Immunocytochemical analysis of syntaxin-1 in rat circumvallate taste buds

Mammalian buds contain a variety of morphological taste cell types, but the type III taste cell is the only cell type that has synapses onto nerve processes. We hypothesize that taste cell synapses utilize the SNARE protein machinery syntaxin, SNAP-25, and synaptobrevin, as is used by synapses in the central nervous system (CNS) for Ca2+-dependent exocytosis. Previous studies have shown that taste cells with synapses display SNAP-25- and synaptobrevin-2-like immunoreactivity (LIR) (Yang et al. [2000a] J Comp Neurol 424:205-215, [2004] J Comp Neurol 471:59-71). In the present study we investigated the presynaptic membrane protein, syntaxin-1, in circumvallate taste buds of the rat. Our results indicate that diffuse cytoplasmic and punctate syntaxin-1-LIR are present in different subsets of taste cells. Diffuse, cytoplasmic syntaxin-1-LIR is present in type III cells while punctate syntaxin-1-LIR is present in type II cells. The punctate syntaxin-1-LIR is believed to be associated with Golgi bodies. All of the synapses associated with syntaxin-1-LIR taste cells are from type III cells onto nerve processes. These results support the proposition that taste cell synapses use classical SNARE machinery such as syntaxin-1 for neurotransmitter release in rat circumvallate taste buds.     

The Plasma Membrane Protein SNAP-25, but not Syntaxin, is Present at Photoreceptor and Bipolar Cell Synapses in the Rat Retina

The two neuronal plasma membrane proteins synaptosomal associated protein 25 kDa (SNAP-25) and syntaxin form, in current models of synaptic vesicle exocytosis, a docking complex on the presynaptic plasma membrane. This docking complex interacts with the integral synaptic vesicle protein, synaptobrevin. We have examined, in the rat retina, the distribution of SNAP-25 and syntaxin using light and electron microscopic immunocytochemistry. SNAP-25 immunoreactivity was present in the outer and inner nuclear layers at the membranes of photoreceptor and amacrine cell somata, in the outer plexiform layer in the terminals of photoreceptor cells and throughout the inner plexiform layer in the terminals of bipolar and amacrine cells. In contrast, syntaxin immunoreactivity was not found in the terminals of photoreceptor and bipolar cells, but only in the terminals of amacrine cells. Because of the absence of detectable syntaxin from the terminals of photoreceptor and bipolar cells, we propose that either a novel syntaxin isoform or a syntaxin–like protein exists at the ribbon synapses formed by these neurons.     

Taste cells with synapses in rat circumvallate papillae display SNAP-25-like immunoreactivity

SNAP-25 is a 25 kDa protein believed to be involved in the processes of membrane fusion and exocytosis associated with neurotransmitter release. In the present study we present evidence that SNAP-25-like immunoreactivity can be used as a marker for taste cells with synapses in rat circumvallate papillae. SNAP-25 immunoreactivity is present in most intragemmal nerve processes and a small subset of taste cells. Intense immunoreactivity is associated with the nerve plexus located below the base of the taste bud. Of a total of 87 taste cells with synapses onto nerve processes, 80 of the presynaptic taste cells had SNAP-25 immunoreactivity. The association of SNAP-25 immunoreactivity with taste cells possessing synapses suggests that these cells may be gustatory receptor cells. Because this SNAP-25 antibody can label taste cells with synapses, it may also serve as a useful tool for future studies correlating structure with function in the taste bud.     

