Syntaxin Immunoreactivity Research Articles

Molecular identification of an N-type Ca 2+ channel in saccular hair cells

We report new molecular evidence for the presence of an N-type (Ca v2.2, α1B) voltage-gated Ca 2+ channel in hair cells of the saccular epithelium of the rainbow trout. The Ca v2.2 amino-acid sequence shows 68% and 63% identity compared with chick and human Ca v2.2, respectively. This channel reveals features that are characteristic of an N-type Ca 2+ channel: an ω-conotoxin GVIA binding domain, G βγ binding regions, and a synaptic protein interaction site. Immunohistochemical studies with a custom antibody show that immunoreactivity for the Ca v2.2 is concentrated in the basolateral and apical regions of hair cells. Whereas trout brain and saccular macula express an 11-amino-acid insert in the second G βγ binding domain of the Ca v2.2 I–II loop, isolated hair cells appear not to express this variant. We constructed fusion polypeptides representing portions of the I–II loop, β1 and β2a auxiliary subunits, the II–III loop, and syntaxin, and examined their intermolecular interactions via immunoprecipitation and surface plasmon resonance. The I–II loop polypeptides bound both β1 and β2a subunits with a preference for β1, and the II–III loop exhibited Ca 2+-dependent syntaxin binding. We demonstrated syntaxin immunoreactivity near afferent endings in hair cells, at hair-cell apices, and in efferent endings on hair cells, the former two sites consistent with binding of syntaxin to Ca v2.2. The present molecular characterization of the Ca v2.2 channel provides novel biochemical evidence for an N-type channel in hair cells, and details molecular interactions of this channel that reflect hair-cell function, such as spontaneous activity and vesicular trafficking. The current work, to our knowledge, represents the first demonstration of a putative N-type channel in hair cells as documented by tissue-specific antibody immunoreactivity and hair-cell-specific cDNA sequence.

Expression of the exocytotic protein syntaxin in mouse oocytes.

Syntaxin is an integral membrane protein that is involved in membrane fusion. The exocytosis of the contents of cortical granules, secretory vesicles located in the cortex of an egg, modify the extracellular environment to block additional spermatozoa from penetrating the newly fertilized egg. The aim of this study was to characterize syntaxin expression in mouse oocytes, and to determine the specific isoform that is expressed. Syntaxin was demonstrated in the mouse ovary and in mouse oocytes by both western blot and reverse transcription-polymerase chain reaction analyses. Syntaxin 4 was specifically expressed in metaphase II oocytes. Syntaxin was also immunolocalized within metaphase II oocytes and one-cell embryos with pronuclei using laser scanning confocal microscopy. In metaphase II oocytes, syntaxin was located on the plasma membrane and in the cortex, where cortical granules are present, but was not seen at sites free of cortical granules. In one-cell embryos, no cytoplasmic region was free of syntaxin immunoreactivity. Immunoelectron microscopy detected syntaxin on both the plasma membrane and the vesicle membranes in mouse metaphase II oocytes. In conclusion the results indicate that syntaxin 4 co-localizes with cortical granules and participates in membrane fusion and exocytosis during the cortical reaction.

Expression of the exocytotic protein syntaxin in mouse oocytes

Syntaxin is an integral membrane protein that is involved in membrane fusion. The exocytosis of the contents of cortical granules, secretory vesicles located in the cortex of an egg, modify the extracellular environment to block additional spermatozoa from penetrating the newly fertilized egg. The aim of this study was to characterize syntaxin expression in mouse oocytes, and to determine the specific isoform that is expressed. Syntaxin was demonstrated in the mouse ovary and in mouse oocytes by both western blot and reverse transcription-polymerase chain reaction analyses. Syntaxin 4 was specifically expressed in metaphase II oocytes. Syntaxin was also immunolocalized within metaphase II oocytes and one-cell embryos with pronuclei using laser scanning confocal microscopy. In metaphase II oocytes, syntaxin was located on the plasma membrane and in the cortex, where cortical granules are present, but was not seen at sites free of cortical granules. In one-cell embryos, no cytoplasmic region was free of syntaxin immunoreactivity. Immunoelectron microscopy detected syntaxin on both the plasma membrane and the vesicle membranes in mouse metaphase II oocytes. In conclusion the results indicate that syntaxin 4 co-localizes with cortical granules and participates in membrane fusion and exocytosis during the cortical reaction.

