Membrane retrieval by macropinocytosis in presynaptic terminals during transmitter release in cat sympathetic ganglia in situ.
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Animals↗
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[Effect of local electrical stimulation and psychotropic drugs on the presynaptic terminals in the hypothalamus of the cat (author's transl)].
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Animals↗
Changes in binomial parameters of quantal release at crustacean motor axon terminals during presynaptic inhibition.
1. The effects of presynaptic inhibition on quantal release of transmitter were investigated at neuromuscular junctions of the motor axon supplying one of the limb muscles of a crab (Pachygrapsus crassipes). 2. Binomial analysis of transmitter release recorded at selected neuromuscular junctions with an extracellular 'macro-patch' electrode indicated high probability of release (p) from a limited number of available sites (n). During presynaptic inhibition, both n and p were reduced. 3. The binomial model provided a good description of results from non-inhibited junctions. During presynaptic inhibition, results from some junctions could be described by the binomial model, while those from other junctions could not. An interpretation of this finding is that presynaptic inhibition differentially affects the probability of release at various release sites of the neuromuscular junctional complex. 4. A morphological study of the region of transmitter release under the macropatch electrode was made. Release-dependent uptake of horseradish peroxidase (HRP) into presynaptic terminals was restricted to the region under the recording electrode, by perfusing the preparation with calcium-free solution containing HRP. Transmitter release, and HRP uptake, occurred only at the site of the electrode, which was filled with a calcium-containing solution. Subsequently, serial sections were prepared for electron microscopy and the region of transmitter release was reconstructed. 5. Numerous axo-axonal synapses were found in the HRP-labelled region. Thus, the morphological prerequisite for presynaptic inhibition exists at the site of transmitter release, and not exclusively at a more remote region. 6. The number of morphologically identified excitatory neuromuscular synapses exceeded the 'release sites' estimated from the binomial model (n) by a wide margin. Morphological differences among synapses were observed. It is proposed that not all morphologically identified synapses participated in transmitter release under the experimental conditions employed. Thus, morphologically defined synapses are likely to be non-uniform in their response properties, including probability of transmitter release (p).
Action Potentials↗
Circulation and turnover of synaptic vesicle membrane in cultured fetal mammalian spinal cord neurons.
Intact neurons in cultures of fetal rodent spinal cord explants show stimulation-dependent uptake of horseradish peroxidase (HRP) into many small vesicles and occasional tubules and multivesicular bodies (MVB) at presynaptic terminals. Presynaptic terminals were allowed to take up HRP during 1 h of strychnine-enhanced stimulation of synaptic transmitter release and then "chased" in tracer-free medium either with strychnine or with 10 mM Mg++ which depresses transmitter release. Tracer-containing vesicles are lost from terminals under both chase conditions; the loss is more rapid (4-8 h) with strychnine than with 10 mM Mg++ (8-16 h). There is a parallel decrease in the numbers of labeled MVB's at terminals. Loss of tracer with 10 mM Mg++ does not appear to be due to the membrane rearrangements (exocytosis coupled to endocytosis) that presumably lead to initial tracer uptake; terminals exposed to HRP and Mg++ for up to 16 h show little tracer uptake into vesicles. Nor is the decrease likely to the due to loss of HRP enzyme activity; HRP is very stable in solution. During the chases there is a striking accumulation of HRP in perikarya that is far more extensive in cultures initially exposed to tracer with strychnine than 10 mM Mg++ regardless of chase conditions. Much of the tracer ends up in large dense bodies. These findings suggest that synaptic vesicle membrane turnover involves retrograde axonal transport of membrane to neuronal perikarya for further processing, including lysosomal degradation. The more rapid (4-8 h) loss of tracer-containing vesicles with strychnine may reflect vesicle membrane reutilization for exocytosis.
Axons↗
A computational model of the ribbon synapse.
A model of the ribbon synapse was developed to replicate both pre- and postsynaptic functions of this glutamatergic juncture. The presynaptic portion of the model is rich in anatomical and physiological detail and includes multiple release sites for each ribbon based on anatomical studies of presynaptic terminals, presynaptic voltage at the terminal, the activation of voltage-gated calcium channels and a calcium-dependent release mechanism whose rate varies as a function of the calcium concentration that is monitored at two different sites which control both an ultrafast, docked pool of vesicles and a release ready pool of tethered vesicles. The postsynaptic portion of the program models diffusion of glutamate and the physiological properties of glutamatergic neurotransmission in target cells. We demonstrate the behavior of the model using the retinal bipolar cell to ganglion cell ribbon synapse. The model was constrained by the anatomy of salamander bipolar terminals based on the ultrastructure of these synapses and presynaptic contacts were placed onto realistic ganglion cell morphology activated by a range of ribbon synapses (46-138). These inputs could excite the cell in a manner consistent with physiological observations. This model is a comprehensive, first-generation attempt to assemble our present understanding of the ribbon synapse into a domain that permits testing our understanding of this important structure. We believe that with minor modifications of this model, it can be fine tuned for other ribbon synapses.