Rab3 Proteins and SNAP-25, Essential Components of the Exocytosis Machinery in Conventional Synapses, are Absent from Ribbon Synapses of the Mouse Retina

GTP-binding rab proteins, present in synaptic vesicles and endocrine secretory granules, have been shown to be involved in the control of regulated exocytosis. We found rab3 proteins in immunoblots of diverse areas of the mouse central nervous system (spinal cord, olfactory bulb, hippocampus, cerebellum and neocortex). Immunohistochemical observations at light- and electron-microscopical levels in the hippocampus and other areas revealed rab3 proteins in virtually all synaptic fields and terminals of the areas investigated. In the retina, rab3A immunoreactivity was confined to the inner and outer plexiform layers. Ultrastructural examination revealed that rab3A was present in conventional terminals in the inner plexiform layer and in horizontal cell processes of the outer plexiform layer. In contrast ribbon synapses, which play a key role in transferring information from the photoreceptor cells to the central nervous system, were immunonegative. We also tested whether other proteins of the rab3 family are present in ribbon synapses. However, using an antibody recognizing rab3B and rab3C in addition to rab3A, we found no immunoreactivity in these synapses. Interestingly, we observed also no immunoreactivity for synaptosomal-associated protein 25 (SNAP-25) in ribbon synapses, but conventional synapses and horizontal cell processes were heavily stained. Our data show that the known rab3 and SNAP-25 isoforms, which are components of the secretory apparatus of conventional synapses, are absent from ribbon synapses of the retina. Our observations suggest different mechanisms of transmitter exocytosis in conventional and ribbon terminals.     

SNAP-25 is present on the Golgi apparatus of retinal neurons

SNAP-25 is a neuronal SNARE protein required for synaptic vesicle exocytosis and neurite outgrowth. Here we show that in addition to synaptic staining, SNAP-25 immunoreactivity is also localized to an intracellular, perinuclear compartment of retinal neurons. Double-labeling with an antibody against the 58 kD resident protein of the trans-golgi network indicates that the intracellular SNAP-25 is localized to the Golgi complex. Immuno-electron microscopic localization of SNAP-25 confirmed its presence on the Golgi apparatus of photoreceptors, bipolar cells, amacrine cells and ganglion cells in the retina. These data implicate SNAP-25 in the trafficking of Golgi-derived vesicles in neurons in addition to the synaptic vesicle cycle.     

Synaptic proteins in rat taste bud cells: appearance in the Golgi apparatus and relationship to alpha-gustducin and the Lewis(b) and A antigens

Taste receptor cells are continuously replaced during the life of the animal, but many of their sensory axons respond primarily to stimuli belonging to a single taste quality. This suggests that a newly arising taste cell must form a synapse with an appropriate sensory axon, requiring cell recognition that is likely to be mediated by surface markers. As an approach to studying this process, we attempted to locate synapses by immunolabeling taste buds of rats for proteins involved in neurotransmitter release. In taste bud cells of vallate papillae and nasoincisor ducts, double-labeling experiments showed that syntaxin-1, SNAP-25, synaptobrevin, and synaptophysin colocalized with the Golgi marker beta COP in elongated cytoplasmic compartments that extended from the perinuclear region into apical and basal processes of the cells. Labeled cells were spindle-shaped, identifying them as light cells. Syntaxin-1 appeared only in taste cells, but SNAP-25, synaptobrevin, and synaptophysin were also seen in nerve fibers. The synaptic vesicle glycoprotein SV2 appeared only in nerve fibers. Taste cells of fungiform papillae did not show immunoreactivity for presynaptic proteins or Golgi markers, but axonal labeling was similar to that in other regions. Taste cells with alpha-gustducin could express either presynaptic proteins or the carbohydrate blood group antigen Lewis(b), but not both. Therefore, Lewis(b) and presynaptic proteins are not expressed during the same period in the life of a taste bud cell. Most taste cells expressing syntaxin-1 (82%) also expressed the A blood group antigen, whether or not they expressed alpha-gustducin.     

