic terminals on ventral longitudinal abdominal muscles in Drosophila larvae

The innervation of ventral longitudinal abdominal muscles (muscles 6, 7, 12, and 13) of third-instar Drosophila larvae was investigated with Nomarski, confocal, and electron microscopy to define the ultrastructural features of synapse-bearing terminals. As shown by previous workers, muscles 6 and 7 receive in most abdominal segments “Type I” endings, which are restricted in distribution and possess relatively prominent periodic terminal enlargements (“boutons”); whereas muscles 12 and 13 have in addition “Type II” terminals, which are more widely distributed and have smaller “boutons.” Serial sectioning of the Type I innervation of muscles 6 and 7 showed that two axons with distinctive endings contribute to it. One axon (termed Axon 1) has somewhat larger boutons, containing numerous synapses and presynaptic dense bodies (putative active zones for transmitter release). This axon also has more numerous intraterminal mitochondria, and a profuse subsynaptic reticulum around or under the synaptic boutons. The second axon (Axon 2) provides somewhat smaller boutons, with fewer synapses and dense bodies per bouton, fewer intraterminal mitochondria, and less-developed subsynaptic reticulum. Both axons contain clear synaptic vesicles, with occasional large dense vesicles. Approximately 800 synapses are provided by Axon 1 to muscles 6 and 7, and approximately 250 synapses are provided by Axon 2. In muscles 12 and 13, endings with predominantly clear synaptic vesicles, generally similar to the Type I endings of muscles 6 and 7, were found, along with another type of ending containing predominantly dense-cored vesicles, with small clusters of clear synaptic vesicles. This second type of ending was found most frequently in muscle 12, and probably corresponds to a subset of the “Type II” endings seen in the light microscope. Type I endings are thought to generate the ?fast’? and ?slow’? junctional potentials seen in electrophysiological recordings, whereas the physiological actions of Type II endings are presently not known. © 1993 John Wiley & Sons, Inc.     

Ultrastructure of orexin-1 receptor immunoreactivities in the spinal cord dorsal horn

The ultrastructural properties of orexin 1-receptor-like immunoreactive (OX1R-LI) neurons in the dorsal horn of the rat spinal cord were examined using light and electron microscopy techniques. At the light microscopy level, the most heavily immunostained OX1R-LI neurons were found in the ventral horn of the spinal cord, while some immunostained profiles, including nerve fibers and small neurons, were also found in the dorsal horn. At the electron microscopy level, OX1R-LI perikarya were identified containing numerous dense-cored vesicles which were more heavily immunostained than any other organelles. Similar vesicles were also found within the axon terminals of the OX1R-LI neurons. The perikarya and dendrites of some of the OX1R-LI neurons could be seen receiving synapses from immunonegative axon terminals. These synapses were found mostly asymmetric in shape. Occasionally, some OX1R-LI axon terminals were found making synapses on dendrites that were OX1R-LI in some cases and immunonegative in others. The synapses made by OX1R-LI axon terminals were found both asymmetric and symmetric in appearance. The results provide solid morphological evidence that OX1R is transported in the dense-cored vesicles from the perikarya to axon terminals and that OX1R-LI neurons in the dorsal horn of the spinal cord have complex synaptic relationships both with other OX1R-LI neurons as well as other neuron types.     

Long-Term Culture of Astrocytes Attenuates the Readily Releasable Pool of Synaptic Vesicles

The astrocyte is a major glial cell type of the brain, and plays key roles in the formation, maturation, stabilization and elimination of synapses. Thus, changes in astrocyte condition and age can influence information processing at synapses. However, whether and how aging astrocytes affect synaptic function and maturation have not yet been thoroughly investigated. Here, we show the effects of prolonged culture on the ability of astrocytes to induce synapse formation and to modify synaptic transmission, using cultured autaptic neurons. By 9 weeks in culture, astrocytes derived from the mouse cerebral cortex demonstrated increases in β-galactosidase activity and glial fibrillary acidic protein (GFAP) expression, both of which are characteristic of aging and glial activation in vitro. Autaptic hippocampal neurons plated on these aging astrocytes showed a smaller amount of evoked release of the excitatory neurotransmitter glutamate, and a lower frequency of miniature release of glutamate, both of which were attributable to a reduction in the pool of readily releasable synaptic vesicles. Other features of synaptogenesis and synaptic transmission were retained, for example the ability to induce structural synapses, the presynaptic release probability, the fraction of functional presynaptic nerve terminals, and the ability to recruit functional AMPA and NMDA glutamate receptors to synapses. Thus the presence of aging astrocytes affects the efficiency of synaptic transmission. Given that the pool of readily releasable vesicles is also small at immature synapses, our results are consistent with astrocytic aging leading to retarded synapse maturation.     

