Vesicle Release Sites Research Articles

Fos and Jun potentiate individual release sites and mobilize the reserve synaptic-vesicle pool at the Drosophila larval motor synapse

In all nervous systems, short-term enhancement of transmitter release is achieved by increasing the weights of unitary synapses; in contrast, long-term enhancement, which requires nuclear gene expression, is generally thought to be mediated by the addition of new synaptic vesicle release sites. In Drosophila motor neurons, induction of AP-1, a heterodimer of Fos and Jun, induces cAMP- and CREB-dependent forms of presynaptic enhancement. Light and electron microscopic studies indicate that this synaptic enhancement is caused by increasing the weight of unitary synapses and not through the insertion of additional release sites. Electrophysiological and optical measurements of vesicle dynamics demonstrate that enhanced neurotransmitter release is accompanied by an increase in the actively cycling synaptic vesicle pool at the expense of the reserve pool. Finally, the observation that AP-1 mediated enhancement eliminates tetanus-induced forms of presynaptic potentiation suggests: (i) that reserve-pool mobilization is required for tetanus-induced short-term synaptic plasticity; and (ii) that long-term synaptic plasticity may, in some instances, be accomplished by stable recruitment of mechanisms that normally underlie short-term synaptic change.

Active zone assembly and synaptic release

Neurotransmitter release at chemical synapses occurs when synaptic vesicles fuse to the presynaptic membrane at a specialized site termed the active zone. The depolarization-induced fusion is highly dependent on calcium ions, and, correspondingly, the transmission characteristics of synapses are thought to be influenced by the spatial arrangement of voltage-gated calcium channels with respect to vesicle release sites. Here, we review the involvement of the Drosophila Bruchpilot (BRP) protein in active zone assembly, a process that is required for the clustering of presynaptic calcium channels to ensure efficient vesicle release.

Brain-Derived Neurotrophic Factor Enhances GABA Release Probability and Nonuniform Distribution of N- and P/Q-Type Channels on Release Sites of Hippocampal Inhibitory Synapses

Long-lasting exposures to brain-derived neurotrophic factor (BDNF) accelerate the functional maturation of GABAergic transmission in embryonic hippocampal neurons, but the molecular bases of this phenomenon are still debated. Evidence in favor of a postsynaptic site of action has been accumulated, but most of the data support a presynaptic site effect. A crucial issue is whether the enhancement of evoked IPSCs (eIPSCs) induced by BDNF is attributable to an increase in any of the elementary parameters controlling neurosecretion, namely the probability of release, the number of release sites, the readily releasable pool (RRP), and the quantal size. Here, using peak-scaled variance analysis of miniature IPSCs, multiple probability fluctuation analysis, and cumulative amplitude analysis of action potential-evoked postsynaptic currents, we show that BDNF increases release probability and vesicle replenishment with little or no effect on the quantal size, the number of release sites, the RRP, and the Ca2+ dependence of eIPSCs. BDNF treatment changes markedly the distribution of Ca2+ channels controlling neurotransmitter release. It enhances markedly the contribution of N- and P/Q-type channels, which summed to >100% ("supra-additivity"), and deletes the contribution of R-type channels. BDNF accelerates the switch of presynaptic Ca2+ channel distribution from "segregated" to "nonuniform" distribution. This maturation effect was accompanied by an uncovered increased control of N-type channels on paired-pulse depression, otherwise dominated by P/Q-type channels in untreated neurons. Nevertheless, BDNF preserved the fast recovery from depression associated with N-type channels. These novel presynaptic BDNF actions derive mostly from an enhanced overlapping and better colocalization of N- and P/Q-type channels to vesicle release sites.

The Presynaptic Release Apparatus Is Functional in the Absence of Dendritic Contact and Highly Mobile within Isolated Axons

Whether contact of an axon with a dendrite is a necessary inductive signal for the assembly of functional presynaptic machinery is controversial. Combining FM1-43 imaging with retrospective immunocytochemistry, we observe many functional synaptic vesicle (SV) release sites lacking postsynaptic specializations in cultured hippocampal neurons. These “orphan” release sites share the same exocytic machinery and mechanisms of endocytic recycling as mature synaptic sites. Moreover, quantitative analysis of FM1-43 destaining at these orphan release sites reveals similar kinetics with slightly lower release probabilities. Time-lapse imaging of FM1-43 reveals that orphans are generated by complete or partial mobilization of synaptic release sites that retain their functionality in transit. Orphan clusters fuse with existing synaptic release sites or form novel release sites onto dendrites. Mobilization and stabilization of orphan boutons to new sites of dendritic contact may represent a necessary presynaptic counterpart to postsynaptic changes observed during development and plasticity in the CNS.

