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PubMed1999

The synaptophysin-synaptobrevin complex: a hallmark of synaptic vesicle maturation.

Exocytosis of synaptic vesicles requires the formation of a fusion complex consisting of the synaptic vesicle protein synaptobrevin (vesicle-associated membrane protein, or VAMP) and the plasma membrane proteins syntaxin and soluble synaptosomal-associated protein of 25 kDa (or SNAP 25). In search of mechanisms that regulate the assembly of the fusion complex, it was found that synaptobrevin also binds to the vesicle protein synaptophysin and that synaptophysin-bound synaptobrevin cannot enter the fusion complex. Using a combination of immunoprecipitation, cross-linking, and in vitro interaction experiments, we report here that the synaptophysin-synaptobrevin complex is upregulated during neuronal development. In embryonic rat brain, the complex is not detectable, although synaptophysin and synaptobrevin are expressed and are localized to the same nerve terminals and to the same pool of vesicles. In contrast, the ability of synaptobrevin to participate in the fusion complex is detectable as early as embryonic day 14. The binding of synaptoporin, a closely related homolog of synaptophysin, to synaptobrevin changes in a similar manner during development. Recombinant synaptobrevin binds to synaptophysin derived from adult brain extracts but not to that derived from embryonic brain extracts. Furthermore, the soluble cytosol fraction of adult, but not of embryonic, synaptosomes contains a protein that induces synaptophysin-synaptobrevin complex formation in embryonic vesicle fractions. We conclude that complex formation is regulated during development and is mediated by a posttranslational modification of synaptophysin. Furthermore, we propose that the synaptophysin-synaptobrevin complex is not essential for exocytosis but rather provides a reserve pool of synaptobrevin for exocytosis that can be readily recruited during periods of high synaptic activity.

Aging↗
PubMed1993

Calcium-dependent serine phosphorylation of synaptophysin.

The phosphorylation of synaptophysin, a major integral membrane protein of small synaptic vesicles, was found to be regulated in a Ca(2+)-dependent manner in rat cerebrocortical slices, synaptosome preparations, and highly purified synaptic vesicles isolated from rat forebrain. K(+)-induced depolarization of slices and synaptosomes prelabeled with 32P-orthophosphate produced a rapid, transient increase in serine phosphorylation of synaptophysin. In synaptosomes, the depolarization-dependent increase in synaptophysin phosphorylation required the presence of external Ca2+ in the incubation medium. The addition of Ca2+ plus calmodulin to purified synaptic vesicles resulted in a 4-fold increase in serine phosphorylation of synaptophysin, and this phosphorylation was antagonized by a peptide inhibitor of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II(. Purified rat forebrain CaM kinase II phosphorylated both purified synaptophysin and endogenous, vesicle-associated synaptophysin, and the resulting 2-dimensional chymotryptic phosphopeptide maps were similar to those derived from synaptophysin phosphorylated in cerebrocortical slices. These data demonstrate that Ca(2+)-dependent phosphorylation of synaptophysin, mediated by CaM kinase II, occurs under physiological conditions.

Animals↗
PubMed1995

Mice lacking synaptophysin reproduce and form typical synaptic vesicles.

Synaptophysin is one of the major integral membrane proteins of the small (30-50nm diameter) electron-translucent transmitter-containing vesicles in neurons and of similar vesicles in neuroendocrine cells. Since its expression is tightly linked to the occurrence of these vesicle types, we mutated the X-chromosomally located synaptophysin gene in embryonic stem cells for the generation of synaptophysin-deficient mice in order to study the consequence of synaptophysin ablation for the formation and function of such vesicles in vivo. The behavior and appearance of mice lacking synaptophysin was indistinguishable from that of their litter mates and reproductive capacity was comparable to normal mice. Furthermore, no drastic compensatory changes were noted in the expression of several other neuronal polypeptides or in the mRNA levels of synaptophysin isoforms, the closely related neuronal synaptoporin/synaptophysinII, and the ubiquitous pantophysin. Immunofluorescence microscopy of several neuronal and neuroendocrine tissues showed that overall tissue architecture was maintained in the absence of synaptophysin, and that the distribution of other synaptic vesicle components was not visibly affected. In electron-microscopic preparations, large numbers of vesicles with a diameter of 39.9nm and an electron-translucent interior were seen in synaptic regions of synaptophysin-deficient mice; these vesicles could be labeled by antibodies against synaptic vesicle proteins, such as synaptobrevin 2.