A survey of the cytology and synaptic organization of the insular trigeminal-cuneatus lateralis nucleus in the rat, including an identification of spinal afferent inputs

The cytology and synaptic organization of the insular trigeminal-cuneatus lateralis (iV-Cul) nucleus was examined in the rat. In addition, the ultrastructural morphology and synaptic connectivity of anterogradely labeled spinal afferent axons terminating in iV-Cul were examined following injection of horseradish peroxidase (HRP) into the cervical spinal cord. The uniformity of the ultrastructural features of iV-Cul neurons supports the presence of a homogeneous neuronal population. The most prominent feature of the iV-Cul neuropil is the presence of numerous interdigitating astrocytic processes, which extensively isolate neuronal somata and processes. iV-Cul contains a heterogeneous population of axonal endings that can be separated into three categories, depending upon whether they contain predominantly spherical-shaped agranular synaptic vesicles (R endings), predominantly pleomorphic-shaped agranular synaptic vesicles (P endings), or a heterogeneous population of dense-core vesicles (DC endings). The R endings represent the majority of axonal endings in iV-Cul and establish asymmetrical axodendritic and axospinous synaptic contacts, primarily along the distal portions of the dendritic tree. P endings establish symmetrical axosomatic, axodendritic, and axospinous synaptic contacts and exhibit a more generalized distribution along the somadendritic tree. DC terminals establish asymmetrical axodendritic synaptic contacts with distal dendritic processes and are the least frequently observed endings in the iV-Cul neuropil. Numerous synaptic glomeruli, exhibiting a single large central R bouton that establishes multiple axodendritic or axospinous synapses, characterize the iV-Cul neuropil. Axoaxonic synapses are conspicuously absent from the iV-Cul neuropil and glomeruli. The anterograde HRP labeling of spinal afferent axons that terminate in iV-Cul indicates that the terminals along these fibers are of the R type and that they are engaged predominantly in synaptic glomeruli. The results of this study indicate that the synaptic organization of iV-Cul is distinctly different from that of neighboring somatosensory nuclei, and supports the recent suggestion that this nucleus should be considered a separate precerebellar spinal relay nucleus in the lateral medulla.     

A Survey of the Cytology and Synaptic Organization of the Insular Trigeminal—Cuneatus Lateralis Nucleus in the Rat,Including an Identification of Spinal Afferent Inputs

The cytology and synaptic organization of the insular trigeminal—cuneatus lateralis (iV-Cul) nucleus was examined in the rat. In addition, the ultrastructural morphology and synaptic connectivity of anterogradely labeled spinal afferent axons terminating in iV-Cul were examined following injection of horseradish peroxidase (HRP) into the cervical spinal cord. The uniformity of the ultrastructural features of iV-Cul neurons supports the presence of a homogeneous neuronal population. The most prominent feature of the iV-Cul neuropil is the presence of numerous interdigitating astrocytic processes, which extensively isolate neuronal somata and processes. iV-Cul contains a heterogeneous population of axonal endings that can be separated into three categories, depending upon whether they contain predominantly spherical-shaped agranular synaptic vesicles (R endings), predominantly pleomorphic-shaped agranular synaptic vesicles (P endings), or a heterogeneous population of dense-core vesicles (DC endings). The R endings represent the majority of axonal endings in iV-Cul and establish asymmetrical axodendritic and axospinous synaptic contacts, primarily along the distal portions of the dendritic tree. P endings establish symmetrical axosomatic, axodendritic, and axospinous synaptic contacts and exhibit a more generalized distribution along the somadendritic tree. DC terminals establish asymmetrical axodendritic synaptic contacts with distal dendritic processes and are the least frequently observed endings in the iV-Cul neuropil. Numerous synaptic glomeruli, exhibiting a single large central R bouton that establishes multiple axodendritic or axospinous synapses, characterize the iV-Cul neuropil. Axoaxonic synapses are conspicuously absent from the iV-Cul neuropil and glomeruli. The anterograde HRP labeling of spinal afferent axons that terminate in iV-Cul indicates that the terminals along these fibers are of the R type and that they are engaged predominantly in synaptic glomeruli. The results of this study indicate that the synaptic organization of iV-Cul is distinctly different from that of neighboring somatosensory nuclei, and supports the recent suggestion that this nucleus should be considered a separate precerebellar spinal relay nucleus in the lateral medulla.     

