Population Of Synaptic Vesicles Research Articles

Vesicular Monoamine and Glutamate Transporters Select Distinct Synaptic Vesicle Recycling Pathways

Previous work has characterized the properties of neurotransmitter release at excitatory and inhibitory synapses, but we know remarkably little about the properties of monoamine release, because these neuromodulators do not generally produce a fast ionotropic response. Since dopamine and serotonin neurons can also release glutamate in vitro and in vivo, we have used the vesicular monoamine transporter VMAT2 and the vesicular glutamate transporter VGLUT1 to compare the localization and recycling of synaptic vesicles that store, respectively, monoamines and glutamate. First, VMAT2 segregates partially from VGLUT1 in the boutons of midbrain dopamine neurons, indicating the potential for distinct release sites. Second, endocytosis after stimulation is slower for VMAT2 than VGLUT1. During the stimulus, however, the endocytosis of VMAT2 (but not VGLUT1) accelerates dramatically in midbrain dopamine but not hippocampal neurons, indicating a novel, cell-specific mechanism to sustain high rates of release. On the other hand, we find that in both midbrain dopamine and hippocampal neurons, a substantially smaller proportion of VMAT2 than VGLUT1 is available for evoked release, and VMAT2 shows considerably more dispersion along the axon after exocytosis than VGLUT1. Even when expressed in the same neuron, the two vesicular transporters thus target to distinct populations of synaptic vesicles, presumably due to their selection of distinct recycling pathways.

Taking a Back Seat: Synaptic Vesicle Clustering in Presynaptic Terminals

Central inter-neuronal synapses employ various molecular mechanisms to sustain neurotransmitter release during phases of high-frequency synaptic activity. One of the features ensuring this property is the presence of a pool of synaptic vesicles (SVs) in the presynaptic terminal. At rest and low rates of stimulation, most of the vesicles composing this pool remain in a tight cluster. They are actively utilized when neurons fire action potentials at higher rates and the capability of the recycling machinery is limited. In addition, SV clusters are capable of migrating between release sites and reassemble into clusters at neighboring active zones (AZs). Within the cluster, thin “tethers” interconnect SVs. These dynamic filamentous structures are reorganized during stimulation thereby releasing SVs from the cluster. So far, one protein family, the synapsins, which bind actin filaments and vesicles in a phosphorylation-dependent manner, has been implicated in SV clustering in vertebrate synapses. As evident from recent studies, many endocytic proteins reside in the SV cluster in addition to synapsin. Here we discuss alternative possible mechanisms involved in the organization of this population of SVs. We propose a model in which synapsins together with other synaptic proteins, a large proportion of which is involved in SV recycling, form a dynamic proteinaceous “matrix” which limits the mobility of SVs. Actin filaments, however, do not seem to contribute to SV crosslinking within the SV cluster, but instead they are present peripherally to it, at sites of neurotransmitter release, and at sites of SV recycling.

Dopamine Release at Individual Presynaptic Terminals Visualized with FFNs

The nervous system transmits signals between neurons via neurotransmitter release during synaptic vesicle fusion. To observe neurotransmitter uptake and release from individual presynaptic terminals directly, we designed fluorescent false neurotransmitters as substrates for the synaptic vesicle monoamine transporter. Using these probes to image dopamine release in the striatum, we made several observations pertinent to synaptic plasticity. We found that the fraction of synaptic vesicles releasing neurotransmitter per stimulus was dependent on the stimulus frequency. A kinetically distinct "reserve" synaptic vesicle population was not observed under these experimental conditions. A frequency-dependent heterogeneity of presynaptic terminals was revealed that was dependent in part on D2 dopamine receptors, indicating a mechanism for frequency-dependent coding of presynaptic selection. Hui Zhang and Niko G. Gubernator contributed equally to this work.

Dopamine Release at Individual Presynaptic Terminals Visualized with FFNs

The nervous system transmits signals between neurons via neurotransmitter release during synaptic vesicle fusion. To observe neurotransmitter uptake and release from individual presynaptic terminals directly, we designed fluorescent false neurotransmitters as substrates for the synaptic vesicle monoamine transporter. Using these probes to image dopamine release in the striatum, we made several observations pertinent to synaptic plasticity. We found that the fraction of synaptic vesicles releasing neurotransmitter per stimulus was dependent on the stimulus frequency. A kinetically distinct "reserve" synaptic vesicle population was not observed under these experimental conditions. A frequency-dependent heterogeneity of presynaptic terminals was revealed that was dependent in part on D2 dopamine receptors, indicating a mechanism for frequency-dependent coding of presynaptic selection.