Adrenal Medulla↗
PubMed1991

Synaptophysin immunoreactivity in the mammalian endocrine pancreas.

Synaptophysin, a major membrane glycoprotein of small presynaptic vesicles in neurons, has also been found in microvesicles of endocrine cells, e.g., of the endocrine pancreas. In the present study, the endocrine pancreas in 9 mammalian species (man, dog, mink, bovine, rabbit, guinea pig, rat, mouse, gerbil) has been investigated immunohistochemically for synaptophysin immunoreactivity. Synaptophysin-positive cells have been identified and localized on semithin plastic sections. Our study demonstrates that, in all species examined, all pancreatic endocrine cell types are consistently synaptophysin-positive independent of their location within the tissue, or the conditions of tissue processing. In addition, a few cells that cannot be hormonally identified show synaptophysin immunoreactivity. Hence, synaptophysin appears to be a regular constituent of all pancreatic endocrine cells in mammals. In several species, a subpopulation of endocrine cells, consisting of glucagon-containing and/or pancreatic-polypeptide-containing cells, exhibits a significantly higher degree of synaptophysin immunoreactivity. In the gerbil, this heterogeneity can readily be detected from the day of birth onwards. Our findings indicate that closely related endocrine cell types may differ with respect to the content of synaptophysin.

Animals↗
PubMed1992

Identification of an alternatively spliced avian member of the synaptophysin gene family.

Synaptic vesicle membrane proteins are important in the release of neurotransmitters and as markers of presynaptic differentiation in neurons, and the synaptophysins are a major class of synaptic vesicle proteins. By low stringency screening of a chick brain cDNA library with a rat synaptophysin probe, we have isolated cDNAs that encode a novel member of the synaptophysin/synaptoporin family. Two different protein-coding forms of the cDNA were found, apparently generated through alternative splicing of a single gene. The deduced proteins, called synaptophysin IIa and synaptophysin IIb, share 258 amino acids (starting from position 10 in IIa and position 30 in IIb), that are most closely related to the rat synaptoporin sequence. The N-terminal sequence of IIa is similar to that of rat synaptoporin, and the N-terminal sequence of IIb is similar to that of rat synaptophysin. Northern blot analysis and nuclease protection experiments demonstrate that IIa and IIb are expressed in a variety of brain regions, the spinal cord, and dorsal root ganglia, but not in non-neuronal tissues. Further, the two splice variants are differentially distributed. In most brain regions the IIb form predominates, and the cerebellum appears to express only the IIb form, but the IIa form is relatively elevated in peripheral neurons. Western blot analysis with an antibody to a synthetic peptide common to both forms demonstrates the expression of synaptophysin II as a 39 kDa protein, apparently distinct from synaptophysin (40 kDa). The results suggest that the regulation and function of the synaptophysin gene family is more complex than had been appreciated.

Amino Acid Sequence↗
PubMed1999

Synaptophysin: A novel marker for human and rat hepatic stellate cells.

Synaptophysin is a protein involved in neurotransmitter exocytosis and is a neuroendocrine marker. We studied synaptophysin immunohistochemical expression in 35 human liver specimens (normal and different pathological conditions), in rat models of galactosamine hepatitis and carbon tetrachloride-induced cirrhosis, and in freshly isolated rat stellate cells. Synaptophysin reactivity was present in perisinusoidal stellate cells in both human and rat normal liver biopsies. The number of synaptophysin-reactive perisinusoidal cells increased in pathological conditions. Double staining for alpha-smooth muscle actin and synaptophysin, detected by confocal laser scanning microscopy, unequivocally demonstrated colocalization of both markers in lobular stellate cells. In addition, freshly isolated rat stellate cells expressed synaptophysin mRNA (detected by polymerase chain reaction) and protein. Finally, electron microscopy showed the presence of small electron translucent vesicles, comparable to the synaptophysin-reactive synaptic vesicles in neurons, in stellate cell projections. We conclude that synaptophysin is a novel marker for quiescent as well as activated hepatic stellate cells. Together with the stellate cell's expression of neural cell adhesion molecule, glial fibrillary acidic protein, and nestin, this finding raises questions about its embryonic origin and its differentiation. In addition, the presence of synaptic vesicles in stellate cell processes suggests a hitherto unknown mechanism of interaction with neighboring cells.

