Expression Of Synaptic Vesicle Proteins Research Articles

Evaluation of the Efficacy of Human Dental Pulp Stem Cell Transplantation in Sprague-Dawley Rats with Sensorial Neural Hearing Loss.

The purpose of the present study was to evaluate the efficacy of spiral ganglion neuron (SGN) regeneration after dental pulp stem cell (DPSC) transplantation in a rat sensorineural hearing loss (HL) model. Sham or experimental HL was induced in adult Sprague-Dawley rats by cochlear round window surgery. An HL rat model was established with a single 10 mM ouabain intratympanic injection. After 7 days, the rats received DPSCs, stem cells from human exfoliated deciduous teeth (SHED), or culture medium in the sutural area to establish four groups: sham, HL-DPSC, HL-SHED, and HL-medium. Histological analyses were performed at 4, 7, and 10 weeks after transplantation, and the number of SGNs, specific SGN protein expression, and the function of SGNs were evaluated. Data were statistically by MS Excel and SPSS v.15.0. Intergroup level of significance was determined via a one-way analysis of variance and Duncan's multiple range test with 95% confidence intervals. New SGN formation was observed in the HL-DPSC and HL-SHED rat groups. The number of SGNs was significantly higher in the HL-DPSC and HL-SHED groups than in the HL-medium group over 4 to 10-week survival period. HL-DPSC rats exhibited higher SGN density compared with that in HL-SHED group, which was statistically significant at week 10. The regenerated SGNs expressed cochlear wiring regulator GATA-binding-protein 3. Moreover, the SGNs from the HL-DPSC group also exhibited a higher expression of synaptic vesicle protein and regulated action potential-dependent neurotransmitter release compared with SGNs from the HL-SHED group. Our findings suggest that DPSCs and SHED repair and regenerate SGNs in rat HL model. Dental pulp stem cells represent a promising treatment strategy for restoring damage to the sensory circuits associated with deafness.

CRHR1 Mediates the Up-Regulation of Synapsin I Induced by Nesfatin-1 Through ERK 1/2 Signaling in SH-SY5Y Cells.

The anorexigenic molecule nesfatin-1 has recently been taken as a potential mood regulator, but the potential mechanisms remain unknown. Results of our previous study have demonstrated that nesfatin-1 could induce anxiety- and depression-like behaviors in rats, accompanied by the hyperactivity of the hypothalamic-pituitary-adrenal axis and the imbalanced mRNA expression of synaptic vesicle proteins. To explore the potential neurobiological mechanism underlying the effect of nesfatin-1 on the synaptic plasticity, the human neuroblastoma SH-SY5Y cells were cultured and treated with different concentrations of nesfatin-1 in the present study. The mRNA and protein expressions of corticotropin-releasing hormone (CRH) were measured via real-time fluorescent quantitative PCR and western blot, respectively. The protein expressions of extracellular signal-regulated kinase 1/2 (ERK1/2), phosphorylated-ERK1/2 (p-ERK1/2), and synapsin I were detected via western blot. The results confirmed that nesfatin-1 (10-9~10-7 mol/L) could up-regulate the expression of CRH. Moreover, nesfatin-1 (10-9~10-7 mol/L) could also increase the protein expressions of p-ERK1/2 and synapsin I, and these effects could be blocked by CP376395, a selective antagonist of CRH type 1 receptor (CRHR1). Furthermore, the increased expression of synapsin I induced by nesfatin-1 could also be reversed by PD98059, a specific inhibitor of the p-ERK. These results indicated that CRHR1 might mediate the effect of nesfatin-1 on the expressions of synapsin I via ERK1/2 signaling pathway.

A novel non-canonical Notch signaling regulates expression of synaptic vesicle proteins in excitatory neurons.