Storage and secretion of β‐NAD, ATP and dopamine in NGF‐differentiated rat pheochromocytoma PC12 cells

In nerve-smooth muscle preparations beta-nicotinamide adenine dinucleotide (beta-NAD) has emerged as a novel extracellular substance with putative neurotransmitter and neuromodulator functions. beta-NAD is released, along with noradrenaline and adenosine 5'-triphosphate (ATP), upon firing of action potentials in blood vessels, urinary bladder and large intestine. At present it is unclear whether noradrenaline, ATP and beta-NAD are stored in and released from common populations of synaptic vesicles. The answer is unattainable in complex systems such as nerve-smooth muscle preparations. Adrenal chromaffin cells are thus used here as a single-cell model to examine mechanisms of concomitant neurosecretion. Using high-performance liquid chromatography techniques with electrochemical and fluorescence detection we simultaneously evaluated secretion of dopamine (DA), ATP, adenosine 5'-diphosphate, adenosine 5'-monophosphate, adenosine, beta-NAD and its immediate metabolites ADP-ribose and cyclic ADP-ribose in superfused nerve growth factor-differentiated rat pheochromocytoma PC12 cells. beta-NAD, DA and ATP were released constitutively and upon stimulation with high-K(+) solution or nicotine. Botulinum neurotoxin A tended to increase the spontaneous secretion of all substances and abolished the high-K(+)-evoked release of beta-NAD and DA but not of ATP. Subcellular fractionation by continuous glycerol and sucrose gradients along with immunoblot analysis of the vesicular marker proteins synaptophysin and secretogranin II revealed that beta-NAD, ATP and DA are stored in both small synaptic-like vesicles and large dense-core-like vesicles. However, the three substances appear to have different preferential sites of release upon membrane depolarization including sites associated with SNAP-25 and sites not associated with SNAP-25.

The Potassium Chloride Cotransporter KCC-2 Coordinates Development of Inhibitory Neurotransmission and Synapse Structure in Caenorhabditis elegans

Chloride influx through GABA-gated chloride channels, the primary mechanism by which neural activity is inhibited in the adult mammalian brain, depends on chloride gradients established by the potassium chloride cotransporter KCC2. We used a genetic screen to identify genes important for inhibition of the hermaphrodite-specific motor neurons (HSNs) that stimulate Caenorhabditis elegans egg-laying behavior and discovered mutations in a potassium chloride cotransporter, kcc-2. Functional analysis indicates that, like mammalian KCCs, C. elegans KCC-2 transports chloride, is activated by hypotonic conditions, and is inhibited by the loop diuretic furosemide. KCC-2 appears to establish chloride gradients required for the inhibitory effects of GABA-gated and serotonin-gated chloride channels on C. elegans behavior. In the absence of KCC-2, chloride gradients appear to be altered in neurons and muscles such that normally inhibitory signals become excitatory. kcc-2 is transcriptionally upregulated in the HSN neurons during synapse development. Loss of KCC-2 produces a decrease in the synaptic vesicle population within mature HSN synapses, which apparently compensates for a lack of HSN inhibition, resulting in normal egg-laying behavior. Thus, KCC-2 coordinates the development of inhibitory neurotransmission with synapse maturation to produce mature neural circuits with appropriate activity levels.

VGLUT1 and VGAT are sorted to the same population of synaptic vesicles in subsets of cortical axon terminals

Glutamate and GABA mediate most of the excitatory and inhibitory synaptic transmission; they are taken up and accumulated in synaptic vesicles by specific vesicular transporters named VGLUT1-3 and VGAT, respectively. Recent studies show that VGLUT2 and VGLUT3 are co-expressed with VGAT. Because of the relevance this information has for our understanding of synaptic physiology and plasticity, we investigated whether VGLUT1 and VGAT are co-expressed in rat cortical neurons. In cortical cultures and layer V cortical terminals we observed a population of terminals expressing VGLUT1 and VGAT. Post-embedding immunogold studies showed that VGLUT1+/VGAT+ terminals formed both symmetric and asymmetric synapses. Triple-labeling studies revealed GABAergic synapses expressing VGLUT1 and glutamatergic synapses expressing VGAT. Immunoisolation studies showed that anti-VGAT immunoisolated vesicles contained VGLUT1 and anti-VGLUT1 immunoisolated vesicles contained VGAT. Finally, vesicles containing VGAT resident in glutamatergic terminals undergo active recycling. In conclusion, we demonstrate that in neocortex VGLUT1 and VGAT are co-expressed in a subset of axon terminals forming both symmetric and asymmetric synapses, that VGLUT1 and VGAT are sorted to the same vesicles and that vesicles at synapses expressing the vesicular heterotransporter participate in the exo-endocytotic cycle.