Enhancement of the Dense-Core Vesicle Secretory Cycle by Glucocorticoid Differentiation of PC12 Cells: Characteristics of Rapid Exocytosis and Endocytosis

The secretory cycle of dense-core vesicles (DCVs) in physiologically stimulated patch-clamped PC12 cells was analyzed using both amperometry and capacitance measurements. Untreated cells had low or undetectable Ca currents and sparse secretory responses to short depolarizations. Dexamethasone (5 microM) treatment for 5-7 d tripled Ca current magnitude and dramatically increased quantal secretion in response to depolarization with action potentials. Such cells expressed L-, N-, and P-type Ca channels, and depolarization evoked rapid catecholamine secretion recorded as amperometric spikes; the average latency was approximately 50 msec. These spikes were much smaller and shorter than those of primary adrenal chromaffin cells, reflecting the smaller size of DCVs in PC12 cells. Depolarizing pulse trains also elicited a rapid increase in membrane capacitance corresponding to exocytosis in differentiated but not in naïve cells. On termination of stimulation, membrane capacitance declined within 20 sec to baseline indicative of rapid endocytosis (RE). RE did not take place when secretion was stimulated in the presence of Ba or Sr, indicating that RE is Ca-specific. RE was blocked when either anti-dynamin antibodies or the pleckstrin homology domain of dynamin-1 was loaded into the cell via the patch pipette. These studies indicate that neuroendocrine differentiation of PC12 cells with glucocorticoids enhances the development of the excitable membrane and increases the coupling between Ca channels and vesicle release sites, leading to rapid exocytosis and endocytosis. Slow catecholamine secretion in undifferentiated cells may be caused in part by a lack of localized secretory machinery rather than being an intrinsic property of dense-core vesicles.

Localization of mGluR6 to dendrites of ON bipolar cells in primate retina.

We prepared antibodies selective for the C-terminus of the human mGluR6 receptor and used confocal and electron microscopy to study the patterns of immunostaining in retina of monkey, cat, and rabbit. In all three species punctate stain was restricted to the outer plexiform layer. In monkey, stain was always observed in the central element of the postsynaptic "triad" of rod and cone terminals. In monkey peripheral retina, stain was seen only in central elements, but in the fovea, stain was also observed in some dendrites contacting the base of the cone terminal. S-cone terminals, identified by staining for S opsin, showed staining of postsynaptic dendrites. These were identified as dendrites of the ON S-cone bipolar cell by immunostaining for the marker cholecystokinin precursor. The staining pattern suggests that all types of ON bipolar cells, despite their marked differences in function, express a single isoform of mGluR6. Ultrastructurally, mGluR6 was located not on the tip of the central element, near the site of vesicle release, but on its base at the mouth of the invagination, 400-800 nm from the release site. Thus, the mGluR6 receptors of ON bipolar cells lie at about the same distance from sites of vesicle release as the iGluR receptors of OFF bipolar cells at the basal contacts.

Foveal Cones form Basal as well as Invaginating Junctions with Diffuse ON Bipolar Cells

The response of a mammalian bipolar cell is generally thought to be determined by the location and morphology of synapses from the cone terminal: ON bipolar cells are believed to be depolarized strictly at invaginating contacts and OFF bipolar cells hyperpolarized at basal contacts. This hypothesis was re-investigated in the macaque fovea (1 deg nasal) using electron micrographs of serial sections. We determined the number of invaginating sites available and then identified the contacts to bipolar cells with axons in the ON level of the inner plexiform layer. A cone terminal forms about 20 active zones marked by ribbons. A few active zones house two invaginating dendrites, so there are 22 invaginating sites per cone. A midget ON bipolar cell collects 18 invaginating contacts from one cone, thus only about four invaginating sites remain for diffuse ON bipolar cells. Two diffuse ON cells were reconstructed; each collects about 25 contacts from an estimated 10 cones. Only three or four of these contacts are invaginating; the rest are basal, adjacent to the triad. This suggests that basal contacts can be depolarizing. The distance from the vesicle release site at active zones to an invaginating contact is 140 ± 40 nm; to a basal contact adjacent to the triad is 500 ± 160 nm, and to the next nearest basal contact is 950 ± 370 nm. Copyright © 1996 Elsevier Science Ltd.