Morphologic characterization of rat taste receptor cells that express components of the phospholipase C signaling pathway

Rat taste buds contain three morphologically distinct cell types that are candidates for taste transduction. The physiologic roles of these cells are, however, not clear. Inositol 1,4,5-triphosphate (IP(3)) has been implicated as an important second messenger in bitter, sweet, and umami taste transductions. Previously, we identified the type III IP(3) receptor (IP(3)R3) as the dominant isoform in taste receptor cells. In addition, a recent study showed that phospholipase Cbeta(2) (PLCbeta(2)) is essential for the transduction of bitter, sweet, and umami stimuli. IP(3)R3 and PLCbeta(2) are expressed in the same subset of cells. To identify the taste cell types that express proteins involved in PLC signal transduction, we used 3,3'diaminobenzidine tetrahydrochloride immunoelectron microscopy and fluorescence microscopy to identify cells with IP(3)R3. Confocal microscopy was used to compare IP(3)R3 or PLCbeta(2) immunoreactivity with that of some known cell type markers such as serotonin, protein gene-regulated product 9.5, and neural cell adhesion molecule. Here we show that a large subset of type II cells and a small subset of type III cells display IP(3)R3 immunoreactivity within their cytoplasm. These data suggest that type II cells are the principal transducers of bitter, sweet, and umami taste transduction. However, we did not observe synapses between type II taste cells and nerve fibers. Interestingly, we observed subsurface cisternae of smooth endoplasmic reticulum at the close appositions between the plasma membrane of type II taste cells and nerve processes. We speculate that some type II cells may communicate to the nervous system via subsurface cisternae of smooth endoplasmic reticulum in lieu of conventional synapses.     

Increase in SNAP-25 immunoreactivity in the mossy fibers following transient forebrain ischemia in the gerbil

SNAP-25 (a synaptosomal-associated protein of 25 kDa) has been shown to be involved both in synaptic vesicle exocytosis and in axonal outgrowth. In the present study, we investigated the changes in SNAP-25 immunoreactivity in the hippocampus of the Mongolian gerbil (Meriones unguiculatus) at different time points after transient forebrain ischemia insult. In parallel, immunostaining for GAP-43, a protein involved in axonal outgrowth, and for syntaxin-1 (stx1A and stx1B), another protein implicated in neurotransmitter release, was also analyzed. The animals were subjected to 2.5 or 5 min of transient forebrain ischemia through bilateral common carotid occlusion, and examined at different intervals after ischemia. SNAP-25 immunoreactivity was increased in the mossy fiber layer as early as 2 days after 5 min of ischemia. Increased SNAP-25 immunoreactivity in mossy fibers was also detected at days 4 and 7 after ischemia. On day 15, SNAP-25 staining was similar to that observed in control non-ischemic animals. In contrast, no changes in GAP-43 and syntaxin-1 immunoreactivity were observed in the mossy fiber layer following 5 min of ischemia. No modifications in SNAP-25, syntaxin-1 or GAP-43 immunoreactivity were observed following 2.5 min of ischemia, the longest period for which no neuronal damage is observed. These results provide evidence of a specific involvement of SNAP-25 in the reactive changes associated with transient forebrain ischemia. Received: 30 June 1997 / Revised, accepted: 26 September 1997     

Expression of exocytosis proteins in rat supraoptic nucleus neurones

In magnocellular neurones of the supraoptic nucleus (SON), the neuropeptides vasopressin and oxytocin are synthesised and packaged into large dense-cored vesicles (LDCVs). These vesicles undergo regulated exocytosis from nerve terminals in the posterior pituitary gland and from somata/dendrites in the SON. Regulated exocytosis of LDCVs is considered to involve the soluble N-ethylmaleimide sensitive fusion protein attachment protein receptor (SNARE) complex [comprising vesicle associated membrane protein 2 (VAMP-2), syntaxin-1 and soluble N-ethylmaleimide attachment protein-25 (SNAP-25)] and regulatory proteins [such as synaptotagmin-1, munc-18 and Ca(2+) -dependent activator protein for secretion (CAPS-1)]. Using fluorescent immunocytochemistry and confocal microscopy, in both oxytocin and vasopressin neurones, we observed VAMP-2, SNAP-25 and syntaxin-1-immunoreactivity in axon terminals. The somata and dendrites contained syntaxin-1 and other regulatory exocytosis proteins, including munc-18 and CAPS-1. However, the distribution of VAMP-2 and synaptotagmin-1 in the SON was limited to putative pre-synaptic contacts because they co-localised with synaptophysin (synaptic vesicle marker) and had no co-localisation with either oxytocin or vasopressin. SNAP-25 immunoreactivity in the SON was limited to glial cell processes and was not detected in oxytocin or vasopressin somata/dendrites. The present results indicate differences in the expression and localisation of exocytosis proteins between the axon terminals and somata/dendritic compartment. The absence of VAMP-2 and SNAP-25 immunoreactivity from the somata/dendrites suggests that there might be different SNARE protein isoforms expressed in these compartments. Alternatively, exocytosis of LDCVs from somata/dendrites may use a different mechanism from that described by the SNARE complex theory.     