Tolerance to the anhedonic effects of lipopolysaccharide is associated with changes in syntaxin immunoreactivity in the nucleus accumbens.

The bacterial endotoxin lipopolysaccharide (LPS) produces a host of effects in mammals known collectively as 'sickness' behaviours. Acute treatment with LPS also results in a loss of hedonic capacity in rodents that can be measured by changes in responding for reinforcing electrical stimulation of the lateral hypothalamus. In contrast, repeated exposure to LPS typically leads to the development of tolerance to many of the physiological and behavioural effects of endotoxin, although the effect of chronic treatment with LPS on anhedonia remains unknown. In the present experiment, rats were trained to respond on an ascending-series current-intensity intracranial self-stimulation (ICSS) protocol, and were then treated with either acute or sub-chronic LPS (100 microg). Compared to vehicle-treated subjects, acute exposure to LPS induced a dramatic loss of ICSS responding; however, with repeated exposure to LPS, rats developed a behavioural tolerance to its anhedonic effects. To investigate a potential molecular substrate for the anhedonic effects of LPS, quantitative immunohistochemistry was used to measure levels of the synaptic proteins syntaxin, SNAP-25 and synaptophysin in the dorsal and ventral striatum of rats treated acutely and sub-chronically with LPS. A single injection of LPS produced a significant decrease in syntaxin immunoreactivity in the nucleus accumbens core and shell, while similar treatment in chronically treated rats that displayed behavioural tolerance had no effect. These results demonstrate a novel molecular substrate for the effects of LPS, and imply that the underlying physiology of the transient anhedonic effects of LPS may differ from that involved in chronic psychiatric disorders in humans.

Effect of prenatal auditory enrichment on developmental expression of synaptophysin and syntaxin 1 in chick brainstem auditory nuclei

Neural activity plays an important role in shaping the developing brain. We have determined the consequence of increased auditory stimulation on the developmental profile of synaptic proteins, synaptophysin and syntaxin 1, in the chick brainstem auditory nuclei, nucleus magnocellularis and nucleus laminaris, by immunohistochemistry and western blotting techniques. The chick embryos were provided with patterned sounds of species-specific calls or musical notes of a sitar, a stringed instrument, in a graded manner from embryonic day 10 (E10) through hatching, for 15 min every hour. During normal synaptogenesis of nucleus magnocellularis and nucleus laminaris, synaptophysin immunoreactivity increased significantly from E8 to E20, in parallel with synapse formation, and reduced at hatching. The embryos receiving species-specific sound stimuli exhibited a similar pattern with higher levels of immunoreactivity, though the difference between the study groups was not statistically significant. The music stimulated embryos showed an earlier peak at E16, followed by a gradual decline until hatching. In all three groups studied, syntaxin immunoreactivity showed a surge at E12, followed by a decline at E16 and subsequent stabilization. The stimulated groups continually expressed higher amounts of syntaxin immunoreactivity. The results suggest that prenatal sound stimulation enhances the normal pattern of synaptic protein expression in these auditory nuclei.

Retention of cleaved synaptosome-associated protein of 25 kDa (SNAP-25) in neuromuscular junctions: a new hypothesis to explain persistence of botulinum A poisoning

Botulinum neurotoxins can block neurotransmitter release for several months. The molecular mechanism of these toxins' action is known, but the persistence of neuromuscular paralysis that they cause is unexplained. At frog neuromuscular junctions, application of botulinum toxin type A caused paralysis and reduced the C-terminus immunoreactivity of SNAP-25, but not that of the remaining N-terminus fragment. Botulinum toxin type C caused paralysis and reduced syntaxin immunoreactivity without affecting that of SNAP-25. Co-application of botulinum A and C reduced syntaxin immunoreactivity, and that of both C and N termini of SNAP-25. Application of hydroxylamine to de-palmitoylate SNAP-25 resulted in a slight reduction of the immunoreactivity of SNAP-25 N terminus, while it had no effect on immunoreactivity of botulinum A cleaved SNAP-25. In contrast, application of hydroxylamine to nerve terminals where syntaxin had been cleaved by botulinum C caused a considerable reduction in SNAP-25 N-terminus immunoreactivity. Hence the retention of immunoreactive SNAP-25 at the neuromuscular junction depends on its interactions with syntaxin and plasma membrane. Persistence of cleaved SNAP-25 in nerve terminals may prevent insertion of new SNAP-25 molecules, thereby contributing to the longevity of botulinum A effects.