Presynaptic metabotropic glutamate receptors in adult and developing neurons: autoexcitation in the olfactory bulb.

The mitral cell of the olfactory bulb is the primary relay neuron that transmits information from the olfactory receptors to the rest of the brain. This excitatory neuron releases glutamate from presynaptic dendrites and axon terminals. All rat mitral cells studied showed strong, selective, and widespread metabotropic glutamate receptor mGluR1 alpha immunoreactivity on the presynaptic membrane of dendrites, often at the synaptic vesicle release site, when examined with light and electron microscopy. The finding of glutamate receptors on mitral cell secondary dendrites supports the conclusion that not all dendritic membrane with glutamate receptors necessarily have gray type I asymmetrical synaptic specializations. In contrast, the metabotropic glutamate receptor mGluR5 was not found in mitral cells but was expressed by granule cells and astrocytes around mitral dendrites. Both mGluR1 alpha and mGluR5 were expressed early in development, with strong immunostaining present by postnatal day 1. MGluR1 alpha staining at birth mirrored the adult staining pattern. MGluR5 staining at birth showed different patterns of immunostaining than that found in the adult, particularly in the external plexiform layer. In vitro olfactory bulb neurons and their dendrites from embryonic day (E) 18 olfactory bulbs responded to t-ACPD and quisqualate, selective and nonselective metabotropic glutamate receptor agonists, and to several ionotropic glutamate agonists with increases in intracellular Ca2+ as studied with fura-2 digital imaging. These data indicate that the receptors were functionally active at an early stage of development. Application of the glutamate receptor blockers d-2-amino-5-phosphonovalerate (AP5) and 6-cyano-7-nitroquinoxaline (CNQX) to E17 olfactory bulb neurons after only 4 days in vitro resulted in a dramatic decrease in Ca2+ levels in 70% of 128 cells tested, suggesting that embryonic neurons after a short time in vitro can actively secrete glutamate. The presence of glutamate receptors on the long mitral cell dendrite suggests that it would be able to respond to release of its own excitatory transmitter, probably at an early stage of development. In the probable absence of other excitatory input to the secondary mitral dendrites, it would be the only excitatory "input." This autoexcitatory response would be modulated by release of GABA from olfactory interneurons occurring milliseconds after glutamate release induced by olfactory nerve activation. This novel type of neuronal microcircuitry would potentially amplify signal transmission and current spread along the long mitral dendrites and could play an important role in lateral inhibition of olfactory neurons.

Evidence for presynaptic N-methyl-D-aspartate autoreceptors in the spinal cord dorsal horn.

The N-methyl-D-aspartate (NMDA) receptor has been implicated in a variety of systems that undergo plastic changes in the central nervous system. We used electron microscopic immunocytochemistry with an antibody directed against an alternatively spliced exon near the C terminus of NMDAR1, the essential functional subunit of the NMDA receptor, to study the distribution of the NMDA receptor in the spinal cord and CA1 region of the hippocampus, two regions where NMDA-mediated long-term plasticity has been demonstrated. In CA1, we found that the NMDA receptor is exclusively expressed on postsynaptic structures. By contrast, in the spinal cord we found that in about one-third of labeled synapses, the receptor is located in the presynaptic terminal, immediately adjacent to the vesicle release site at the active zone. Using combined postembedding immunocytochemistry, we also showed that > 70% of the NMDA receptor immunoreactive terminals are glutamate positive, which suggests that the presynaptic NMDA receptor is an autoreceptor. Nerve ligation studies demonstrated that the receptor is transported in dorsal roots and sciatic nerve to the spinal cord and periphery, respectively. These data indicate that an NMDA autoreceptor is located in terminals of primary afferent fibers, where it could facilitate the transmission of inputs to the spinal cord by increasing the release of neurotransmitter from the primary afferent terminal.

Three types of Ca2+ channel trigger secretion with different efficacies in chromaffin cells.