Electron microscopic study on the innervation of the pancreas of the domestic fowl

Summary The innervation of the pancreas of the domestic fowl was studied electron microscopically. The extrapancreatic nerve is composed mostly of unmyelinated nerve fibers with a smaller component of myelinated nerve fibers. The latter are not found in the parenchyma. The pancreas contains ganglion cells in the interlobular connective tissue. The unmyelinated nerve fibers branch off along blood vessels. Their synaptic terminals contact with the exocrine and endocrine tissues. The synaptic terminals can be divided into four types based on a combination of three kinds of synaptic vesicles. Type I synaptic terminals contain only small clear vesicles about 600 Å in diameter. Type II terminals are characterized by small clear and large dense core vesicles 1,000 Å in diameter. Type III terminals contain small clear vesicles and small dense core vesicles 500 Å in diameter. Type IV terminals are characterized by small and large dense core vesicles. The exocrine tissue receives a richer nervous supply than the endocrine tissue. Type II and IV terminals are distributed in the acinus, and they contact A and D cells of the islets. B cells and pancreatic ducts are supplied mainly by Type II terminals, the blood vessels by Type IV terminals.This work was supported by a scientific research grant (No. 144017) and (No. 136031) from the Ministry of Education of Japan to Prof. M. Yasuda     

Excitatory Transmission in Insect Neuromuscular Systems

Many, but not all, visceral muscles in insects are innervatedby neurosecretory axons. The neurosecretory junctions with theheart muscle of the American cockroach, Periplaneta americana,show ultrastructural and electrophysiological evidence of chemicallytransmitting synapses, and cytochemical evidence for the presenceof monoamines. Electron microscopy of nerve terminals showsthat synaptic vesicles may be formed directly from electron-dense"neurosecretory" granules Neurotomy of motor axons to skeletal muscles in insects leadsto aggregation and clumping of synaptic vesicles after 48 hours.Treatment of in vitro nerve-muscle preparations with variousrespiratory poisons caused aggregation similar to that developedin neurotomized animals. This suggested that vesicle aggregationin both cases may have resulted from a decrease in availableadenosine triphosphate in the nerve terminal with subsequentalteration in the normal charge density which supports a repulsiveforce between the vesicles.     

Interneuronal synapses in the local ganglia of the cat urinary bladder.

The interneuronal synapses of the urinary bladder in the cat were studied by electron microscopy. The great majority of the fibres containing vesicles are found within the ganglia occurring in the trigonum area. Morphologically differentiated synaptic contacts could be observed on the surface of the local neurons and between the different nerve processes. The presynaptic terminals can be divided into three types based on a combination of synaptic vesicles. Type I terminals, presumably cholinergic synaptic terminals, contain only small clear vesicles of 40-50 nm in diameter. Type II terminals, presumably adrenergic terminals, are characterized by small granulated vesicles of 40-60 nm in diameter. Type III terminals, probably of local origin, contain a variable number of large granulated vesicles of 80-140 nm in diameter. Occasionally, a single nerve fibre contacted several (two or four) other nerve processes forming a typical synapse. In other cases, on one nerve cell soma or on other nerve processes there are two or three different-type nerve terminals establishing synapses. It might be inferred from these observations that convergence and divergence can occur in the local ganglia and that cholinergic and adrenergic synaptic terminals can modulate the ganglionic activity. However, a local circuit also can play an important role in coordinating the function of the bladder.     

Ultrastructure of the pineal gland of the eastern chipmunk (Tamias striatus)

The ultrastructure of the pineal gland of the wild-captured eastern chipmunk (Tamias striatus) was examined. A homogenous population of pinealocytes was the characteristic cellular element of the chipmunk pineal gland. Often, pinealocytes showed a folliclelike arrangement. Mitochondria, Golgi apparatus, granular endoplasmic reticulum, lysosomes, centrioles, dense-core vesicles, clear vesicles, glycogen particles, and microtubules were consistent components of the pinealocyte cytoplasm. The extraordinary ultrastructural feature of the chipmunk pinealocyte was the presence of extremely large numbers of “synaptic” ribbons. The number of “synaptic” ribbons in this species exceeded by a factor of five to 30 times that found in any species previously reported. In addition to pinealocytes, the pineal parenchyma contained glial cells (oligodendrocytes and fibrous astrocytes). Capillaries of the pineal gland of the chipmunk consisted of a fenestrated endothelium. Adrenergic nerve terminals were relatively sparse.     