Notch signaling plays crucial roles for cellular differentiation during development through γ-secretase-dependent intramembrane proteolysis followed by transcription of target genes. Although recent studies implicate that Notch regulates synaptic plasticity or cognitive performance, the molecular mechanism how Notch works in mature neurons remains uncertain. Here we demonstrate that a novel Notch signaling is involved in expression of synaptic proteins in postmitotic neurons. Levels of several synaptic vesicle proteins including synaptophysin 1 and VGLUT1 were increased when neurons were cocultured with Notch ligands-expressing NIH3T3 cells. Neuron-specific deletion of Notch genes decreased these proteins, suggesting that Notch signaling maintains the expression of synaptic vesicle proteins in a cell-autonomous manner. Unexpectedly, cGMP-dependent protein kinase (PKG) inhibitor, but not γ-secretase inhibitor, abolished the elevation of synaptic vesicle proteins, suggesting that generation of Notch intracellular domain is dispensable for this function. These data uncover a ligand-dependent, but γ-secretase-independent, non-canonical Notch signaling involved in presynaptic protein expression in postmitotic neurons.

Depression-like Behavior Induced by Nesfatin-1 in Rats: Involvement of Increased Immune Activation and Imbalance of Synaptic Vesicle Proteins.

Depression is a multicausal disorder and has been associated with metabolism regulation and immuno-inflammatory reaction. The anorectic molecule nesfatin-1 has recently been characterized as a potential mood regulator, but its precise effect on depression and the possible mechanisms remain unknown, especially when given peripherally. In the present study, nesfatin-1 was intraperitoneally injected to the rats and the depression-like behavior and activity of the hypothalamic-pituitary-adrenal (HPA) axis were evaluated. The plasma concentrations of nesfatin-1, interleukin 6 (IL-6), and C-reactive protein (CRP); and the hypothalamic expression levels of nesfatin-1, synapsin I, and synaptotagmin I mRNA were evaluated in nesfatin-1 chronically treated rats. The results showed that both acute and chronic administration of nesfatin-1 increased immobility in the forced swimming test (FST), and resulted in the hyperactivity of HPA axis, as indicated by the increase of plasma corticosterone concentration and hypothalamic expression of corticotropin-releasing hormone (CRH) mRNA. Moreover, after chronic nesfatin-1 administration, the rats exhibited decreased activity and exploratory behavior in the open field test (OFT) and increased mRNA expression of synapsin I and synaptotagmin I in the hypothalamus. Furthermore, chronic administration of nesfatin-1 elevated plasma concentrations of IL-6 and CRP, which were positively correlated with despair behavior, plasma corticosterone level, and the hypothalamic mRNA expression of synapsin I and synaptotagmin I. These results indicated that exogenous nesfatin-1 could induce the immune-inflammatory activation, which might be a central hug linking the depression-like behavior and the imbalanced mRNA expression of synaptic vesicle proteins in the hypothalamus.

Visualizing presynaptic function

Synaptic communication in the nervous system is initiated by the fusion of synaptic vesicles with the presynaptic plasma membrane and subsequent neurotransmitter release. In the 1980s, this process was characterized by electron microscopy, albeit without the ability to follow processes in living cells. In the last two decades, fluorescence imaging methods have been developed that report synaptic vesicle fusion, endocytosis and recycling. These probes have provided unprecedented insight into synaptic vesicle trafficking in individual synaptic terminals and revealed heterogeneity in recycling pathways as well as synaptic vesicle populations. These methods either take advantage of uptake of fluorescent probes into recycling vesicles or exogenous expression of synaptic vesicle proteins tagged with a pH-sensitive fluorescent marker at regions facing the vesicle lumen. We provide an overview of these methods, with particular emphasis on the challenges associated with their use and the opportunities for future investigations.