Juvenile neuroaxonal dystrophy in a Rottweiler: accumulation of synaptic proteins in dystrophic axons

Dystrophic axons in a 2-year-old male Rottweiler with neuroaxonal dystrophy have shown synaptophysin, synapsin-I, synaptosomal-associated protein of 25 kDa (SNAP-25), Rab 3a, and alpha-synuclein immunoreactivity. Similar findings have been observed in isolated dystrophic axons in the nuclei gracillis and cunneatus in five dogs aged between 14 and 18 years. Abnormal expression of integral synaptic vesicle, synaptic vesicle-associated presynaptic plasma membrane and cytosolic proteins, which participate in the trafficking, docking and fusion of the synaptic vesicle to the plasma membrane, suggest severe disruption of axonal transport in dystrophic axons in canine neuroaxonal dystrophy.     

Synaptic protein expression by regenerating adult photoreceptors.

Regeneration of functionally normal synapses is required for functional recovery after degenerative central nervous system insults and requires proper expression and targeting of presynaptic proteins by regenerating neurons. The reconstitution of presynaptic terminals by regenerating adult neurons is poorly understood, however. We examined the intrinsic ability of regenerating adult retinal photoreceptors to reconstitute properly differentiated presynaptic terminals in the absence of target contact. The expression and localization of vesicle-associated membrane protein (VAMP), synaptic vesicle protein 2 (SV2), synaptophysin, synapsin I, and synaptosomal-associated protein of 25 kDa (SNAP-25) was assessed immunocytochemically. Photoreceptor terminals in the intact retina contain VAMP, SV2, synaptophysin, and SNAP-25, but not synapsin I. Isolated, regenerating adult photoreceptors intrinsically expressed the proper complement of synaptic vesicle proteins in the absence of target contact: VAMP, SV2, and synaptophysin were present at all stages of regenerative growth; synapsin I was never expressed. At early stages of regenerative growth, VAMP, SV2, and synaptophysin were diffusely localized in the cell, with prominent VAMP labeling distributed along the plasma membrane. SV2 and synaptophysin rapidly localized to regenerated terminals, but VAMP accumulated much more slowly, indicating that these proteins are trafficked independently. In contrast, labeling for SNAP-25, which is associated with the presynaptic plasma membrane, was undetectable in regenerating photoreceptors, suggesting that SNAP-25 expression is target-regulated. Thus, regenerating photoreceptors can intrinsically regulate the expression of the proper set of synaptic vesicle proteins. Proper expression of other presynaptic proteins, such as SNAP-25, and proper subcellular localization of synaptic proteins such as VAMP, however, may require extrinsic cues such as target contact.     

Heterogeneous expression of SNAP-25 and synaptic vesicle proteins by central and peripheral inputs to sympathetic neurons