Animals↗
PubMed2000

Normal cellular levels of synaptophysin mRNA expression in the prefrontal cortex of subjects with schizophrenia.

BACKGROUND: Previous studies have reported that the 38-kd synaptic vesicle-associated protein, synaptophysin, is decreased in the prefrontal cortex of subjects with schizophrenia. METHODS: To determine whether the decreased protein levels reflect diminished expression of the synaptophysin gene by prefrontal cortex neurons, we used in situ hybridization histochemistry to determine the cellular levels of synaptophysin messenger RNA in prefrontal cortex area 9 from 10 matched pairs of schizophrenic and normal control subjects. RESULTS: Neither the density of neurons with detectable levels of synaptophysin messenger RNA nor the mean level of synaptophysin messenger RNA expression per neuron differed between schizophrenic and control subjects in any cortical layer. CONCLUSIONS: These findings indicate that the expression of synaptophysin messenger RNA is not altered in this brain region in schizophrenia. Consequently, reduced levels of synaptophysin protein in the prefrontal cortex of subjects with schizophrenia are more likely to reflect either posttranscriptional abnormalities of synaptophysin in prefrontal cortex neurons or a diminished number of axonal projections to the prefrontal cortex from other brain regions.

Adult↗
PubMed2001

Synaptic vesicle alterations in rod photoreceptors of synaptophysin-deficient mice.

The abundance of the integral membrane protein synaptophysin in synaptic vesicles and its multiple possible functional contributions to transmitter exocytosis and synaptic vesicle formation stand in sharp contrast to the observed lack of defects in synaptophysin knockout mice. Assuming that deficiencies are compensated by the often coexpressed synaptophysin isoform synaptoporin, we now show that retinal rod photoreceptors, which do not synthesize synaptoporin either in wild-type or in knockout mice, are affected by the loss of synaptophysin. Multiple pale-appearing photoreceptors, as seen by electron microscopy, possess reduced cytoplasmic electron density, swollen mitochondria, an enlarged cell surface area, and, most importantly, a significantly reduced number of synaptic vesicles with an unusually bright interior. Quantification of the number of synaptic vesicles per unit area, not only in these, but also in all other rod terminals of knockout animals, reveals a considerable reduction in vesicles that is even more pronounced during the dark period, i.e., at times of highest synaptic activity. Moreover, activity-dependent reduction in synaptic vesicle diameter, typically occurring in wild-type mice, is not detected in knockout animals. The large number of clathrin-coated pits and vesicles in dark-adapted synaptophysin knockout mice is taken as an indication of compensatory usage of synaptophysin-independent pathway(s), and, conversely, in view of the overall reduction in the number of synaptic vesicles, as an indication for the presence of another synaptophysin-dependent synaptic vesicle recycling pathway. Our results provide in vivo evidence for the importance of the integral membrane protein synaptophysin for synaptic vesicle recycling and formation.

Action Potentials↗
PubMed1987

Synaptophysin expressed in the bronchopulmonary tract: neuroendocrine cells, neuroepithelial bodies, and neuroendocrine neoplasms.

Synaptophysin is an integral membrane glycoprotein with an Mr of 38,000 that occurs in the small, clear vesicles present in neuronal cells and tumors as well as in pancreatic islet cells and various neuroendocrine (NE) carcinomas. We found that synaptophysin is also expressed in normal NE cells of the lungs of newborn rabbits and mice as well as of human fetuses. In bronchial ganglion cells and in nerves, synaptophysin is coexpressed with neurofilament proteins (NFPs), whereas in solitary NE cells and in at least some of the neuroepithelial bodies (NEBs) of the bronchial mucosal lining, synaptophysin coexists with cytokeratins. We also studied a series of NE neoplasms of the lung covering the entire spectrum of differentiation (i.e., from carcinoids to small-cell NE carcinomas), and found that synpatophysin was present in the majority of them. In these tumors, synaptophysin was invariably coexpressed with cytokeratin filaments and desmoplakin, as well as, occasionally, with NFP. Synaptophysin was identified throughout, the whole range of these NE neoplasms, i.e., from benign to low-grade to aggressive and rapidly metastasizing carcinomas; its presence was unaffected by the highly variable expression of serotonin and/or neuropeptides in these neoplasms, and was unrelated to the presence or absence of associated endocrine syndromes. Our findings indicate that synaptophysin occurs in the neural as well as in the epithelial components of the dispered NE system of the lung as well as in the majority of NE neoplasms of this organ, and that the expression of this protein is therefore independent of the cytoskeletal characteristics and other differentiation features of both normal and transformed NE cells of the lung. We emphasize the value of synaptophysin as an immunocytochemical marker of NE differentiation.