Animals↗
Neuronal calcium sensor 1 and activity-dependent facilitation of P/Q-type calcium currents at presynaptic nerve terminals.
P/Q-type presynaptic calcium currents (IpCa) undergo activity-dependent facilitation during repetitive activation at the calyx of the Held synapse. We investigated whether neuronal calcium sensor 1 (NCS-1) may underlie this phenomenon. Direct loading of NCS-1 into the nerve terminal mimicked activity-dependent IpCa facilitation by accelerating the activation time of IpCa in a Ca2+-dependent manner. A presynaptically loaded carboxyl-terminal peptide of NCS-1 abolished IpCa facilitation. These results suggest that residual Ca2+ activates endogenous NCS-1, thereby facilitating IpCa. Because both P/Q-type Ca2+ channels and NCS-1 are widely expressed in mammalian nerve terminals, NCS-1 may contribute to the activity-dependent synaptic facilitation at many synapses.
Action Potentials↗
A syntaxin 1, Galpha(o), and N-type calcium channel complex at a presynaptic nerve terminal: analysis by quantitative immunocolocalization.
Presynaptic Ca(V)2.2 (N-type) calcium channels are subject to modulation by interaction with syntaxin 1 and by a syntaxin 1-sensitive Galpha(O) G-protein pathway. We used biochemical analysis of neuronal tissue lysates and a new quantitative test of colocalization by intensity correlation analysis at the giant calyx-type presynaptic terminal of the chick ciliary ganglion to explore the association of Ca(V)2.2 with syntaxin 1 and Galpha(O). Ca(V)2.2 could be localized by immunocytochemistry (antibody Ab571) in puncta on the release site aspect of the presynaptic terminal and close to synaptic vesicle clouds. Syntaxin 1 coimmunoprecipitated with Ca(V)2.2 from chick brain and chick ciliary ganglia and was widely distributed on the presynaptic terminal membrane. A fraction of the total syntaxin 1 colocalized with the Ca(V)2.2 puncta, whereas the bulk colocalized with MUNC18-1. Galpha(O,) whether in its trimeric or monomeric state, did not coimmunoprecipitate with Ca(V)2.2, MUNC18-1, or syntaxin 1. However, the G-protein exhibited a punctate staining on the calyx membrane with an intensity that varied in synchrony with that for both Ca channels and syntaxin 1 but only weakly with MUNC18-1. Thus, syntaxin 1 appears to be a component of two separate complexes at the presynaptic terminal, a minor one at the transmitter release site with Ca(V)2.2 and Galpha(O), as well as in large clusters remote from the release site with MUNC18-1. These syntaxin 1 protein complexes may play distinct roles in presynaptic biology.
Animals↗
Distribution of glutamate receptor subunits at neurochemically characterized synapses in the entopeduncular nucleus and subthalamic nucleus of the rat.
Glutamatergic neurotransmission in the subthalamic nucleus (STN) and in the output nuclei of the basal ganglia is critical in the expression of basal ganglia function, and increased glutamate transmission in these nuclei has been implicated in the pathology of Parkinson's disease. In order to determine the precise spatial relationship of subunits of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and N-methyl-D-aspartate (NMDA) glutamate receptors to nerve terminals enriched in glutamate or gamma-aminobutyric acid (GABA) in one of the output nuclei, the entopeduncular nucleus (EP), and the STN, postembedding immunolabelling for glutamate receptor subunits and for glutamate and GABA was carried out in the rat. Immunolabelling for the AMPA glutamate receptor subunits 1, 2/3, and 4 (GluR1, GluR2/3, and GluR4) and the NMDA receptor subunit 1 (NR1) was localized predominantly within asymmetrical synapses in both the EP and STN. Quantitative analysis revealed that, on average for the whole population, each of the receptor subunits was evenly distributed along the synaptic specialization. Multiple AMPA receptor subunits and the GluR2/3 and NMDA (NR1) subunits were co-localized within individual synapses. The combination of immunolabelling for glutamate and GABA with the receptor immunolabelling revealed that the majority of axon terminals presynaptic to the receptor-immunoreactive synapses were enriched in glutamate immunoreactivity and were GABA-immunonegative. However, at some NR1- and GluR2/3-positive synapses, the level of glutamate immunoreactivity was low in the presynaptic terminal and, in the STN, some of them were GABA-immunopositive. It is concluded that glutamatergic transmission at individual synapses of different origins in the EP and STN is mediated by a combination ofAMPA and NMDA glutamate receptors.