Neurons in prevertebral sympathetic ganglia receive convergent synaptic inputs from peripheral enteric neurons in addition to inputs from spinal preganglionic neurons. Although all inputs are functionally cholinergic, inputs from these two sources have distinctive neurochemical and functional profiles. We used multiple-labeling immunofluorescence, quantitative confocal microscopy, ultrastructural immunocytochemistry, and intracellular electrophysiologic recordings to examine whether populations of inputs to the guinea pig coeliac ganglion express different levels of synaptic proteins that could influence synaptic strength. Boutons of enteric intestinofugal inputs, identified by immunoreactivity to vasoactive intestinal peptide, showed considerable heterogeneity in their immunoreactivity to synaptosome-associated protein of 25 kDa (SNAP-25), synapsin, synaptophysin, choline acetyltransferase, and vesicular acetylcholine transporter. Mean levels of immunoreactivity to these proteins were significantly lower in terminals of intestinofugal inputs compared with terminals of spinal preganglionic inputs. Nevertheless, many boutons with undetectable levels of SNAP-25 immunoreactivity formed morphologically normal synapses with target neurons. Treatment with botulinum neurotoxin type A (20-50 nM for 2 hours in vitro) generated significant cleavage of SNAP-25 and produced similar dose- and time-dependent inhibitions of synaptic transmission from all classes of inputs, regardless of their mean level of SNAP-25 expression. The simplest interpretation of these results is that only synaptic boutons with detectable levels of SNAP-25 immunoreactivity contribute significantly to fast cholinergic transmission. Consequently, the low synaptic strength of intestinofugal inputs to final motor neurons in sympathetic pathways may be due in part to the low proportion of their boutons that express SNAP-25 and other synaptic proteins.     

Ultrastructural localization of mint1 at synapses in mouse hippocampus

Mint1 and mint2 were isolated in the course of seeking the protein ligands to munc18-1, a neuronal protein essential for synaptic vesicle exocytosis. The mint family of proteins has been highly conserved in the course of evolution, being retained from C. elegans to mammals. Several lines of biochemical and genetic evidence have suggested that mint1 and LIN-10, its homologue in C. elegans, function at synapses in the brain. Because the precise subcellular location of mint1 is incompletely known, we used immunostaining to examine the distribution of mint1 in the mouse brain including ultrastructural localization in synapses. Strong, finely punctate mint1 immunolabeling was detected throughout the brain, including cerebral cortex, striatum, hippocampus, thalamus, basal ganglia and cerebellum. At the most synapses in the molecular layer, mint1 was particularly abundant at the active zone and to a lesser extent in association with synaptic vesicles in the presynaptic terminals. In contrast, a very few synapses showed mint1 immunoreactivity in the postsynaptic density and there was no synapse double-positive in presynaptic and postsynaptic terminals. Mint1 distribution within presynaptic terminals overlapped that of munc18-1. These localization results are consistent with previously demonstrated biochemical interactions and strongly support functions of mint1 in synaptic vesicle exocytosis and synaptic organization in the central nervous system.     

Phosphorylation of cysteine string protein in the brain: developmental, regional and synaptic specificity

Protein phosphorylation modulates regulated exocytosis in most cells, including neurons. Cysteine string protein (CSP) has been implicated in this process because its phosphorylation on Ser10 alters its interactions with syntaxin and synaptotagmin, and because the effect of CSP overexpression on exocytosis kinetics in chromaffin cells requires phosphorylatable Ser10. To characterize CSP phosphorylation in the brain, we raised phosphospecific antibodies to Ser10. Western blotting revealed that the proportion of phosphorylated CSP (P-CSP) varies between distinct brain regions and also exhibits developmental regulation, with P-CSP highest early in development. Immunohistochemical analysis of the cerebellar cortex revealed a novel pool of P-CSP that did not colocalize with synaptic vesicle markers during early development. Strikingly, in the adult cerebellar granular layer P-CSP was highly enriched in a subset of glutamatergic synapses but undetectable in neighbouring GABA-ergic synapses. In view of the functional consequences of CSP phosphorylation, such differences could contribute to the synapse-specific regulation of neurotransmitter release.     

Synaptic vesicle protein 2A: basic facts and role in synaptic function

In recent years, there has been considerable interest in determining the function of synaptic vesicle protein 2A and its role as a target for antiepileptic drugs. Although it is known that synaptic vesicle protein 2A is involved in normal synaptic vesicle function, its participation in synaptic vesicle cycling and neurotransmitter release in normal and pathological conditions is unclear. However, the experimental evidence suggests that synaptic vesicle protein 2A could be a vesicular transporter, regulate synaptic exocytosis as a gel matrix, or modulate synaptotagmin‐1 activity. This review describes and discusses the participation of synaptic vesicle protein 2A in synaptic modulation in normal and pathological conditions.     