Decreased expression of synaptic proteins in a mouse model of posttraumatic stress disorder

Posttraumatic stress disorder (PTSD) is a common and debilitating psychiatric disease. Although hippocampal atrophy and several other factors have already been found to be associated with PTSD, the pathomechanisms underlying this anxiety disorder still remain elusive. Clinical fMRI studies and the fact that the hippocampus is a known player in fear extinction processes fuelled the assumption that alterations of hippocampal function most likely play a central role in PTSD pathogenesis. Aiming at identifying pathomechanisms and potential PTSD biomarkers, we compared the expression levels of several synaptic, glial and stress-associated proteins in hippocampal tissue of either traumatized or unstressed control mice. In traumatized mice, we found a decreased protein expression of hippocampal synaptic vesicle proteins while neuronal marker proteins remained unchanged, indicating a synapse specific effect and excluding the possibility of an overall reduction of neuronal cell number. Reduced expression of synaptic vesicle proteins in posttraumatic stress disorder could implicate impaired synaptic transmission either by depletion of neurotransmitter due to reduced vesicle number or by synapse retraction.

SV2 Acts via Presynaptic Calcium to Regulate Neurotransmitter Release

Synaptic vesicle 2 (SV2) proteins, critical for proper nervous system function, are implicated in human epilepsy, yet little is known about their function. We demonstrate, using direct approaches, that loss of the major SV2 isoform in a central nervous system nerve terminal is associated with an elevation in both resting and evoked presynaptic Ca(2+) signals. This increase is essential for the expression of the SV2B(-/-) secretory phenotype, characterized by changes in synaptic vesicle dynamics, synaptic plasticity, and synaptic strength. Short-term reproduction of the Ca(2+) phenotype in wild-type nerve terminals reproduces almost all aspects of the SV2B(-/-) secretory phenotype, while rescue of the Ca(2+) phenotype in SV2B(-/-) neurons relieves every facet of the SV2B(-/-) secretory phenotype. Thus, SV2 controls key aspects of synaptic functionality via its ability to regulate presynaptic Ca(2+), suggesting a potential new target for therapeutic intervention in the treatment of epilepsy.

The effects of GABA transporter inhibition on synaptophysin and synaptotagmin expression in diazepam tolerance

Background. In light of the differential interactions seen between benzodiazepine, GABA transporter (GAT) inhibition and drug tolerance, the locomotor effects of a GAT1-specific inhibitor (SKF89976A) following diazepam tolerance were analysed and compared with the concomitant expression of synaptic vesicle proteins implicated in synaptic plasticity. Methods. Male PVG/OlaHsd rats were chronically dosed with diazepam to produce tolerance, and the expression of mRNA for synaptophysin and synaptotagmin were analysed in the hippocampus by means of in situ hybridisation. The action of the GAT inhibitor SKF89976A on the expression of these mRNAs, and on open field behaviour was subsequently observed. Results. The results show an unexpected sedative effect of GAT-inhibition in diazepam-tolerant rats. The expression data show a significant effect of diazepam treatment on synaptophysin expression, which is reversible by SKF89976A treatment. Conclusions. The increased synaptophysin expression in the hippocampus of diazepam-tolerant rats may indicate a role for modulation of transmitter release, synaptic plasticity and learning in pharmacological tolerance. The reversibility of this effect following acute GAT inhibition suggests a complicated relationship between the benzodiazepine-binding site and other synaptic GABA-binding sites. Furthermore, the sedative behavioural effect of the GAT inhibitor in diazepam-tolerant rats is an unusual observation with implications for the treatment of drug-tolerant individuals.

Male and female stress in serotonin transporter knockout mice

Serotonin (5-HT) is an important modulator of many physiological, behavioural and developmental processes and it plays an important role in stress coping reactions. Anxiety disorders and depression are stress-related disorders and they are associated with malfunction of the serotonin system, in which the serotonin transporter (5-HTT) plays an important role. 5-Htt knockout (KO) mice represent an artificially hyperserotonergic environment, show an increased anxiety-like behaviour and seem to be a good model to investigate the role of the serotonin system concerning stress reactions and anxiety disorders. As synaptic vesicle proteins (SVPs) seem to be involved in stress reactions we studied the effect of acute immobilization stress on SVP expression in the 5-Htt KO mouse model as well as the expression of the two immediate early genes (IEGs) FBJ osteosarcoma oncogene (c-Fos) and fos-like antigen 2 (Fra-2). Based on gender- and genotype-dependent differences in corticosterone levels we looked for expression differences in the brain by performing a quantitative real time-PCR study using primer pairs specific for the SVPs Synaptotagmin (Syt) I, Syt IV and Syntaxin (Stx) 1a and for the IEGs c-Fos and Fra-2 in five different brain regions in 5-Htt KO and wildtype mice. We found mainly gender-dependent differences but less stress effects on the expression of SVPs. Regarding the expression of IEGs we found stress-, gender- and genotype-dependent differences mainly in hypothalamus.