Animals↗
Reinforcing properties of cocaine in the medical prefrontal cortex: primary action on presynaptic dopaminergic terminals.
The presynaptic mechanisms involved in the initiation of cocaine reinforcement were investigated using neurotoxin lesions. Rats were trained to intracranially self-administer cocaine (50 to 90 pmol) into the medial prefrontal cortex and after dose-effect analyses were completed, each rat received a unilateral 6-hydroxydopamine lesion (4 micrograms in 0.2 microliter) at the self-administration site. The lesion selectively decreased dopamine content in the medial prefrontal cortex (-45%) and decreased cocaine-maintained responding to vehicle levels. Lever-pressing could be reinstated by substituting dopamine (300 pmol) but not serotonin for cocaine. Dopamine self-administration was attenuated by including equimolar concentrations of the D2 dopaminergic antagonist sulpiride in the injectate. These results suggest that the initiation of reinforcing neuronal activity in the medial prefrontal cortex appears to result in part through the direct interaction of cocaine with presynaptic reuptake sites associated with dopaminergic nerve endings. The resulting increased synaptic concentration of the neurotransmitter may then interact with postsynaptic D2 binding sites to activate neuronal systems involved in the mediation of this reinforcement.
Animals↗
Dynamics of dendritic spines and their afferent terminals: spines are more motile than presynaptic boutons.
Previous work has established that dendritic spines, sites of excitatory input in CNS neurons, can be highly dynamic, in later development as well as in mature brain. Although spine motility has been proposed to facilitate the formation of new synaptic contacts, we have reported that spines continue to be dynamic even if they bear synaptic contacts. An outstanding question related to this finding is whether the presynaptic terminals that contact dendritic spines are as dynamic as their postsynaptic targets. Using multiphoton time-lapse microscopy of GFP-labeled Purkinje cells and DiI-labeled granule cell parallel fiber afferents in cerebellar slices, we monitored the dynamic behavior of both presynaptic terminals and postsynaptic dendritic spines in the same preparation. We report that while spines are dynamic, the presynaptic terminals they contact are quite stable. We confirmed the relatively low levels of presynaptic terminal motility by imaging parallel fibers in vivo. Finally, spine motility can occur when a functional presynaptic terminal is apposed to it. These analyses further call into question the function of spine motility, and to what extent the synapse breaks or maintains its contact during the movement of the spine.
Animals↗
GABAergic terminals are presynaptic to primary afferent terminals in the substantia gelatinosa of the rat spinal cord.
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Afferent Pathways↗
Syntaxin-1A is excluded from recycling synaptic vesicles at nerve terminals.
At presynaptic terminals, intermixing during cycles of exocytosis and endocytosis challenges the molecular identity of the plasma and synaptic vesicle membranes. Although synaptic vesicle components are retrieved during recycling, the extent to which plasma membrane proteins enter the synaptic vesicle recycling pathway has not been examined. The target-SNARE (N-ethylmaleimide-sensitive factor attachment protein receptor) syntaxin-1 was shown previously to be present on putative synaptic vesicular membranes (Koh et al., 1993; Walch-Solimena et al., 1995; Kretzschmar et al., 1996), suggesting that syntaxin may cycle between the synaptic vesicle pool and the cell surface (Walch-Solimena et al., 1995). This implies that the molecular identity of the two membranes is not maintained during synaptic activity. Because the main role of syntaxin-1 is as a target-SNARE for vesicle fusion, appearance on synaptic vesicles could lead to futile interactions with vesicle-SNARE proteins. We investigated whether the subcellular localization of syntaxin-1A, tagged with the pH-sensitive fluorescent tag pHluorin, is regulated during neurotransmission using laser-scanning microscopy. We report here that syntaxin-1A is predominantly localized to the plasma membrane, with a small proportion present in an intracellular compartment with a lumenal pH consistent with synaptic vesicles. However, the internal fraction of syntaxin-1A is excluded from synaptic vesicles that undergo action potential-dependent recycling. These data indicate that the molecular identity of opposing exocytotic membranes is preserved by the sorting of syntaxin-1A from recycling synaptic vesicles.