Expression of Rab3A GTPase and other synaptic proteins is induced in differentiated NT2N neurons

Postmitotic NT2N cells, which are derived from human NT2 teratocarcinoma cells by treatment with retinoic acid (RA) and mitotic inhibitors, are viewed as a good in vitro model for mature neurons of the human central nervous system. Although NT2N cells exhibit many morphological and biochemical characteristics of neurons, the expression of key protein components involved in regulated exocytosis have not been firmly established. Here we show by immunoblot analysis that mature morphologically differentiated NT2N cells contain readily detectable quantities of the synaptic vesicle-associated proteins, synaptobrevin, synapsin, and synaptophysin. They also express the presynaptic plasma membrane protein, SNAP-25, and a Rab GTPase implicated in the control of Ca²⁺-dependent exocytosis, Rab3A. These proteins were not detected in untreated NT2 cells or cells exposed to RA for only 6 d. The induction of an array of proteins known to be involved in the docking and fusion of synaptic vesicles with the plasma membrane provides further support for the validity of NT2N cells as a model for human cortical neurons and suggests that these cells may be useful for in vitro molecular studies of the Ca²⁺-regulated exocytic pathway in nerve terminals.     

Selective reduction in synaptic proteins involved in vesicle docking and signalling at synapses in the ataxic mutant mouse stargazer

The spontaneous recessive mutant mouse stargazer has a specific and pronounced deficit in brain-derived neurotrophic factor (BDNF) mRNA expression in the cerebellum. Cerebellar granule cells, in particular, show a selective and near-total loss of BDNF. The mutation involves a defect in the calcium channel subunit Cacng2. This severely reduces expression of stargazin. A stargazin-induced failure in BDNF expression is thought to underlie the cerebellar ataxia with which the mutant presents. BDNF is known to regulate plasticity at cerebellar synapses. However, relatively little is known about the mechanism involved. We previously demonstrated that the stargazer mutation affects the phenotype of cerebellar glutamatergic neurons. Stargazer neurons have less glutamate and proportionally fewer docked vesicles at presynaptic sites than controls. In the current study, we investigate the mechanism underlying BDNF-induced synaptic changes by analyzing alterations in synaptic signalling proteins in the stargazer cerebellum. Expression levels of synaptic proteins were evaluated by measuring relative density of immunogold label over granule cell terminals in ultrathin sections from ataxic stargazer mutants compared with matched nonataxic littermates. We show that there is a selective and marked depletion in the levels of vesicle-associated proteins (synaptobrevin, synaptophysin, synaptotagmin, and Rab3a) but not of plasma membrane-associated protein (SNAP-25) in the terminals of the BDNF-deficient granule cells. Changes are restricted to the cerebellum; levels in the hippocampus are unaltered. These data suggest that the BDNF deficits in the cerebellum of stargazer affect synaptic vesicle docking by selectively altering synaptic-protein distribution and abundance.     

The SNARE complex in neuronal and sensory cells

Transmitter release at synapses ensures faithful chemical coding of information that is transmitted in the sub-second time frame. The brain, the central unit of information processing, depends upon fast communication for decision making. Neuronal and neurosensory cells are equipped with the molecular machinery that responds reliably, and with high fidelity, to external stimuli. However, neuronal cells differ markedly from neurosensory cells in their signal transmission at synapses. The main difference rests in how the synaptic complex is organized, with active zones in neuronal cells and ribbon synapses in sensory cells (such as photoreceptors and hair cells). In exocytosis/neurosecretion, SNAREs (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors) and associated proteins play a critical role in vesicle docking, priming, fusion and synchronization of neurotransmitter release. Recent studies suggest differences between neuronal and sensory cells with respect to the molecular components of their synaptic complexes. In this review, we will cover current findings on neuronal and sensory-cell SNARE proteins and their modulators. We will also briefly discuss recent investigations on how deficits in the expression of SNARE proteins in humans impair function in brain and sense organs.