THE ELECTRON MICROSCOPIC LOCALIZATION OF CHOLINESTERASE ACTIVITY IN THE CENTRAL NERVOUS SYSTEM OF AN INSECT, PERIPLANET A AMERICANAL.

The distribution of esterase activity in the last abdominal ganglion, the connectives and the cereal nerves of the cockroach Periplaneta americana has been investigated cytochemically. Activity of an unspecific eserine-insensitive esterase (or esterases) has been found in glial elements in these regions of the nerve cord. In addition, sites of cholinesterase (eserine-sensitive) activity have been found in association with (a) the glial sheaths of the axons in the cereal nerves and connectives, (b) the glial folds encapsulating the neuron perikarya in the ganglion, and (c) in localized areas along the membranes of axon branches within the neuropile, often flanked by focal clusters of synaptic vesicles. These results are discussed with particular reference to the previously reported insensitivity of the insect nerve cord to applied acetylcholine, and to the probable existence of a cholinergic synaptic mechanism in the central nervous system of this insect.     

A Polychromatic Stain for Use in Parasitology

In sensory systems, insight into synaptic arrangements on cells of known physiological response properties has helped our understanding of the structural basis for these properties. To carry out these types of studies, however, synaptic types in the region of interest must be defined. Unfortunately, defining synaptic types in the brainstem has proved to be a challenging enterprise. Our study was done to classify synapses in the gustatory part of the nucleus solitarius using objective quantitative criteria and a cluster analysis procedure. Cluster analysis allows classification of a population of objects, such as synaptic terminals, into groups that exhibit similar characteristics. Six terminal types were identified using cluster analysis and subsequent analyses of variance and post hoc tests. Unlike classification schemes used for the cerebral cortex, where synaptic apposition density thickness and shape of vesicles is useful (Gray's Type I and II synapses), the concentration of vesicles in a terminal was a more useful measurement with which to classify terminals in the nucleus solitarius. To validate that vesicle density (vesicles/μm²) is a useful defining characteristic to classify terminals in the nucleus solitarius, terminals of a known type were used. GABAergic terminals were identified using postembedding immunohistochemical techniques, and their vesicle density was determined. GABAergic terminals fall into the range of two of the terminal types defined by the cluster analysis and, based on vesicle density, two types of GABAergic terminals were identified. We conclude that vesicle density is a helpful means to identify synapses in this brainstem nucleus.     

Gap Junctions Contribute to the Regulation of Walking-Like Activity in the Adult Mudpuppy (Necturus Maculatus)

Although gap junctions are widely expressed in the developing central nervous system, the role of electrical coupling of neurons and glial cells via gap junctions in the spinal cord in adults is largely unknown. We investigated whether gap junctions are expressed in the mature spinal cord of the mudpuppy and tested the effects of applying gap junction blocker on the walking-like activity induced by NMDA or glutamate in an in vitro mudpuppy preparation. We found that glial and neural cells in the mudpuppy spinal cord expressed different types of connexins that include connexin 32 (Cx32), connexin 36 (Cx36), connexin 37 (Cx37), and connexin 43 (Cx43). Application of a battery of gap junction blockers from three different structural classes (carbenexolone, flufenamic acid, and long chain alcohols) substantially and consistently altered the locomotor-like activity in a dose-dependent manner. In contrast, these blockers did not significantly change the amplitude of the dorsal root reflex, indicating that gap junction blockers did not inhibit neuronal excitability nonselectively in the spinal cord. Taken together, these results suggest that gap junctions play a significant modulatory role in the spinal neural networks responsible for the generation of walking-like activity in the adult mudpuppy.     