Action Potentials↗
Regulatory roles of complexins in neurotransmitter release from mature presynaptic nerve terminals.
Complexins are presynaptic proteins whose functional roles in synaptic transmission are still unclear. In cultured rat hippocampal neurons, complexins are distributed throughout the cell bodies, dendrites and axons, whereas synaptotagmin I and synaptobrevin/VAMP-2, essential proteins for neurotransmitter release, accumulated in the synaptic-releasing sites as early as 1 week in culture. With a maturation of synapses in vitro, complexins also accumulated in the synaptic release sites and co-localized with synaptotagmin I and synaptobrevin/VAMP-2 after 3-4 weeks in culture. Complexins I and II were expressed in more than 90 and 70% of the cultured neurons, respectively; however, they were largely distributed in different populations of synaptic terminals. In the developing rat brain, complexins were distributed in neuronal cell bodies in the early stage of postnatal development, but gradually accumulated in the synapse-enriched regions with development. In mature presynaptic neurons of Aplysia buccal ganglia, injection of anticomplexin II antibody caused a stimulation of neurotransmitter release. Injection of recombinant complexin II and alphaSNAP caused depression and facilitation of neurotransmitter release from nerve terminals, respectively. The effect of complexin was reversed by a subsequent injection of recombinant alphaSNAP, and vice versa. These results suggest that complexins are not essential but have some regulatory roles in neurotransmitter release from presynaptic terminals of mature neurons.
Adaptor Proteins, Vesicular Transport↗
Highwire regulates synaptic growth in Drosophila.
The formation, stabilization, and growth of synaptic connections are dynamic and highly regulated processes. The glutamatergic neuromuscular junction (NMJ) in Drosophila grows new boutons and branches throughout larval development. A primary walking behavior screen followed by a secondary anatomical screen led to the identification of the highwire (hiw) gene. In hiw mutants, the specificity of motor axon pathfinding and synapse formation appears normal. However, NMJ synapses grow exuberantly and are greatly expanded in both the number of boutons and the extent and length of branches. These synapses appear normal ultrastructurally but have reduced quantal content physiologically. hiw encodes a large protein found at presynaptic terminals. Within presynaptic terminals, HIW is localized to the periactive zone surrounding active zones; Fasciclin II (Fas II), which also controls synaptic growth, is found at the same location.
Animals↗
Calcium dependence of presynaptic calcium current and post-synaptic response at the squid giant synapse.
1. Neurotransmitter release has a non-linear dependence upon the external Ca concentration, [Ca]o. This may be due to a 'co-operative' action of Ca in triggering release. The dependence of presynaptic Ca currents and post-synaptic currents (p.s.c.s) upon [Ca]o was examined at voltage-clamped 'giant' synapses of squid to determine whether this 'co-operativity' occurs during or after influx of Ca into the presynaptic terminal. 2. Presynaptic Ca current was proportional to [( Ca]o/(1 + [Ca]o/KD]n, where n, the order of the function, was roughly 1 and KD, the apparent dissociation constant for Ca, was approximately 80 mM. 3. P.s.c.s also could be described by the same function, but had an n of 3-4 and a lower KD. 4. These results suggest that the 'co-operative' action of Ca occurs at a step or steps beyond entry of Ca into the presynaptic terminal. 5. Synaptic transfer curves relating presynaptic Ca currents, elicited by depolarizations to different potentials, to resultant p.s.c.s were power functions whose exponent depended upon [Ca]o. Maximum exponents were as high as 4 at [Ca]o of 3 mM. The dependence of these curves upon [Ca]o helps to explain why previous determinations, which were performed at a variety of [Ca]o levels, yielded a variety of transfer curve exponent values. 6. Transfer curves generated from responses to constant presynaptic depolarizations, with Ca current varied by [Ca]o changes, also were power functions with exponents of approximately 4. Thus p.s.c.s were high-exponent power functions of Ca current regardless of whether Ca current was modified by changes in membrane potential or in [Ca]o.
Action Potentials↗
Calcium entry and transmitter release at voltage-clamped nerve terminals of squid.