Why Do Hair Cells Have Ribbons? Focus on “Synaptic Ribbon Enables Temporal Precision of Hair Cell Afferent Synapse by Increasing the Number of Readily Releasable Vesicles: A Modeling Study”

Hair cells, like retinal photoreceptors and bipolar cells, encode stimuli with graded changes in membrane potential and transmit that signal through ribbon synapses to drive action potentials in associated afferent neurons ([Sterling and Matthews 2005][1]). Thus one would expect transmitter release

Vesicular Glutamate and GABA Transporters Sort to Distinct Sets of Vesicles in a Population of Presynaptic Terminals

Vesicular glutamate transporters (VGLUTs) 1 and 2 are expressed by neurons generally accepted to release glutamate as a neurotransmitter, whereas VGLUT3 appears in populations usually associated with a different classical transmitter. We now demonstrate VGLUT2 as well as the vesicular GABA transporter (VGAT) in a subset of presynaptic terminals in the dentate gyrus of the rat hippocampal formation. The terminals are distributed in a characteristic band overlapping with the outer part of the granule cell layer and the inner zone of the molecular layer. Within the terminals, which make asymmetric as well as symmetric synapses onto the somatodendritic compartment of the dentate granule cells, the 2 transporters localize to distinct populations of synaptic vesicles. Moreover, the axons forming these terminals originate in the supramammillary nucleus (SuM). Our data reconcile previous apparently conflicting reports on the physiology of the dentate afferents from SuM and demonstrate that both glutamate and GABA may be released from a single nerve terminal.

Vesicle Pool Heterogeneity at Hippocampal Glutamate and GABA Synapses

Glutamate and GABA are the major fast excitatory and inhibitory neurotransmitters, respectively, in the CNS. Although glutamate and GABA have clearly distinct postsynaptic actions, we are just beginning to appreciate that presynaptic differences between glutamatergic and GABAergic neurons may contribute to distinct functions of these transmitter systems. We therefore probed possible differences between the functional synaptic vesicle populations of glutamatergic and GABAergic neurons. We examined superecliptic synaptopHluorin (SpH) fluorescence during 20 Hz electrical stimulation in transfected hippocampal neurons and identified the phenotype of SpH-fluorescent synapses with post hoc immunostaining. With 200 stimuli (10 s), individual glutamate synapses displayed considerably more variability in peak SpH fluorescence than GABA synapses, without a strong difference in the mean SpH fluorescence increase. This spatial heterogeneity could not be accounted for by differences in endocytosis, which was nearly constant over these short time periods across glutamate and GABA synapses. Instead, variability in vesicle exocytosis correlated with variability in total vesicle staining and in measures of the total recycling pool size. Differences were also evident using FM1-43 [N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl) pyridinium dibromide] uptake. These data support the idea that the population of glutamate synapses exhibits more heterogeneity in release properties than the population of GABA synapses, possibly correlated with glutamatergic synaptic malleability.

Neurotrophic factors acutely alter the sorting of the vesicular acetyl choline transporter and the vesicular monoamine transporter 2 in bimodal sympathetic neurons

Individual sympathetic neurons in co-cultures with cardiac myocytes store acetylcholine and noradrenaline in two different populations of synaptic vesicles and release both neurotransmitters from single presynaptic terminals. Neurotrophic factors selectively and acutely stimulate differential release of the two types of neurotransmitters from these bimodal neurons. Here we investigated the acute effects of neurotrophic factors on two pivotal marker proteins for catecholaminergic and cholinergic synaptic vesicle populations: the vesicular monoamine transporter 2 and the vesicular acetylcholine transporter. We observed that separation of the two fluorescence labeled transporters is not restricted to the varicosities, but can also be observed in the neurites as well as in the cell soma. Application of nerve growth factor, brain-derived neurotrophic factor and ciliary neuronotrophic factor caused acute alterations in transporter segregation. These results point to a novel function of neurotrophic factors during the short-term regulation of synaptic protein sorting in neurons.