To determine whether the different types of Ca2+ channels present in the same secretory cell contribute equally to secretion, we used chromaffin cells to analyse the coupling between three distinct types of Ca2+ channel and exocytosis. These are omega-conotoxin-GVIA-sensitive N-type channels, omega-agatoxin-IVA-sensitive P-type Ca2+ channels and dihydropyridine-sensitive facilitation Ca2+ channels, which are normally quiescent but are activated by depolarizing pre-pulses, repetitive depolarizations to physiological potentials, or agents that raise cyclic AMP. We have simultaneously monitored changes in capacitance as an assay of catecholamine secretion, and Ca2+ currents. Although all three types of Ca2+ channel trigger secretion individually, facilitation channels produce much greater secretion for a given size of Ca2+ current, indicating that they are coupled more efficiently to exocytosis. These results indicate that facilitation Ca2+ channels may be physically nearer vesicle release sites. They also show that low efficiency P- and N-type channels could trigger mild release and that high-efficiency facilitation channels may underlie the massive catecholamine release that occurs during the 'fight or flight' response.

Feedback regulation of acetylcholine release at individual endplates in experimental myasthenia gravis: J.J. Plomp, G.Th.H. van Kempen and P.C. Molenaar. Division of Physiological Pharmacology, Department of Physiology, Sylvius Laboratory, Post Box 9503, 2300 RA Leiden, The Netherlands

The synapse between motor neurons and skeletal muscle is known as the neuromuscular junction (NMJ). Proper alignment of presynaptic and post-synaptic structures of motor neurons and muscle fibers, respectively, is essential for efficient motor control of skeletal muscles. The synaptic cleft between these two cells is filled with basal lamina. Laminins are heterotrimer extracellular matrix molecules that are key members of the basal lamina. Laminin α4, α5, and β2 chains specifically localize to NMJs, and these laminin isoforms play a critical role in maintenance of NMJs and organization of synaptic vesicle release sites known as active zones. These individual laminin chains exert their role in organizing NMJs by binding to their receptors including integrins, dystroglycan, and voltage-gated calcium channels (VGCCs). Disruption of these laminins or the laminin-receptor interaction occurs in neuromuscular diseases including Pierson syndrome and Lambert–Eaton myasthenic syndrome (LEMS). Interventions to maintain proper level of laminins and their receptor interactions may be insightful in treating neuromuscular diseases and aging related degeneration of NMJs.

Effect of 2‐(4‐Phenylpiperidino)cyclohexanol (AH 5183) on the Veratridine‐Induced Increase in Acetylcholine Synthesis by Rat Hippocampal Tissue

The intent of this study was to determine whether the drug 2-(4-phenylpiperidino)cyclohexanol (AH 5183 or vesamicol) might inhibit the veratridine-induced increase in acetylcholine (ACh) synthesis by reducing the veratridine-induced activation of a detergent-soluble choline-O-acetyltransferase (EC 2.3.1.6; ChAT) fraction associated with a vesicle-bound store of ACh. When minces of rat hippocampal tissue were loaded with [14C]choline and subsequently depolarized with veratridine, an increase in the synthesis of [14C]ACh occurred that could be abolished by L-AH 5183 (75 nM). When minces were depolarized with veratridine in the presence of L-AH 5183 (75 nM), the depolarization-induced activation of a detergent-soluble ChAT fraction associated with a vesicle-bound store of ACh was blocked. Conversely, the veratridine-induced activation of a water-soluble ChAT fraction believed to be cytosolic was not. AH 5183 also blocked the repletion of the vesicle-bound store with newly synthesized ACh following veratridine-induced depletion of ACh, a result that appeared to be mediated by an effect on the synthesis of ACh at the vesicular surface. These results suggest that veratridine depolarization of rat hippocampal nerve terminals stimulates the synthesis of ACh by activating a detergent-soluble fraction of ChAT closely associated with synaptic vesicle release sites. ACh synthesis and transport at the vesicular surface may be influenced by a common AH 5183-sensitive regulatory protein.

Are the presynaptic membrane particles the calcium channels?

The number of large intramembrane particles associated with sites of synaptic vesicle release at the squid giant synapse was determined and compared to the average maximal presynaptic calcium current in order to derive an estimate of the conductance each particle would have if it were a calcium channel. This value, 0.21 pS, compares favorably with conductances of calcium channels in other preparations, substantiating the idea that the large intramembrane particles, which are concentrated at "active zones," represent calcium channels.