The effects of GABA transporter inhibition on synaptophysin and synaptotagmin expression in diazepam tolerance

In light of the differential interactions seen between benzodiazepine, GABA transporter (GAT) inhibition and drug tolerance, the locomotor effects of a GAT1-specific inhibitor (SKF89976A) following diazepam tolerance were analysed and compared with the concomitant expression of synaptic vesicle proteins implicated in synaptic plasticity. Male PVG/OlaHsd rats were chronically dosed with diazepam to produce tolerance, and the expression of mRNA for synaptophysin and synaptotagmin were analysed in the hippocampus by means of in situ hybridisation. The action of the GAT inhibitor SKF89976A on the expression of these mRNAs, and on open field behaviour was subsequently observed. The results show an unexpected sedative effect of GAT-inhibition in diazepam-tolerant rats. The expression data show a significant effect of diazepam treatment on synaptophysin expression, which is reversible by SKF89976A treatment. The increased synaptophysin expression in the hippocampus of diazepam-tolerant rats may indicate a role for modulation of transmitter release, synaptic plasticity and learning in pharmacological tolerance. The reversibility of this effect following acute GAT inhibition suggests a complicated relationship between the benzodiazepine-binding site and other synaptic GABA-binding sites. Furthermore, the sedative behavioural effect of the GAT inhibitor in diazepam-tolerant rats is an unusual observation with implications for the treatment of drug-tolerant individuals.

Differential expression of synaptic vesicle proteins after repeated electroconvulsive seizures in rat frontal cortex and hippocampus

Electroconvulsive therapy (ECT) remains the treatment of choice for patients with severe or drug-resistant depressive disorders, yet the mechanism behind its efficacy and the effect on neurotransmission is essentially unknown. As synaptic vesicle proteins (SVPs) are required for vesicle fusion and neurotransmitter release, we have examined the effect of single and repeated electroconvulsive seizures (ECS), an animal model of ECT, on the expression of 14 SVPs in the rat frontal cortex and the hippocampus using quantitative real-time polymerase chain reaction (real-time qPCR). Only in the frontal cortex, the mRNA level of synapsin II was significantly upregulated after repeated ECS. In contrast, the mRNA levels of 6 of the 14 SVPs were significantly regulated in the hippocampus after ECS. We found that SNAP29 was upregulated and synaptotagmin III was downregulated after one single ECS in the hippocampus. Furthermore, SNAP29, synapsin I, synapsin III, VAMP2, and VAMP5 were significantly upregulated, whereas synaptotagmin III was significantly downregulated after repeated ECS in the hippocampus. We suggest that these genes are highly important in the long-term therapeutic effect of ECS, and thus it can be hypothesized that the SVPs are involved in the pathophysiology of depression.

The effect of methylphenidate treatment on the expression of synaptic vesicle proteins in a mouse model for Attention-Deficit/Hyperactivity Disorder

Mice with a genetic deletion of the dopamine transporter (DAT) demonstrate several key features of Attention-Deficit/Hyperactivity Disorder (ADHD) such as hyperactivity, cognitive impairment and paradoxical calming responses towards methylphenidate (MPH), the most potent drug used to treat ADHD patients. Thus, the DAT knockout (KO) mouse model represents a practical mouse model for ADHD. To achieve a better understanding of processes involved in altered dopaminergic neurotransmission, we carried out gene expression analyses of DAT KO and wild-type (WT) mice by employing quantitative Real time-PCR e.g. on genes involved in neurotransmission like the synaptic proteins synaptotagmin (Syt) 1 and 4 and Syntaxin 1A. We could found significantly reduced Syt 1 and 4 expression in striatum of DAT KO mice. In addition, we focused on the mechanisms underlying the effectiveness of MPH. We investigated expression levels of Syt 1 and 4 and Syntaxin 1A in MPH treated DAT-KO and WT control animals as synaptic proteins have already been shown to be involved in the action of psychostimulants. By the use of an acute, a chronic, and a combined acute and chronic treatment paradigm we could reveal selective and differential effects of MPH on the expression of investigated synaptic proteins in different brain regions. Our data further support the critical role of different synaptic proteins in the adaptive plasticity of dopaminergic circuits – with and without psychostimulant treatment.