Synaptic Vesicles: Turning Reluctance Into Action

Vesicle availability partly determines the efficacy of synaptic communication in the CNS. The authors recently found that some hippocampal glutamate vesicles exhibit reluctance to exocytose during short, high-frequency action potential trains. These same vesicles can be “coaxed” into exocytosis by increased Ca2+entry, by direct depolarization of synaptic terminals, or by challenge with hypertonic sucrose, a tool used to cause fusion of the population of release-ready synaptic vesicles. Interestingly, the authors did not find evidence of reluctance at hippocampal GABA synapses, suggesting that vesicle reluctance might be a negative feedback mechanism to prevent runaway excitation. It is also possible that synapses exhibit reluctance to retain a dormant population of readily accessible vesicles, ready to respond to triggers such as enhanced Ca2+ influx or neuromodulators. Recent work from the calyx of Held synapse suggests that reluctance might arise from inactivation of Ca2+ channels. The authors review this work, along with several other potential mechanisms of reluctance.

Nicotinamide adenine dinucleotide is released from sympathetic nerve terminals via a botulinum neurotoxin A-mediated mechanism in canine mesenteric artery

Using high-performance liquid chromatography techniques with fluorescence and electrochemical detection, we found that beta-nicotinamide adenine dinucleotide (beta-NAD) is released in response to electrical field stimulation (4-16 Hz, 0.3 ms, 15 V, 120 s) along with ATP and norepinephrine (NE) in the canine isolated mesenteric arteries. The release of beta-NAD increases with number of pulses/stimulation frequencies. Immunohistochemistry analysis showed dense distribution of tyrosine hydroxylase-like immunoreactivity (TH-LI) and sparse distribution of TH-LI-negative nerve processes, suggesting that these blood vessels are primarily under sympathetic nervous system control with some contribution of other (e.g., sensory) neurons. Exogenous NE (3 micromol/l), alpha,beta-methylene ATP (1 micromol/l), neuropeptide Y (NPY, 0.1 micromol/l), CGRP (0.1 micromol/l), vasoactive intestinal peptide (VIP, 0.1 micromol/l), and substance P (SP, 0.1 micromol/l) had no effect on the basal release of beta-NAD, suggesting that the overflow of beta-NAD is evoked by neither the sympathetic neurotransmitters NE, ATP, and NPY, nor the neuropeptides CGRP, VIP, and SP. Botulinum neurotoxin A (BoNTA, 0.1 micromol/l) abolished the evoked release of NE, ATP, and beta-NAD at 4 Hz, suggesting that at low levels of neural activity, release of these neurotransmitters results from N-ethylmaleimide-sensitive factor attachment protein receptor/synaptosomal-associated protein of 25 kDa-mediated exocytosis. At 16 Hz, however, the evoked release of NE, ATP, and beta-NAD was reduced by BoNTA by approximately 90, 60, and 80%, respectively, suggesting that at higher levels of neural activity, beta-NAD is likely to be released from different populations of synaptic vesicles or different populations of nerve terminals (i.e., sympathetic and sensory terminals).

Intracellular chloride channels: determinants of function in the endosomal pathway.

Endosomes, and related subcellular compartments, contain various Cl- channels in the ClC family. In this review, we describe the known roles of intracellular Cl- channels and also explore some of the functional implications of transmembrane Cl- flux in these organelles. Cl- influx acts to control intralumenal pH, both by shunting the effects of the proton pump on membrane potential and, possibly, through direct effects of Cl- on the proton pump. Changes in intralumenal pH likely help regulate membrane trafficking. We propose that changes in intralumenal Cl- concentration ([Cl-]) could theoretically play a direct role in regulating membrane trafficking and organellar function through effects on chloride-sensitive proteins in the vesicular membrane, which could transduce information about intralumenal [Cl-] to the outside of the vesicle and thereby recruit various signaling molecules. We present a model in which regulation of cytosolic [Cl-] and vesicular Cl- conductance could help control the amount or type of neurotransmitter stored in a particular population of synaptic vesicles.

Glycine transporter isoforms show differential subcellular localization in PC12 cells.

The subcellular localization of glycine transporters one (GLYT1) and two (GLYT2) stably expressed in PC12 cells has been studied. To facilitate visualization, enhanced green fluorescent protein (GFP) was fused to the amino terminus of both glycine transporters. Functional analysis of the GFP-GLYT1 and GFP-GLYT2 stable cell lines demonstrated that they exhibited high affinity for glycine and the characteristic properties of both glycine transporter subtypes. The GFP-coupled transporters were differently distributed throughout the cell. GFP-GLYT1 was mainly localized on the plasma membrane, whereas most of GFP-GLYT2 was present on large dense-core vesicles and endosomes. Both transporters were absent from the synaptic vesicle population in PC12 cells.

