Axonal transport of synaptic vesicle proteins in the rat optic nerve

The optic nerve, as a part of the central nervous system (CNS), has been used to study axonal transport for decades. The present study has concentrated on the axonal transport of synaptic vesicle proteins in the optic nerve, using the “stop-flow/nerve crush” method. After blocking fast axonal transport, distinct accumulations of synaptic vesicle proteins developed during the first hour after crush-operation and marked increases were observed up to 8 h postoperative. Semiquantitative analysis, using cytofluorimetric scanning (CFS) of immunoincubated sections, revealed that the ratio between distal accumulations (organelles in retrograde transport) and proximal accumulations (organelles in anterograde transport) was much higher (up to 80–90%) for the transmembrane proteins than that for surface adsorbed proteins (only 10–20%). The pattern of axonal transport in the optic nerve was comparable to that in the sciatic nerve. However, clathrin and Rab3a immunoreactivities were accumulated in much lower amounts than that in the sciatic nerve. Most synaptic vesicle proteins were colocalized in the axons proximal to the crush. A differential distribution of synaptobrevin I and II, however, was observed in the optic nerve axons; synaptobrevin I was present in large-sized axons, while synaptobrevin II immunoreactivity was present in most axons, including the large ones. The two isoforms were, thus, partially colocalized. The results demonstrate that (1) cytofluorimetric scanning techniques could be successfully used to study axonal transport not only in peripheral nerves, but also in the CNS; (2) synaptic vesicles are transported with fast axonal transport in this nerve; and (3) some differences were noted compared with the sciatic nerve, especially for Rab3a and clathrin. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 237–250, 1997.     

Optical monitoring of synaptic vesicle trafficking in ribbon synapses

Synaptic transmission constitutes the major basis of communication among nerve cells. Upon nerve terminal depolarisation, calcium influx triggers the exocytosis of synaptic vesicles at active zones. Vesicles are then retrieved by endocytosis, recycled and refilled with neurotransmitter. Fluorescent styryl dyes have proven very useful as tools for studying several aspects of the synaptic vesicle cycle. Here, we review recent imaging studies using styryl FM dyes and bipolar cells of goldfish retina, which have a giant synaptic terminal containing ribbon-type active zones. Optical techniques applied to this unique synaptic terminal have provided novel insights into the trafficking of synaptic vesicles during and following strong stimulation.     

Structures of yeast vesicle trafficking proteins

In protein transport between organelles, interactions of v- and t-SNARE proteins are required for fusion of protein-containing vesicles with appropriate target compartments. Mammalian SNARE proteins have been observed to interact with NSF and SNAP, and yeast SNAREs with yeast homologues of NSF and SNAP proteins. This observation led to the hypothesis that, despite low sequence homology, SNARE proteins are structurally similar among eukaryotes. SNARE proteins can be classified into two groups depending on whether they interact with SNARE binding partners via conserved glutamine (Q-SNAREs) or arginine (R-SNAREs). Much of the published structural data available is for SNAREs involved in exocytosis (either in yeast or synaptic vesicles). This paper describes circular dichroism, Fourier transform infrared spectroscopy, and dynamic light scattering data for a set of yeast v- and t-SNARE proteins, Vti1p and Pep12p, that are Q-SNAREs involved in intracellular trafficking. Our results suggest that the secondary structure of Vti1p is highly alpha-helical and that Vti1p forms multimers under a variety of solution conditions. In these respects, Vti1p appears to be distinct from R-SNARE proteins characterized previously. The alpha-helicity of Vti1p is similar to that of Q-SNARE proteins characterized previously. Pep12p, a Q-SNARE, is highly alpha-helical. It is distinct from other Q-SNAREs in that it forms dimers under many of the solution conditions tested in our experiments. The results presented in this paper are among the first to suggest heterogeneity in the functioning of SNARE complexes.     

Cell proliferation in the developing rat pineal gland. A bromodeoxyuridine immunohistochemical study

The immunohistochemical detection of bromodeoxyuridine (BrdU) was used to study the cell proliferation in the developing rat pineal gland, from the appearance of pineal primordium in the embryonic day 15 (E15) until 30 days after birth. The results showed three different proliferative phases. From E15 to E21, the pineal gland shows a phase of rapid proliferation. The second phase corresponds to the first postnatal week, in which the number of labeled cells per surface unit decreases suddenly to values between 20% to 10% of those of embryonic period. From the second postnatal week onwards, the number of BrdU-positive cells progressively decreases.     