Animals↗
PubMed1988

In adrenal medulla synaptophysin (protein p38) is present in chromaffin granules and in a special vesicle population.

We have analyzed the properties and subcellular localization of synaptophysin (protein p38) in bovine adrenal medulla. In one-dimensional immunoblotting the adrenal antigen appears identical to synaptophysin of rat synaptic vesicles. In two-dimensional immunoblotting it migrates as a heterogeneous band varying in pI from 4.5 to 5.8. Subcellular fractionation by various sucrose gradients revealed that synaptophysin was present in two different cell particles. More than half of the antigens present in adrenal medulla were confined to special membranes that sedimented both with the "large granules" and with microsomal elements. These membranes could be removed from the large granule sediment by washing. In gradients it equilibrated in regions of low sucrose density. These membranes did not contain any markers for chromaffin granules. Less than half of the amount of synaptophysin present in adrenal medulla copurified with chromaffin granules. Despite several variations in the fractionation scheme synaptophysin could not be removed from chromaffin granules. After washing of granule membranes with alkaline solution synaptophysin still cosedimented in gradients with typical granule markers. The concentration of synaptophysin in membranes of chromaffin granules is low (less than 10%) when compared with synaptic vesicles. It is concluded that in adrenal medulla synaptophysin is present in special membranes, probably in high concentration, and in membranes of chromaffin granules, either in a low concentration in all or in a higher concentration in some of them.

Adrenal Medulla↗
PubMed1993

Neuronotypic differentiation results in reduced levels and altered distribution of synaptophysin in PC12 cells.

Several synaptic vesicle proteins including synaptophysin and p65/synaptotagmin are expressed by the pheochromocytoma cell line PC12. Stimulation of these cells with nerve growth factor for 7 days induces morphologic neuronotypic differentiation, but the levels of synaptophysin are markedly reduced. Stimulation with cyclic AMP analogs also produces neuronotypic differentiation of PC12 cells, and the degree of morphologic differentiation induced by these agents parallels their ability to effect reduction in synaptophysin levels. By contrast, levels of p65/synaptotagmin are increased following neuronotypic differentiation. The contrasting effects of neuronotypic differentiation on levels of synaptophysin and p65/synaptotagmin indicate potential differences in the regulation of these proteins in PC12 cells. Immunocytochemical labeling of undifferentiated PC12 cells reveals concentrations of synaptophysin in the perinuclear region. After neuronotypic differentiation, there is reduction in perinuclear labeling and concentration of label in swellings along PC12 cell processes. At the ultrastructural level, synaptophysin labeling is found on similar organelles in both undifferentiated and nerve growth factor-stimulated PC12 cells. Although the highest labeling densities were seen on small clear vesicles, specific labeling was also seen on dense core vesicles. The presence of synaptophysin on both small clear vesicles and dense core vesicles indicates potential functional similarities in these vesicle types. The changes in the levels and immunocytochemical distribution of synaptophysin after neuronotypic differentiation suggest possible functional heterogeneity among morphologically similar populations of small clear vesicles.

Animals↗
PubMed2000

Effect of a memory-enhancing drug, AIT-082, on the level of synaptophysin.