Depletion of rabphilin 3A in a transgenic mouse model (R6/1) of Huntington's disease, a possible culprit in synaptic dysfunction

Huntington's disease (HD) is a hereditary neurodegenerative disorder characterized by progressive psychiatric, cognitive, and motor disturbances. We studied the expression of synaptic vesicle proteins in the R6/1 transgenic mouse model of HD. We observed that the levels of rabphilin 3A, a protein involved in exocytosis, is substantially decreased in synapses of most brain regions in R6/1 mice. The appearance of the reduction coincides with the onset of motor deficits and behavioral disturbances. Double immunohistochemistry did not show colocalization between rabphilin 3A and huntingtin aggregates in the HD mice. Using in situ hybridization, we demonstrated that rabphilin 3A mRNA expression was substantially reduced in the R6/1 mouse cortex compared to wild-type mice. Our results indicate that a decrease in mRNA levels underlie the depletion of protein levels of rabphilin 3A, and we suggest that this reduction may be involved in causing impaired synaptic transmission in R6/1 mice.

Importance of vesicle proteins in the diagnosis and treatment of neuroendocrine tumors.

We have analyzed the expression of synaptic vesicle proteins in human neuroendocrine tumors and the potential use of vesicle proteins in the diagnosis and treatment of neuroendocrine tumors. Biopsies from endocrine and nonendocrine tumors of the gastrointestinal tract, pancreas, and adrenals were examined by immunocytochemistry using antibodies against synaptic vesicle protein 2 (SV2), vesicular monoamine transporter 1 and 2 (VMAT1 and 2), and neuroendocrine secretory protein 55 (NESP55). SV2 was expressed in all endocrine tumors of the gastrointestinal tract and pancreas as well as in gastrointestinal stromal tumors (GISTs). None of the adenocarcinomas expressed SV2. VMAT1 and 2 were expressed in amine-producing tumors of the gastrointestinal tract (ECL cell and EC cell carcinoids) and in a small number of peptide-producing pancreatic endocrine tumors. NESP55 was expressed in neuroblastomas and adrenal pheochromocytomas as well as in a subgroup of pancreatic endocrine tumors. The importance of VMAT1 and 2 for the uptake of 123I-MIBG in tumor cells was demonstrated. It was concluded that neuroendocrine tumors express multiple synaptic vesicle proteins that are useful in the histopathological diagnosis and classification of tumors. Vesicle proteins may prove to be useful for targeting tumor therapy.

Persistent, noncytolytic infection of neurons by Borna disease virus interferes with ERK 1/2 signaling and abrogates BDNF-induced synaptogenesis.

Infection of the central nervous system by Borna disease virus (BDV) provides a unique model to study the mechanisms whereby a persistent viral infection can impair neuronal function and cause behavioral diseases reminiscent of mood disorders, schizophrenia, or autism in humans. In the present work, we studied the effect of BDV infection on the response of hippocampal neurons, the main target for this virus, to the neurotrophin BDNF. We showed that persistent infection did not affect neuronal survival or morphology. However, it blocked BDNF-induced ERK 1/2 phosphorylation, despite normal expression of the TrkB BDNF receptor. In addition, BDNF-induced expression of synaptic vesicle proteins was abrogated, which resulted in severely impaired synaptogenesis and defects in synaptic organization. Thus, we provide the first evidence that a virus can interfere specifically with neurotrophin-regulated neuroplasticity, thereby hampering proper neuronal connectivity. These results may help to understand the behavioral disorders associated with BDV infection.