Visualizing recycling of synaptic vesicle proteins

The use of modern techniques (in particular, novel fluorescence markers of a few molecular participants of the exo-and endocytotic processes, including pH-sensitive agents, immuno-electron and laser-scanning microscopy) allows experimenters to visualize different stages of recycling of synaptic vesicle proteins. Neirofiziologiya/Neurophysiology, Vol. 39, Nos. 4/5, pp. 350–351, July–October, 2007.     

Okadaic acid disrupts synaptic vesicle trafficking in a ribbon-type synapse

Protein phosphorylation plays an essential role in regulating synaptic transmission and plasticity. However, regulation of vesicle trafficking towards and away from the plasma membrane is poorly understood. Furthermore, the extent to which phosphorylation modulates ribbon-type synapses is unknown. Using the phosphatase inhibitor okadaic acid (OA), we investigated the influence of persistent phosphorylation on vesicle cycling in goldfish bipolar cells. We followed uptake of FM1-43 during vesicle recycling in control and OA-treated cells. FM1-43 fluorescence spread to the center of control synaptic terminals after depolarization elicited Ca2+ influx. However, OA (1-50 nm) impaired this spatial spread of FM1-43 in a dose-dependent manner. Capacitance measurements revealed that OA (50 nm) did not modify either the amount or kinetics of exocytosis and endocytosis evoked by depolarizing pulses. The extremely low concentrations of OA (1-5 nm) sufficient to observe the inhibition of vesicle mobility implicate phosphatase 2A (PP2A) as a major regulator of vesicle trafficking after endocytosis. These results contrast with those at the neuromuscular junction where OA enhances lateral movement of vesicles between distinct vesicle clusters. Thus, our results suggest that phosphatases regulate vesicle translocation at ribbon synapses in a different manner than conventional active zones.     

Cholinergic signaling in the rat pineal gland

Summary 1. Innervation of the mammalian pineal gland is mainly sympathetic. Pineal synthesis of melatonin and its levels in the circulation are thought to be under strict adrenergic control of serotoninN-acetyltransferase (NAT). In addition, several putative pineal neurotransmitters modulate melatonin synthesis and secretion.2. In this review, we summarize what is currently known on the pineal cholinergic system. Cholinergic signaling in the rat pineal gland is suggested based on the localization of choline acetyltransferase (ChAT) and acetylcholinesterase (AChE), as well as muscarinic and nicotinic ACh binding sites in the gland.3. A functional role of ACh may be regulation of pineal synaptic ribbon numbers and modulation of melatonin secretion, events possibly mediated by phosphoinositide (PI) hydrolysis and activation of protein kinase C via muscarinic ACh receptors (mAChRs).4. We also present previously unpublished data obtained using primary cultures of rat pinealocytes in an attempt to get more direct information on the effects of cholinergic stimulus on pinealocyte melatonin secretion. These studies revealed that the cholinergic effects on melatonin release are restricted mainly to intact pineal glands since they were not readily detected in primary pinealocyte cultures.     

Effects of adrenergic agonists and antagonists on the numbers of synaptic ribbons in the rat pineal gland

In the pineal gland numbers of synaptic ribbons (SR) undergo day/night changes which parallel the rhythm of melatonin synthesis. Since pineal biosynthetic activity is controlled by activation of adrenoreceptors, we investigated the effects of adrenergic agonists and antagonists on pineal synaptic ribbon numbers and N-acetyltransferase (NAT) activity, the key enzyme of melatonin synthesis in rats. In vivo application of the beta-adrenergic antagonist propranolol decreased melatonin synthesis when given during the dark phase but did not affect SR numbers. Treatment during daytime with the beta-adrenergic agonist isoproterenol increased pineal NAT activity whereas SR numbers did not change. Norepinephrine stimulated NAT activity in vitro in a dose-dependent manner, but did not elevate SR numbers. Incubation with an analog of the second messenger cyclic adenosine monophosphate increased both NAT activity and SR numbers. These results suggest that the beta-adrenergic system does not play a decisive role in the regulation of the nocturnal increase in SR numbers observed in the rat pineal gland.     