Differences in the retrograde axonal transport of nerve growth factor and dopamine beta‐hydroxylase

There are two potential populations of vesicles in the sympathetic nervous system that may contribute to the retrograde axonal transport of signals from the target tissue. These are derived from the population of recycled synaptic vesicles and endosomes derived from receptor mediated endocytosis of growth factors. We have analysed the retrograde axonal transport of two l125‐labelled marker proteins, nerve growth factor (NGF) and dopamine beta‐hydroxylase (d.b.h.), to determine whether these two populations are processed in the same way in the nerve terminal. Two models were used in the rat, either from the anterior eye chamber to the superior cervical ganglion and trigeminal ganglion or the ligated sciatic nerve after injection into the footpad. The results show that there are significant differences in the regulatory processes that control the targeting of these proteins for retrograde transport. Many inhibitors of the second messenger cascades inhibit the transport of both molecules, for example Latrunculin A and wortmannin. However, the retrograde axonal transport of NGF is inhibited by phenylarsine oxide but not by bis‐tyrphostin, FK506, dibutyryl‐cAMP, thapsigargin, cadmium and nickel whereas the transport of d.b.h. is the reverse, suggesting the mechanism of endocytosis and targeting the vesicles in the nerve terminal for retrograde axonal transport is different. We have isolated vesicles labelled with these iodinated proteins from the sciatic nerve and have shown that the density of the NGF containing vesicles is significantly lighter than those containing d.b.h. This suggests that there are at least two distinct pathways for retrograde axonal transport of vesicles.

Evidence for a gamma-hydroxybutyrate (GHB) uptake by rat brain synaptic vesicles.

gamma-Hydroxybutyrate (GHB) is an endogenous metabolite of mammalian brain which is derived from GABA. Much evidence favours its role as an endogenous neuromodulator, synthesized, stored and released at particular synapses expressing specific receptors. One key step for GHB involvement in neurotransmission is its uptake by a specific population of synaptic vesicles. We demonstrate that this specific uptake exists in a crude synaptic vesicle pool obtained from rat brain. The kinetic parameters and the pharmacology of this transport are in favour of an active vesicular uptake system for GHB via the vesicular inhibitory amino acid transporter. This result supports the idea that GABA and GHB accumulate together and are coliberated in some GABAergic synapses of the rat brain, where GHB acts as a modulatory factor for the activity of these synapses following stimulation of specific receptors.

Neurotransmitter release through the V0 sector of V-ATPase.

Neurotransmitter release through the V0 sector of V-ATPase.

Influence of long-term experimental orthostatic body position on innervation density in extremity vessels.

The aim of this study was to quantitate the density of nerve terminals as well as their synaptic vesicle population in the adventitia of saphenous (SV and SA) and brachial veins and arteries (BV and BA) obtained from rats maintained in a horizontal control or a tilted position. Adult animals were kept individually in tube-like cages in a 45 degrees head-up position. After 2 wk, both tilted and control animals were anesthetized, and the whole body was perfused with fixative solution at physiological pressure. Vessels segments were then excised for electron microscopy and immunohistochemistry. The nerve terminal density (NTD) of SA was 8.20 +/- 1.46 nerve terminals/100 microm(2) cross section of adventitia and that of SV was 4.53 +/- 0.61 nerve terminals/100 microm(2) cross section of adventitia in control rats. Tilting caused a significant increase in NTD of both SA (70%) and SV (52%). The synaptic microvesicle density (SyVD) was larger in SA than SV in control rats (30.48 +/- 4.41 vs. 13.38 +/- 2.61 synaptic vesicles/10 terminal sections), but tilting resulted in more pronounced changes in SyVD of SV (95%) than SA (54%). No significant changes in NTD and SyVD of BA were found after tilt (-3.6% relative to 4.99 +/- 0.33 compared with 0.4% relative to 24.89 +/- 3.7, respectively). Whereas NTD of BV exhibited a tendency to increase (3.73 +/- 0.86 vs. 2.31 +/- 0.29 nerve terminals/100 microm(2) cross section of adventitia), SyVD did not change significantly (18.96 +/- 2.74 vs. 22.85 +/- 3.17 synaptic vesicles/10 terminal sections). A large number of nerve terminals of all vessels were tyrosine hydroxylase immunoreactive (containing norepinephrine). These findings support the hypothesis that long-term gravitational load causes adaptive morphological and functional remodeling of sympathetic innervation in blood vessels of the extremities.

Morphological correlates of functionally defined synaptic vesicle populations.

By combining photoconversion of FM1-43-stained vesicles and electron microscopy of hippocampal synapses, we find evidence that the population of morphologically docked synaptic vesicles corresponds to the release-ready neurotransmitter quanta. Furthermore, those synaptic vesicles that are participating in cycles of exo- and endocytosis tend to be closer to the active zone than vesicles that are being held in reserve.