大鼠脊髓胶状质内GABA能神经元突触联系的超微结构研究

本文用免疫电镜方法对脊髓胶状质内ＧＡＢＡ能神经元的突触联系进行了超微结构研究。结果表明;脊髓胶状质内有许多ＧＡＢＡ能神经元胞体和末梢分布;标记的ＧＡＢＡ能神经末梢可作为突触前成分与未标记的ＧＡＢＡ形成输一树突触。未标记的末梢可与标记的ＧＡＢＡ末梢形成输一轴突触。此外,标记的ＧＡＢＡ能神经末梢还可作为突触前成分与标记的ＧＡＢＡ能轴突、树突或胞体形成输－轴、轴－树或轴－体突触,即自调节突触。上述结果揭示：ＧＡＢＡ能末梢可对脊髓胶状质内其它神经元产生抑制或脱抑制作用。值得注意的是胶状质内含ＧＡｎＡ的神经结构可形成各种形式的自调节突触,并借此实现其对脊髓功能的复杂调节。     

Roles of glial cells in the formation,function, and maintenance of the neuromuscular junction

Like other vertebrate synapses, the neuromuscular junction (NMJ) has glial cells that are closely associated with the pre- and post-synaptic components. These “perisynaptic Schwann cells” (PSCs) cover nerve terminals and are in close proximity to the synapse, yet their role at the NMJ has remained mysterious for decades. In this review we explore historical perspectives on PSCs and highlight key developments in recent years that have provided novel insight into PSC functions at the NMJ. First among these developments is the generation of specific antibody probes for PSCs. Using one such antibody and the principle of complement-mediated cell lysis, we have developed a novel technique to selectively ablate PSCs en masse from frog NMJs in vivo. Applying this approach, we have shown that PSCs are essential for the long-term maintenance of synaptic structure and function. In addition, PSCs are essential for the growth and maintenance of NMJs during development. Probes for PSCs also allow us to observe in vivo that processes extended by PSCs guide nerve terminals during synapse development, remodeling, and regeneration. PSCs may therefore dictate the pattern of innervation at the NMJ. Finally, PSCs may also induce postsynaptic acetylcholine receptor expression and aggregation. This wealth of recent findings about PSCs suggests that these synapse-associated glial cells are a more integral and essential component of the NMJ than previously appreciated. New approaches currently being applied at the NMJ may further support the emerging view that glial cells help make bigger, stronger, and more stable synapses.     

The ultrastructural characteristics of the motor neuron synaptic organization in the spinal cord of the frog Rana ridibunda

Electron microscopic studies on the spinal motor nuclei in amphibians indicate significant diversity in chemical synapses formed on motoneurones by axonal endings of supra- and intraspinal systems. High ultrastructural specialization was observed among axosomatic, axodendritic and axoaxonal synapses. Several types of axo-spine synapses and axodentritic synaptic complexes of the "glomerular" type were revealed. New data on ultrastructural peculiarities of chemical synapses presented in this paper, together with earlier detailed data on morphologically mixed and electrotonic synapses, increase our knowledge of evolutionary trends in synaptic organization of motoneurones in the spinal cord and suggest the existence of a complex mechanism of integration of synaptic influences in the spinal cord of lower vertebrates.     

Polyunsaturated fatty acids influence synaptojanin localization to regulate synaptic vesicle recycling

The lipid polyunsaturated fatty acids are highly enriched in synaptic membranes, including synaptic vesicles, but their precise function there is unknown. Caenorhabditis elegans fat-3 mutants lack long-chain polyunsaturated fatty acids (LC-PUFAs); they release abnormally low levels of serotonin and acetylcholine and are depleted of synaptic vesicles, but the mechanistic basis of these defects is unclear. Here we demonstrate that synaptic vesicle endocytosis is impaired in the mutants: the synaptic vesicle protein synaptobrevin is not efficiently retrieved after synaptic vesicles fuse with the presynaptic membrane, and the presynaptic terminals contain abnormally large endosomal-like compartments and synaptic vesicles. Moreover, the mutants have abnormally low levels of the phosphoinositide phosphatase synaptojanin at release sites and accumulate the main synaptojanin substrate phosphatidylinositol 4,5-bisphosphate at these sites. Both synaptobrevin and synaptojanin mislocalization can be rescued by providing exogenous arachidonic acid, an LC-PUFA, suggesting that the endocytosis defect is caused by LC-PUFA depletion. By showing that the genes fat-3 and synaptojanin act in the same endocytic pathway at synapses, our findings suggest that LC-PUFAs are required for efficient synaptic vesicle recycling, probably by modulating synaptojanin localization at synapses.     