Immunoisolation and subfractionation of synaptic vesicle proteins

Within recent years, the advances in proteomics techniques have resulted in considerable novel insights into the protein expression patterns of specific tissues, cells, and organelles. The information acquired from large-scale proteomics approaches indicated, however, that the proteomic analysis of whole cells or tissues is often not suited to fully unravel the proteomes of individual organellar constituents or to identify proteins that are present at low copy numbers. In addition, the identification of hydrophobic proteins is still a challenge. Therefore, the development of techniques applicable for the enrichment of low-abundance membrane proteins is essential for a comprehensive proteomic analysis. In addition to the enrichment of particular subcellular structures by subcellular fractionation, the spectrum of techniques applicable for proteomics research can be extended toward the separation of integral and peripheral membrane proteins using organic solvents, detergents, and detergent-based aqueous two-phase systems with water-soluble polymers. Here, we discuss the efficacy of a number of experimental protocols. We demonstrate that the appropriate selection of physicochemical conditions results in the isolation of synaptic vesicles of high purity whose proteome can be subfractionated into integral membrane proteins and soluble proteins by several phase separation techniques.     

Newly produced synaptic vesicle proteins are preferentially used in synaptic transmission

Aged proteins can become hazardous to cellular function, by accumulating molecular damage. This implies that cells should preferentially rely on newly produced ones. We tested this hypothesis in cultured hippocampal neurons, focusing on synaptic transmission. We found that newly synthesized vesicle proteins were incorporated in the actively recycling pool of vesicles responsible for all neurotransmitter release during physiological activity. We observed this for the calcium sensor Synaptotagmin 1, for the neurotransmitter transporter VGAT, and for the fusion protein VAMP2 (Synaptobrevin 2). Metabolic labeling of proteins and visualization by secondary ion mass spectrometry enabled us to query the entire protein makeup of the actively recycling vesicles, which we found to be younger than that of non‐recycling vesicles. The young vesicle proteins remained in use for up to ~ 24 h, during which they participated in recycling a few hundred times. They were afterward reluctant to release and were degraded after an additional ~ 24–48 h. We suggest that the recycling pool of synaptic vesicles relies on newly synthesized proteins, while the inactive reserve pool contains older proteins.     

Precise annual changes in the numbers of "synaptic" ribbons and spherules in the rat pineal gland

Although pineal "synaptic" ribbons (SR) are frequently examined by means of quantitative electron microscopy, their functional significance remains unclear. The same is true for related structures--"synaptic" spherules (SPH). In the course of such studies, it has been noted that SR counts may differ from laboratory to laboratory. Because seasonal changes may play a role, a 2-year study was performed on male rats kept under routine laboratory conditions and killed at monthly intervals during daytime or nighttime. Both structures examined showed distinct day-night differences throughout the year, with higher numbers at night than during the day. There were significant annual changes in both SR and SPH during both daytime and nighttime. The comparison of the curves from the 2 years showed that they were virtually identical both during daytime and nighttime. The numbers of SR were the highest in October and the lowest in April; the numbers of SPH had two plateaus, one with lower values from November to April, and the other with higher values from May to October. It appears from the present study that SR and SPH numbers in the rat pineal gland show statistically significant and precisely timed seasonal changes that may well account for the variations of SR numbers in the different publications.     

Expression of N-acetylglucosamine residues in developing rat fundic gland cells

The development of rat fundic gland was studied by immunohistochemistry using a recently developed monoclonal antibody, HIK 1083, at both light and electron microscope levels. Antibody HIK 1083 recognized oligosaccharides with a non-reducing terminal -linked N-acetylglucosamine (GlcNAc) residue. In the developing rat fundic gland, cells expressing -GlcNAc residues were discernible from day 19.5 of gestation and continued to exist till adult. The distribution of the -GlcNAc expressing cells was consistent with that described previously for cells reacting to Griffonia simplicifolia lectin (GSA-II) in all developmental stages. These cells were located at the bottom of the fundic gland when they first appeared. With the elongation and maturation of the gland, these cells moved upwards and were finally restricted in the neck region of the gland. Combining previous reports and the present electron microscopical observations, HIK 1083-positive cells in the adult rat fundic gland are mucous neck cells. The interaction between antibody HIK 1083 and GSA-II lectin was investigated. GSA-II prevented the subsequent binding of HIK 1083, while HIK 1083 did not prevent GSA-II binding to mucous neck cells. Our results suggested that -GlcNAc residues exist in rat fundic gland from day 19.5 of gestation and continue to exist till adult. Cells expressing -GlcNAc residues appeared as typical mucous neck cells from postnatal four weeks.     