Our objective is to study the effect of AIT-082 on the level of synaptophysin in cultured pheochromocytoma (PC12) cells. The drug AIT-082, a unique purine hypoxanthine derivative, is under development for the treatment of Alzheimer's disease (AD). We analyzed synaptophysin protein as an index of synaptic numbers and density and indirectly neuronal transmission. PC12 cells were treated with nerve growth factor (NGF) (50 ng/ml) and/or different doses of AIT-082 (5-50 ng/ml) obtained from NeoTherapeutics, CA. In the western immunoblots of conditioned media and cell lysates, we detected synaptophysin as 36-40 kDa protein bands. When PC12 cells were treated with NGF and samples were analyzed at 24 or 48 hours after treatment, the secretion of synaptophysin was drastically reduced in the conditioned medium. A significant reduction in the intracellular levels of synaptophysin in NGF-treated samples was also noted. By contrast, when PC12 cells were treated with AIT-082, the secretion of synaptophysin was increased in the conditioned medium as compared to the control. There was also a significant increase in the intracellular levels of synaptophysin in AIT-082-treated cultures. NGF treatment resulted in sympathetic neuronal phenotypes in PC12 cells. As it is known that the immunoreactivity of the synaptophysin protein correlates with the density of the synaptic terminal, our results suggest that treatment by AIT-082 could enhance neurotransmitter release at the presynaptic terminal, which may play a role in the improvement of cognition seen in AD subjects.

Adrenal Gland Neoplasms↗
PubMed1987

Synaptophysin expression in neuroendocrine neoplasms as determined by immunocytochemistry.

Synaptophysin is an integral membrane glycoprotein originally isolated from presynaptic vesicles of bovine neurons. The authors have studied a wide spectrum of neuroendocrine (NE) neoplasms by immunofluorescence microscopy on cryostat sections of freshly frozen tissues using a monoclonal antibody to this protein (SY 38). Without exception, they found the identical--or a very similar--protein expressed in all neuroblastomas, ganglioneuroblastomas, ganglioneuromas, pheochromocytomas, and paragangliomas studied. In these "neural" type NE neoplasms, synaptophysin was coexpressed with neurofilament proteins. Synaptophysin was also demonstrated in NE neoplasms of "epithelial" type in which it was predominantly coexpressed with cytokeratins and desmoplakin. It was invariably found in all variants of islet cell neoplasms and in all medullary thyroid carcinomas. Synaptophysin was also demonstrated in several adenomas of the hypophysis and parathyroids, in the majority of carcinoids of the bronchopulmonary and gastrointestinal tracts, and in many, though not all, NE carcinomas of the same sites, and of the skin. Conversely, SY 38 did not immunostain any of a large number of benign and malignant non-NE epithelial neoplasms; nor was any immunostaining obtained in a group of mesenchymal tumors. It is remarkable that SY 38 did not immunostain a number of malignant melanomas, including several that were immunostained for neuron-specific enolase (NSE) and several neuropeptides. Parallel studies conducted on conventionally fixed, paraffin-embedded tissue sections immunostained by the use of the avidin-biotin complex technique yielded very similar results. The findings indicate that synaptophysin is expressed in the whole range of NE neoplasms without detectable relation to the expression of other NE markers such as NSE, serotonin, and neuropeptides. Nor could the expression of synaptophysin by these tumors be correlated with their epithelial and/or neural cytoskeletal characteristics, their clinical aggressiveness, or the presence or absence of endocrinologic abnormalities. While the consistent expression of synaptophysin by the "neural" type of NE neoplasms would seem predictable its presence in diverse benign and malignant NE tumors of "epithelial" type is remarkable. It is concluded that synaptophysin is a significant as well as novel NE marker, and the use of antibody SY 38 as a broad range marker for the study and diagnosis of NE neoplasms is proposed.

Endocrine System Diseases↗
PubMed1997

Reduction of synaptophysin immunoreactivity in the prefrontal cortex of subjects with schizophrenia. Regional and diagnostic specificity.