Neuronal protein NP 185 is developmentally regulated,initially expressed during synaptogenesis,and localized in synaptic terminals

Evidence is presented here that demonstrates the presence of NP185 (AP₃) in neuronal cells, specifically within syn-aptic terminals of the central nervous system and in the peripheral nervous system, particularly in the neuro-muscular junction of adult chicken muscle. Biochemical results obtained in our laboratories indicate that NP185 is associated with brain synaptic vesicles, with clathrin-coated vesicles, and with the synaptosomal plasma membrane. Also, NP185 binds to tubulin and clathrin light chains and the binding is regulated by phosphorylation (Su et al., 1991). Based on these properties and the data reported here, we advance the postulate that NP185 fulfills multiple functions in synaptic terminals. One function is that of a plasma membrane docking or channel protein, another of a signaling molecule for brain vesicles to reach the synaptic terminal region, and a third is that of a recycling molecule by binding to protein components on the lipid bilayer of the synaptic plasma membrane during the process of endocytosis. In support of these premises, a thorough study of NP185 using the developing chick brain, adult mouse brain, and chicken straited muscle was begun by temporally and spatially mapping the expression and localization of NP185 in evolving and mature nerve endings. To achieve these objectives, monoclonal antibodies to NP185 were used for immunocytochemistry in tissue sections of chicken and mouse cerebella. The distribution of NP185 was compared with those of other cytoskeletal and cytoplasmic proteins of axons and synapses, namely synaptophysin, vimentin, neurofilament NF68, and the intermediate filaments of glial cells (GFAP). The data indicate that expression of NP185 temporally coincides with synaptogenesis, and that the distribution of this protein is specific for synaptic terminal buttons of the CNS and the PNS.     

Tomosyn Inhibits Synaptic Vesicle Priming in Caenorhabditis elegans

Caenorhabditis elegans TOM-1 is orthologous to vertebrate tomosyn, a cytosolic syntaxin-binding protein implicated in the modulation of both constitutive and regulated exocytosis. To investigate how TOM-1 regulates exocytosis of synaptic vesicles in vivo, we analyzed C. elegans tom-1 mutants. Our electrophysiological analysis indicates that evoked postsynaptic responses at tom-1 mutant synapses are prolonged leading to a two-fold increase in total charge transfer. The enhanced response in tom-1 mutants is not associated with any detectable changes in postsynaptic response kinetics, neuronal outgrowth, or synaptogenesis. However, at the ultrastructural level, we observe a concomitant increase in the number of plasma membrane-contacting vesicles in tom-1 mutant synapses, a phenotype reversed by neuronal expression of TOM-1. Priming defective unc-13 mutants show a dramatic reduction in plasma membrane-contacting vesicles, suggesting these vesicles largely represent the primed vesicle pool at the C. elegans neuromuscular junction. Consistent with this conclusion, hyperosmotic responses in tom-1 mutants are enhanced, indicating the primed vesicle pool is enhanced. Furthermore, the synaptic defects of unc-13 mutants are partially suppressed in tom-1 unc-13 double mutants. These data indicate that in the intact nervous system, TOM-1 negatively regulates synaptic vesicle priming.     

Expression and Localization of sPLA2-III in the Rat CNS

Phospholipases A₂ (PLA₂) are enzymes that cleave the sn-2 bond of membrane phospholipids to yield free fatty acids and lysophospholipids. Secretory PLA₂-III (sPLA₂-III) has been suggested to be important for neuronal differentiation, growth and survival, and is highly expressed in the spinal cord. The aim of this study is to elucidate its expression and distribution in different regions of the adult rat CNS. Quantitative RT-PCR analyses showed high levels of sPLA₂-III mRNA expression in the brainstem and spinal cord and low expression in the olfactory bulb. Western blot analyses showed high level of expression in the brainstem, spinal cord and cerebral neocortex. A dense band corresponding to the catalytically active, mature/cleaved form, and a faint band corresponding to the full length sPLA₂-III were detected in post-mitochondrial supernatants, from different parts of the CNS. Subcellular fractionation of spinal cord homogenates showed that sPLA₂-III protein is present in the ‘light membrane/cytosol’ fraction, but not the nucleus, synaptosomal membrane or synaptic vesicle-enriched fractions. sPLA₂-III was immunolocalized to neurons in the cerebral neocortex, Purkinje neurons in the cerebellar cortex, periaqueductal gray, red nucleus, spinal trigeminal nucleus and dorsal horn of the spinal cord. Electron microscopy of the spinal cord and cerebral neocortex showed that sPLA₂-III was localized in dendrites or dendritic spines, that formed asymmetrical synapses with unlabeled, putatively glutamatergic, axon terminals. The localization of mature/cleaved form of sPLA₂-III in postsynaptic structures suggest a physiological role of the enzyme in neurotransmission or synaptic plasticity.