Presynaptic and post-synaptic cells of the squid giant synapse were voltage-clamped simultaneously to study the relationship between presynaptic Ca current and transmitter-induced post-synaptic current (p.s.c.). Local Ca application was used to restrict Ca current and transmitter release to a limited region of the presynaptic terminal and thus minimize errors due to spatial heterogeneity of presynaptic membrane potential. Presynaptic terminals were depolarized by brief (3-6 ms) voltage-clamp pulses of varying amplitude to collect graded series of presynaptic Ca current and p.s.c. records. During presynaptic depolarization at 14 degrees C, Ca current activation preceded initial onset of p.s.c. (on-p.s.c.) by an interval of approximately 1 ms. The main component of on-p.s.c. followed Ca current activation by about 2 ms. The delay between a brief Ca tail current and peak response of the p.s.c. produced after pulse termination (off-p.s.c.) was also approximately 2 ms. Curves relating both Ca current and p.s.c. magnitudes to presynaptic potential were bell shaped with peaks near -10 mV, but the p.s.c. curve showed stronger voltage dependence on both sides of the peak. With very small and very large presynaptic command pulses, Ca current could be observed without measureable p.s.c. Synaptic transfer curves, plotting p.s.c. as a function of presynaptic Ca current, resembled third-power functions. On the average, p.s.c.s fit a curve representing the 2.9 power of Ca current (range 2.4-3.5 in eighteen experiments). Transfer curves consisted of two limbs: one from presynaptic pulses below -10 mV and the other from more positive pulses. These two limbs were similar and generally resembled power functions of identical exponent. It is thus likely that the third-power function accurately reflects synaptic current transfer, rather than interference from some other voltage-dependent process. Power functions fitting small-pulse and large-pulse limbs of some transfer curves had different scale coefficients, even though exponent values were the same. Consideration of synaptic transmission kinetics suggests that the voltage dependence of Ca channel opening rates can probably explain the difference in transfer curve limbs. Our experiments provide no evidence for an intrinsic voltage dependence of the transmitter release process.
Action Potentials↗
Ca(2+) changes induced by different presynaptic nicotinic receptors in separate populations of individual striatal nerve terminals.
Presynaptic nicotinic acetylcholine receptors likely play a modulatory role in the nerve terminal. Using laser-scanning confocal microscopy, we have characterized physiological responses obtained on activation of presynaptic nicotinic receptors by measuring calcium changes in individual nerve terminals (synaptosomes) isolated from the rat corpus striatum. Nicotine (500 nM) induced Ca(2+) changes in a subset (10-25%) of synaptosomes. The Ca(2+) responses were dependent on extracellular Ca(2+) and desensitized very slowly (several minutes) on prolonged exposure to agonist. The nicotine-induced Ca(2+) responses were dose-dependent and were completely blocked by dihydro-beta-erythroidine (5 microM), differentially affected by mecamylamine (10 microM) and alpha-conotoxin MII (100 nM), and not affected by alpha-bungarotoxin (500 nM). Immunocytochemical studies using well-characterized monoclonal antibodies revealed the presence of the alpha4 and alpha3/alpha5 nicotinic subunits. The nicotine-induced responses were unaffected by prior depolarization or by a mixture of Ca(2+) channel toxins including omega-conotoxin MVIIC (500 nM), omega-conotoxin GVIA (500 nM) and agatoxin TK (200 nM). Our results indicate that nicotinic receptors present on striatal nerve terminals induce Ca(2+) entry largely without involving voltage-gated Ca(2+) channels, most likely by direct permeation via the receptor channel itself. In addition, at least two subpopulations of presynaptic nicotinic receptors reside on separate terminals in the striatum, suggesting distinct modulatory roles.
Animals↗
Analysis of repolarization of presynaptic motor terminals in Drosophila larvae using potassium-channel-blocking drugs and mutations.
In Drosophila melanogaster muscles and neuronal cell bodies at least four different potassium currents have been identified whose activity shapes the electrical properties of these cells. Potassium currents also control repolarization of presynaptic terminals and, therefore, exert a major effect on transmitter release and synaptic plasticity. However, because of the small size of presynaptic terminals in Drosophila, it has not been possible to analyze the potassium currents they express. As a first approach to characterizing the ionic currents present at presynaptic motor terminals of Drosophila larvae, we recorded synaptic currents at the neuromuscular junction. From the alterations in evoked synaptic currents caused by various drugs and by mutations known to affect potassium currents in other tissues, we suggest that the repolarizing mechanism in presynaptic terminals consists of at least four distinct currents. One is affected by aminopyridines or Sh mutations, a second component is affected by the slo mutation, a third is sensitive to quinidine and one or more additional components are blocked by tetraethylammonium. Depolarization depends on a presynaptic calcium current, which displays only slight voltage-dependent inactivation. Because the mechanism of repolarization exerts a major effect on synaptic activity, this analysis provides a framework for further genetic and molecular dissection of the basic processes involved in the regulation of transmitter release.
Animals↗