Retention of cleaved synaptosome-associated protein of 25 kDa (SNAP-25) in neuromuscular junctions: a new hypothesis to explain persistence of botulinum A poisoning

Botulinum neurotoxins can block neurotransmitter release for several months. The molecular mechanism of these toxins' action is known, but the persistence of neuromuscular paralysis that they cause is unexplained. At frog neuromuscular junctions, application of botulinum toxin type A caused paralysis and reduced the C-terminus immunoreactivity of SNAP-25, but not that of the remaining N-terminus fragment. Botulinum toxin type C caused paralysis and reduced syntaxin immunoreactivity without affecting that of SNAP-25. Co-application of botulinum A and C reduced syntaxin immunoreactivity, and that of both C and N termini of SNAP-25. Application of hydroxylamine to de-palmitoylate SNAP-25 resulted in a slight reduction of the immunoreactivity of SNAP-25 N terminus, while it had no effect on immunoreactivity of botulinum A cleaved SNAP-25. In contrast, application of hydroxylamine to nerve terminals where syntaxin had been cleaved by botulinum C caused a considerable reduction in SNAP-25 N-terminus immunoreactivity. Hence the retention of immunoreactive SNAP-25 at the neuromuscular junction depends on its interactions with syntaxin and plasma membrane. Persistence of cleaved SNAP-25 in nerve terminals may prevent insertion of new SNAP-25 molecules, thereby contributing to the longevity of botulinum A effects.

Presynaptic protein interactions in vivo: evidence from botulinum A, C, D and E action at frog neuromuscular junction.

The present study examines the paralytic action of botulinum neurotoxins at their natural target, the neuromuscular junction. We asked whether syntaxin, synaptosome-associated protein of 25 kDa (SNAP-25) and vesicle-associated membrane protein (VAMP/synaptobrevin), the proteins proteolysed by botulinum, are susceptible to cleavage in frog nerve terminals, and whether they form complexes in vivo. In control terminals, the three SNAREs were distributed in broad bands at 1 micrometer intervals, at sites consistent with presynaptic Ca2+ channels. Within 3 h, botulinum A, C, D and E (BoNT/A/C/D/E) blocked nerve-evoked muscle contractions but their effects on substrate immunoreactivity varied. The effect of BoNT/A on either C-terminus or N-terminus immunoreactivity of SNAP-25 was undetectable after 3-h incubation, although C-terminus immunoreactivity was reduced after 24 h; N-terminus immunoreactivity was not affected even after 36 h. BoNT/E reduced C-terminus immunoreactivity of SNAP-25 1.5 h after toxin application when transmitter release was blocked, but required 24 h to reduce N-terminus immunoreactivity. BoNT/C reduced syntaxin immunoreactivity after 24-h incubation but did not affect SNAP-25. BoNT/D reduced VAMP immunoreactivity at 3 h while it increased SNAP-25 C-terminal staining fourfold. BoNT/A and BoNT/C applied together for 24 h reduced syntaxin immunoreactivity and that of both C- and N-terminus of SNAP-25, indicating that retention of SNAP-25 N-terminus after cleavage by BoNT/A depended on intact syntaxin. Therefore, we infer that SNAP-25 interacts with VAMP and with syntaxin in vivo. Neurotoxin action abolished only 40-60% of SNAP-25, VAMP or syntaxin immunoreactivity suggesting that distinct pools of these proteins, not immediately involved in triggered exocytosis, are resistant to proteolysis.

Developmental expression of synaptophysin, synapsin I and syntaxin in the rat retina

Expression of synaptophysin, synapsin I and syntaxin was studied immunocytochemically in the developing rat retina using indirect immunoperoxidase technique. In the inner plexiform layer (IPL), syntaxin immunoreactivity appeared at postnatal day 1 (P1) whereas synaptophysin and synapsin I staining were first observed at P2. In the outer plexiform layer (OPL), synaptophysin appeared at P4, while synapsin I and syntaxin appeared at P8. In the case of synaptophysin, a punctate pattern of staining was observed from the time of its appearance (P4) in the OPL and from P12 onwards in the IPL. Synapsin I and syntaxin immunoreactivity in the OPL were of a low intensity throughout the development and in the adult stage. These findings are discussed in relation to synaptogenesis in the rat retina.