Differential distribution and developmental expression of synaptic vesicle protein 2 isoforms in the mouse retina.

Synaptic vesicle protein 2 (SV2), a ubiquitous synaptic vesicle protein, is known to participate in the regulation of Ca(2+)-mediated synaptic transmission, although its precise function has not been established. Three SV2 isoforms (SV2A, SV2B, SV2C) have been identified recently, each of which has a unique distribution in brain, suggesting synapse-specific functions. To determine if SV2A, -B, and -C are differentially distributed among synapses in the retina and the sequence of their development, we examined their distribution and expression patterns immunocytochemically in adult and developing mouse retina. The three SV2 isoforms were differentially distributed in the synapses of the two plexiform layers in the adult retina. SV2A was present in cone, but not rod, terminals in the outer plexiform layer (OPL) and in many synaptic terminals in the inner plexiform layer (IPL). SV2B was present only in the ribbon synapse-containing terminals of rod and cone photoreceptors and bipolar cells. SV2C was present in starburst amacrine cells, other conventional synapses in the IPL of unknown origin, and in presumptive interplexiform cell terminals in the INL and OPL. Each SV2 isoform was expressed in its distinct presynaptic terminals early and throughout postnatal development. In addition, SV2A was transiently expressed by developing horizontal cells. The unique distribution of each isoform suggests potentially distinct functions at different types of synapses, with SV2B having ribbon synapse-specific functions, and SV2C being important for the functions of starburst amacrine cells. Rod and cone terminals contain different complements of SV2 isoforms, indicating that ribbon synapses are not all identical. The early expression of SV2 isoforms prior to initiation of synapse formation suggests that they may have important synapse-specific roles during synaptogenesis.

Synaptotagmin I and IV are differentially regulated in the brain by the recreational drug 3,4-methylenedioxymethamphetamine (MDMA)

3,4-Methylenedioxymethamphetamine (MDMA or Ecstasy) is a widely abused drug. In brains of mice exposed to MDMA, we recently detected altered expression of several cDNAs and genes by using the differential display polymerase chain reaction (PCR) method. Expression of one such cDNA, which exhibited 98% sequence homology with the synaptic vesicle protein synaptotagmin IV, decreased 2 h after MDMA treatment. Herein, the effect of MDMA on expression of both synaptotagmin I and IV was studied in detail, since the two proteins are functionally interrelated. PCR analyses (semi-quantitative and real-time) confirmed that upon treatment with MDMA, expression of synaptotagmin IV decreased both in the midbrain and frontal cortex of mice. Decreases in the protein levels of synaptotagmin IV were confirmed by Western immunoblotting with anti-synaptotagmin IV antibodies. In contrast, the same exposure to MDMA increased expression of synaptotagmin I in the midbrain, a region rich in serotonergic neurons, but not in the frontal cortex. This differential expression was confirmed at the protein level with anti-synaptotagmin I antibodies. MDMA did not induce down- or up-regulation of synaptotagmin IV and I, respectively, in serotonin transporter knockout mice (−/−) that are not sensitive to MDMA. Therefore, psychoactive drugs, such as MDMA, appear to modulate expression of synaptic vesicle proteins, and possibly vesicle trafficking, in the brain.

Expression of presynaptic proteins is closely correlated with the chronotopic pattern of axons in the retinotectal system of the chick