BACKGROUND: Multiple lines of evidence indicate that the prefrontal cortex is a site of dysfunction in schizophrenia. However, the apparent absence of gross structural abnormalities in this area suggests that the pathophysiological characteristics of schizophrenia may involve more subtle disturbances in prefrontal cortical circuitry, such as alterations in synaptic connectivity and transmission. In this study, immunoreactivity for synaptophysin, an integral membrane protein of small synaptic vesicles, was used to assess the integrity of cortical synaptic circuitry in schizophrenia. METHODS: Using immunocytochemical techniques and adjusted optical density measurements, we examined synaptophysin immunoreactivity in prefrontal cortical areas 9 and 46 and in area 17 (the primary visual cortex) from 10 pairs of case subjects with schizophrenia and control subjects. matched on a pairwise basis for age, sex, race, and postmortem interval, and in 5 matched pairs of nonschizophrenic psychiatric case subjects and normal control subjects. RESULTS: Compared with levels found in matched control subjects, synaptophysin immunoreactivity in areas 46 and 9 was significantly decreased (P < .001 and P < .008, respectively) across all cortical layers in the case subjects with schizophrenia. In contrast, no differences were observed in area 17. In addition, levels of synaptophysin immunoreactivity in areas 46, 9, and 17 did not differ between 5 nonschizophrenic psychiatric case subjects and their matched controls, suggesting that decreased synaptophysin levels in the prefrontal cortex of patients with schizophrenia may be specific to that disorder. CONCLUSION: Additional studies are required to determine if the decrease in levels of synaptophysin immunoreactivity is caused by a decrease in the number or size of presynaptic terminals, a decrease in the number of synaptic vesicle per terminal, or a decrease in the expression of synaptophysin. However, all of these potential explanations are consistent with a disturbance in synaptic transmission in the prefrontal cortex of patients with schizophrenia.

Adult↗
PubMed1997

Reduction of synaptophysin immunoreactivity in the prefrontal cortex of subjects with schizophrenia. Regional and diagnostic specificity.

BACKGROUND: Multiple lines of evidence indicate that the prefrontal cortex is a site of dysfunction in schizophrenia. However, the apparent absence of gross structural abnormalities in this area suggests that the pathophysiological characteristics of schizophrenia may involve more subtle disturbances in prefrontal cortical circuitry, such as alterations in synaptic connectivity and transmission. In this study, immunoreactivity for synaptophysin, an integral membrane protein of small synaptic vesicles, was used to assess the integrity of cortical synaptic circuitry in schizophrenia. METHODS: Using immunocytochemical techniques and adjusted optical density measurements, we examined synaptophysin immunoreactivity in prefrontal cortical areas 9 and 46 and in area 17 (the primary visual cortex) from 10 pairs of case subjects with schizophrenia and control subjects matched on a pairwise basis for age, sex, race, and postmortem interval, and in 5 matched pairs of nonschizophrenic psychiatric case subjects and normal control subjects. RESULTS: Compared with levels found in matched control subjects, synaptophysin immunoreactivity in areas 46 and 9 was significantly decreased (P < .001 and P < .008, respectively) across all cortical layers in the case subjects with schizophrenia. In contrast, no differences were observed in area 17. In addition, levels of synaptophysin immunoreactivity in areas 46, 9, and 17 did not differ between 5 nonschizophrenic psychiatric case subjects and their matched controls, suggesting that decreased synaptophysin levels in the prefrontal cortex of patients with schizophrenia may be specific to that disorder. CONCLUSIONS: Additional studies are required to determine if the decrease in levels of synaptophysin immunoreactivity is caused by a decrease in the number or size of presynaptic terminals, a decrease in the number of synaptic vesicles per terminal, or a decrease in the expression of synaptophysin. However, all of these potential explanations are consistent with a disturbance in synaptic transmission in the prefrontal cortex of patients with schizophrenia.

Adult↗
PubMed1999

Patterns of synaptophysin expression during development of the inner ear in the chick.

The onset of active neural connections between the periphery and the central nervous system is integral to the development of sensory systems. This study presents patterns of synaptogenesis in the chick basilar papilla (i.e., cochlea) by examining the immunohistochemical expression of synaptophysin with a specific monoclonal antibody, SBI 20.10. The initial onset of synaptophysin expression occurs in nerve fibers and ganglion cell bodies at a time when neurites reach the basement membrane of the chick cochlea on embryonic day 6-7 (ED 6-7). By ED 8, synaptophysin positive fibers invade the neural side of the entire length of the cochlea, so that by ED 9-10, fibers are forming multiple terminals on the basolateral ends of retracting receptor or hair cells. In contrast, on the abneural side, immunoreactive terminals are seen first as small, punctate contacts and then as large, synaptophysin positive calyceal endings beneath short hair cells. These terminals are sparse during early development, more numerous by ED 17-19, but still incomplete after 2 weeks posthatching. In comparison, hair cells show synaptophysin immunoreactivity in both supra- and infranuclear regions by ED 11-12, a time when efferent innervation is incomplete. Thus, during development, synaptophysin is expressed at both synaptic and nonsynaptic sites, is relatively selective in its regional distribution, and is expressed in hair cells at a time when auditory function begins. Our results present a framework with which to understand the potential role of synaptophysin in early synaptogenesis of the cochlea.