Cultured astrocytes express proteins involved in vesicular glutamate release

Bradykinin induces receptor-mediated calcium-dependent release of glutamate from cultured astrocytes through a mechanism that is neither due to cell-swelling mechanism nor due to the reversal of the glutamate transporter. Astrocytes may thus release glutamate using a mechanism resembling the neuronal vesicular release of neurotransmitters. Synaptobrevin is a vesicular protein that together with plasma membrane proteins syntaxin and SNAP-25 participate in formation of the anchoring core complex required for initiation of exocytosis. Here, we demonstrate that synaptobrevin II is present in cultured astrocytes. Furthermore, we demonstrate that botulinus toxin type B and tetanus toxin cause a decrease in synaptobrevin II immunoreactivity and abolish bradykinin-induced release of glutamate from cultured astrocytes. While we were not able to demonstrate the presence of SNAP-25 or syntaxin immunoreactivity in cultured astrocytes, pretreatment with BoTx-A (which cleaves SNAP-25) and BoTx-C (which cleaves syntaxins) result in a decrease in the baseline release of glutamate and diminish the bradykinin-evoked release of glutamate from cultured astrocytes. These findings strongly support the notion that astrocytes may release neurotransmitters using a mechanism similar to the neuronal secretory process.

Cingulate cortex synaptic terminal proteins and neural cell adhesion molecule in schizophrenia

The neuronal organization and patterns of afferent innervation are abnormal in the cingulate cortex in schizophrenia, and associated changes in synaptic terminals could be present. A panel of monoclonal antibodies was defined with biochemical and fusion protein studies as detecting syntaxin (antibody SP6), synaptophysin (antibody SP4) and synaptosomal-associated protein-25 (antibody SP12). These antibodies and a polyclonal antibody reactive with neural cell adhesion molecule were used to investigate the cingulate cortex in schizophrenia. Immunocytochemistry indicated that syntaxin immunoreactivity had a considerably wider distribution than synaptophysin. Overall, multivariate analysis indicated increased synaptic terminal protein immunoreactivity in schizophrenia compared to controls ( P=0.004). Controlled for age and post mortem interval, syntaxin immunoreactivity was significantly elevated in schizophrenia ( P=0.004), and neural cell adhesion molecule immunoreactivity was also elevated ( P=0.05). The neural cell adhesion molecule to synaptophysin ratio was increased ( P=0.005), possibly indicating the presence of less mature synapses in schizophrenia. Elevated syntaxin immunoreactivity is consistent with increased glutamatergic afferents to the cingulate cortex in schizophrenia, and combined with the neural cell adhesion molecule to synaptophysin ratio results suggests that synaptic function in this region in schizophrenia may be abnormal.

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.

Synaptotagmin I, synaptobrevin II, and syntaxin I are coexpressed in rat and gerbil pinealocytes.

Recent studies have shown that mammalian pinealocytes contain a compartment of synaptic-like microvesicles that may serve secretory functions; however, knowledge of the molecular composition of these microvesicles is still incomplete. Therefore, we have analyzed rat and gerbil pineal glands for the presence of synaptotagmin I, synaptobrevin I and II, syntaxin I, and synaptoporin (synaptophysin II) by immunoblot analyses and immunostaining of serial semithin sections. These proteins, which are components of the synaptic vesicle membrane or presynaptic plasmalemma, are thought to be essential for synaptic vesicle trafficking and exocytosis. Antibodies against synaptotagmin I, synaptobrevin II, and syntaxin I label pinealocytes (identified with an antiserum directed against synaptophysin I) in pineal glands of both species, the coexpression of the latter proteins being demonstrable at the single cell level. In contrast, pinealocytes are not or only weakly stained by the synaptoporin antibody. Immunoreactivity for synaptobrevin I is restricted to intrapineal nerve terminals, thus indicating a differential expression of synaptic vesicle protein isoforms within endocrine tissues. Immunogold staining has been performed in the gerbil pineal and reveals that synaptobrevin II and synaptotagmin I can be localized to the synaptic-like microvesicles that are concentrated in pinealocyte process terminals. Syntaxin immunoreactivity is associated with clear microvesicles and with the plasma membrane. Our findings corroborate the hypothesis that the synaptic-like microvesicles of pinealocytes can be considered as the endocrine equivalent of neuronal synaptic vesicles. Since pinealocytes of several mammalian species contain abundant clear microvesicles, the pinealocyte may serve as a paradigm for studies aimed at elucidating the biogenesis and functions of synaptic-like microvesicles in neuroendocrine cells.