Newly synthesized presynaptic integral membrane proteins in neurons are transported in precursor vesicles from the site of protein biosynthesis in the cell body by fast axonal flow to the presynaptic terminal. We followed the path that presynaptic proteins travel on the way to their central targets of the highly ordered primary visual pathway of the chick and analyzed the developmental changes in the expression of synaptic vesicle protein 2 (SV2), synaptotagmin, and syntaxin. Immunofluorescences revealed that: (1) the onset of protein expression in the retinal ganglion cells occurs in a central to peripheral developmental pattern from embryonic day 4 (E4) onward; (2) the proteins were found first in the inner and later in the outer plexiform layer of the retina; and (3) they were redistributed from the photoreceptor inner segments and cell bodies to the terminals in the outer plexiform layer. From E4 onward, immunopositive axons for SV2, synaptotagmin, and syntaxin were found in the optic nerve, disappearing after E9 for SV2 and synaptotagmin. The optic tract was stained for SV2 and synaptotagmin between E7 and E12, for syntaxin until the posthatching period. Finally, immunoreactivities for the investigated proteins were present at the surface of the tectum from E8 onward, when first retinal axons arrived there. The present study revealed that SV2 and synaptotagmin, but not syntaxin, are, expressed in a transient wave that follows the advancement of optic axons and the proteins towards the optic tectum.

Increase in synaptic vesicle proteins accompanies long-term potentiation in the dentate gyrus

Maintenance of long-term potentiation in synapses formed by the perforant path on to granule cells of the dentate gyrus is accompanied by a sustained increase in the extracellular concentration of glutamate, 3,4 the presumed transmitter at this excitatory hippocampal pathway. Quantal analysis 2,12,13,19,27 indicates that, at least in the first hour of induction, this reflects an increase in transmitter release rather than a decrease in glutamate uptake, while biochemical studies 4,17,18 have suggested that the increase in release persists for several hours. Morphological studies have described early but persistent increases in the spine number 5,14 and area. 28 Increases in the number of segmented/perforated synapses persisting for at least 1 h after induction of long-term potentiation, have also been reported. 9,24 These morphological changes suggest both presynaptic and postsynaptic modifications. 15 Increases in synaptic vesicle number 20 and distribution 1 lasting for at least 1 h specifically indicate presynaptic changes. To explore further the role of the presynaptic terminal in long-term potentiation, we have investigated changes in three synaptic vesicle proteins, synapsin, synaptotagmin and synaptophysin, in control tissue and in tissue prepared from potentiated dentate gyrus 45 min and 3 h after induction of long-term potentiation. We found that there was an increase in the concentration of the three proteins 3 h after induction of long-term potentiation. No such increase was observed 45 min after induction or in tissue prepared from animals in which an intraventricular injection of the N-methyl- d-aspartate receptor antagonist, D(−)-2-amino-5-phosphonopentanoic acid, blocked induction of long-term potentiation. This finding demonstrates an increased expression of synaptic vesicle proteins in long-term potentiation and implies the existence of distinct temporal phases of long-term potentiation during which different synaptic mechanisms for increased transmitter release are engaged.

Coordinate and Noncoordinate Regulation of Synaptic Vesicle Protein Genes during Embryonic Development

Because the formation of a mature nerve terminal requires the accumulation of large quantities of synaptic vesicles, the expression of synaptic vesicle proteins would be expected to correlate with synaptogenesis. However, previous studies have provided conflicting evidence on this point. We have examined the developmental pattern of expression of mRNA and protein for three RNAs derived from two genes coding for synaptic vesicle membrane proteins. For these experiments, we cloned a chick p65 (synaptotagmin) cDNA using a reduced stringency screen with a rat p65 cDNA probe. We examined p65 expression in chick forebrain in conjunction with that of synaptophysin II. RNase protection assays for p65 and the two isoforms of synaptophysin II (Bixby, 1992) show essentially coordinate increases of these three mRNAs in embryonic forebrain during the peak period of synaptogenesis (E17 to E20). However, each of the three mRNAs has a distinct temporal pattern of expression during the early stages of embryogenesis. In the ciliary ganglion, upregulation of synaptophysin II mRNA correlates very well with synaptogenesis. Our results suggest that the regulation of expression of vesicle membrane protein mRNA can serve as a marker for synaptogenesis, despite temporal differences in early expression patterns. In contrast to mRNA expression, assays for vesicle protein expression show a relatively steady rise in both p65 and synaptophysin II throughout the embryonic period, without a sharp increase corresponding to that seen in message levels. These results suggest that the expression both of the p65 and of the synaptophysin II proteins is post-transcriptionally regulated.