Ultracytochemistry of the synaptic ribbons in the rat pineal organ

Summary The synaptic complexes of the rat pinealocytes are neither cholinergic nor adrenergic. In the synaptic vesicles, a neurotransmitter carrier substance of lipid nature reacting with OsO₄-Zn I₂ mixture (similar to that present in both cholinergic and adrenergic vesicles) was not found.In addition, there were no indications of glucose-6-phosphatase or thiamine-pyrophosphatase activity in the synaptic vesicles. Thus, it appears that the synaptic vesicles do not originate from the rough or smooth endoplasmic reticulum.The synaptic ribbons do not contain carbohydrates, are of protein nature and possess some chemical resemblance to microtubules and microtubular bouquets.Appropriate ultracytochemical reactions have not shown detectable quantities of sodium and calcium ions in pinealocyte synaptic complexes.Grateful acknowledgment is made to Mr. P.-A. Milliquet for technical assistance and to Dr. T. Jalanti (C.M.E., Lausanne) for his help in the use of the X-ray microanalyser.Dedicated to Professor Dr. med. G. Töndury on the occasion of his 70th birthday.     

Role of synaptic proteins in neurotransmitter release-related vesicular trafficking

As is known, regulated exocytosis of synaptic vesicles constitutes a primary means of communication between neurons, and it is subjected to substantial alterations in a number of brain pathologies. Recent investigations showed that vesicular transport events in neuroendocrine cells and presynaptic terminals are realized by a family of specialized membrane proteins of the vesicle (v-SNAREs) and another family located in the target cytoplasmic membrane (t-SNAREs). A variety of such proteins has already been described in different preparations; however, their precise localization and role in vesicular trafficking during functional changes in the cells remain ambiguous. In addition, new synaptic proteins appear to be involved in the vesicular cycle; the functions of these proteins remain unclear. The role of synaptic proteins in the course of cell excitation, in particular functions of core SNARE synaptic proteins (vesicular synaptobrevin/VAMPs and plasma membrane syntaxins/SNAP-25), as well as those of novel presynaptic proteins (Munc-13, Munc-18, CAPS proteins, and others), are discussed in this review. Neirofiziologiya/Neurophysiology, Vol. 40, No. 2, pp. 155–159, March–April, 2008.     

Rhythmic control of endocannabinoids in the rat pineal gland

Endocannabinoids modulate neuroendocrine networks by directly targeting cannabinoid receptors. The time-hormone melatonin synchronizes these networks with external light condition and guarantees time-sensitive and ecologically well-adapted behaviors. Here, the endocannabinoid arachidonoyl ethanolamide (AEA) showed rhythmic changes in rat pineal glands with higher levels during the light-period and reduced amounts at the onset of darkness. Norepinephrine, the essential stimulus for nocturnal melatonin biosynthesis, acutely down-regulated AEA and other endocannabinoids in cultured pineal glands. These temporal dynamics suggest that AEA exerts time-dependent autocrine and/or paracrine functions within the pineal. Moreover, endocananbinoids may be released from the pineal into the CSF or blood stream.     

Circadian and photoperiodic correlation between the number of pineal gland synaptic ribbons and serum melatonin levels in the rat

A study is made of the number of pineal gland synaptic ribbons in 35 male Wistar rats over a 24-hour period during the months of September and February, in correlation to the serum melatonin levels during the same periods and photophases. The results of the study confirm those reported by others authors and suggest that the synaptic ribbons may be the stimuli-transmitting organs facilitating pineal secretory function.     

Prenatal development of "synaptic" ribbons in the guinea pig pineal gland

Pineal "synaptic" ribbons are a heterogeneous population of organelles. "Synaptic" ribbons (SR) sensu stricto, "synaptic" spherules (SS), and intermediate forms (IMF) are present. Their function and origin are unknown, and a knowledge of their prenatal development is lacking. Thus the pineal glands of prenatal, neonatal, and adult guinea pigs were prepared for electron microscopy. "Synaptic" ribbons were studied morphologically and quantitatively. The three categories of "synaptic" ribbons reported in adult pineal glands were also present in prenatal pineal glands. Their structural features, distribution, grouping, and composition patterns are similar to those in adults. "Synaptic" ribbons were first detected in pinealocytes of the distal region of a 42-day postcoitus (PC) pineal gland and were comparable with those in adults. They increased in number with age and reached a peak at 63 days PC, followed by a steep decline at 66 and 67 days PC. By day 69 PC, the numbers increased again and showed a dramatic increase after birth. Several true ribbon synapses were seen at day 63 PC between pinealocyte cell processes or between pinealocyte cell process and pinealocyte cell body. Since true ribbon synapses have not been found in adult guinea pig pinealocytes, their synaptic nature could have been lost during development. No precursors for the "synaptic" ribbons were found. The endoplasmic reticulum cisternae may be the origin for the ribbon vesicles because of their close association with the "synaptic" ribbons.