Animals↗
PubMed1993

Synaptophysin immunohistochemistry reveals inside-out pattern of early synaptogenesis in ferret cerebral cortex.

Synaptogenesis in the ferret cerebral cortex was examined from the day of birth to adulthood with an antibody against synaptophysin at the light and electron microscopic levels. Due to the premature birth of ferrets, the generation of cells destined to the upper cortical layers and their subsequent migration to their final positions in the cortical plate are largely postnatal events. Throughout the newborn ferret cerebral cortex, a high amount of synaptophysin immunoreactivity was present within the marginal zone and subplate region. Staining was also conspicuous within the forming cortical plate. The typical layering pattern of synaptophysin immunoreactivity in the developing cortical plate correlated with the migration pattern of cortical neurons. The synaptic density was lowest directly below the marginal zone, where the youngest neurons just stopped their migration. Below this zone, the density of the synaptic staining increased gradually toward lower (and older) cortical plate layers. As the cortex expanded, the synaptophysin immunoreactivity pattern closely followed the expansion, suggesting that synapses were formed in a given layer shortly after the cells migrating to this layer reached their final position. As soon as cell migration had finished, the entire cortical plate contained dense synaptophysin immunoreactivity, in a pattern similar to that observed in the adult animal. During cortical development, a rostrocaudal and a laterodorsal gradient of synaptogenesis was observed. At any given time, rostral and lateral regions of the cerebral cortex were more advanced in their development than caudal and dorsal regions. Electron microscopic examination of synaptophysin immunoreactivity in the developing cerebral cortex of ferrets confirmed that labeling was solely associated with synaptic vesicles. These vesicles were typically, but not exclusively, confined to synaptic boutons. Especially around the end of the first postnatal week, long fiber profiles loaded with synaptic vesicles were occasionally detected. As some of these fibers also showed en passant synapses along their course, we concluded that synaptic vesicle labeling may be reliably used to study synaptogenesis at the light microscopic level. A systematic analysis of samples from postnatal days 0 and 7 corroborated this conclusion, showing that synaptic profile distribution completely matched the distribution of synaptophysin immunoreactivity seen in the light microscope. In conclusion, synaptogenesis begins as soon as migratory cells reach their final position in the cortical plate. As long as cell migration continues, synaptogenesis is under the constraints of neurogenesis, following its gradients.

Aging↗
PubMed1986

Molecular characterization of synaptophysin, a major calcium-binding protein of the synaptic vesicle membrane.

Synaptophysin, a mol. wt 38 000 glycopolypeptide of the synaptic vesicle membrane, was solubilized using Triton X-100 and purified by immunoaffinity or ion-exchange chromatography. From gel permeation and sucrose-density centrifugation in H2O/D2O, a Stokes radius of 7.3 nm, a partial specific volume of 0.830 and a total mol. wt of 119 000 were calculated for the native protein. Cross-linking of synaptic vesicles with glutaraldehyde, dimethylsuberimidate, or Cu2+ -o-phenantroline, resulted in the formation of a mol. wt 76 kd dimer of synaptophysin. Crosslinking of the purified protein in addition produced tri- and tetrameric adducts of the polypeptide. Native synaptophysin thus is a homooligomeric protein. Synaptophysin is N-glycosylated, since cultivation of the rat phaeochromocytoma cell line PC12 in the presence of tunicamycin reduced its mol. wt by about 6 kd. Upon transfer to nitrocellulose and incubation with 45Ca2+, synaptophysin behaved as one of the major calcium-binding proteins of the synaptic vesicle membrane. Pronase treatment of intact synaptic vesicles abolished this 45Ca2+ binding indicating that the Ca2+ binding site of synaptophysin must reside on a cytoplasmic domain of the transmembrane polypeptide. Based on these data, we propose that synaptophysin may play an important role in Ca2+-dependent neurotransmitter release.

Animals↗