Stephen Meriney
University of Pittsburgh, Neuroscience, Faculty Member
Research Interests: Genetics()
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Research Interests: Channels()
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We have studied voltage-dependent inhibition of N-type calcium currents to investigate the effects of G-protein modulation-induced alterations in channel gating on action potential-evoked calcium current. In isolated chick ciliary... more
We have studied voltage-dependent inhibition of N-type calcium currents to investigate the effects of G-protein modulation-induced alterations in channel gating on action potential-evoked calcium current. In isolated chick ciliary ganglion neurons, GTPgammaS produced voltage-dependent inhibition that exhibited slowed activation kinetics and was partially relieved by a conditioning prepulse. Using step depolarizations to evoke calcium current, we measured tail current amplitudes on abrupt repolarization to estimate the time course of calcium channel activation from 1 to 30 ms. GTPgammaS prolonged significantly channel activation, consistent with the presence of kinetic slowing in the modulated whole cell current evoked by 100-ms steps. Since kinetic slowing is caused by an altered voltage dependence of channel activation (such that channels require stronger or longer duration depolarization to open), we asked if GTPgammaS-induced modulation would alter the time course of calcium channel activation during an action potential. Using an action potential waveform as a voltage command to evoke calcium current, we abruptly repolarized to -80 mV at various time points during the repolarization phase of the action potential. The resulting tail current was used to estimate the relative number of calcium channels that were open. Using action potential waveforms of either 2.2- or 6-ms duration at half-amplitude, there were no differences in the time course of calcium channel activation, or in the percent activation at any time point tested during the repolarization, when control and modulated currents were compared. It is also possible that modulated channels might open briefly and that these reluctant openings would effect the time course of action potential-evoked calcium current. However, when control and modulated currents were scaled to the same peak amplitude and superimposed, there was no difference in the kinetics of the two currents. Thus voltage-dependent inhibition did not alter the kinetics of action potential-evoked current. These results suggest that G-protein-modulated channels do not contribute significantly to calcium current evoked by a single action potential.
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Cysteine string proteins (CSPs) are evolutionarily conserved proteins that are associated with synaptic vesicles and other regulated secretory organelles. To investigate the role of CSPs in vertebrate neuromuscular transmission, we... more
Cysteine string proteins (CSPs) are evolutionarily conserved proteins that are associated with synaptic vesicles and other regulated secretory organelles. To investigate the role of CSPs in vertebrate neuromuscular transmission, we introduced anti-CSP antibodies into the cell bodies of Xenopus spinal motor neurons that form synapses with embryonic muscle cells in culture. These antibodies produced a rapid (within 3-6 min), and in most cases complete, inhibition of stimulus-dependent neurotransmitter secretion. However, spontaneous neurotransmitter release was stable (both in frequency and amplitude) throughout the period of antibody exposure. Several control experiments validated the specificity of the anti-CSP antibody effects. First, the anti-CSP antibody actions were not mimicked either by antibodies against another synaptic vesicle protein SV2, or by nonspecific immunoglobins. Second, heat treatment of the anti-CSP antibodies eliminated their effect on evoked secretion. Third, immunoblot experiments showed that the anti-CSP and anti-SV2 antibodies were highly selective for their respective antigens in these Xenopus cultures. We conclude from these results that CSPs are vital constituents of the pathway for regulated neurotransmitter release in vertebrates. Moreover, the selective inhibition of evoked, but not spontaneous transmitter release by anti-CSP antibodies indicates that there is a fundamental difference in the machinery that mediates these secretory processes.
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Voltage-dependent calcium (Ca2+) currents were characterized and modulatory effects of somatostatin were measured in acutely dissociated chick ciliary ganglion neurons at embryonic stages 34, 37, and 40. This developmental time period... more
Voltage-dependent calcium (Ca2+) currents were characterized and modulatory effects of somatostatin were measured in acutely dissociated chick ciliary ganglion neurons at embryonic stages 34, 37, and 40. This developmental time period coincides with the period of synapse formation between ciliary ganglion neurons and peripheral eye muscles. At all three developmental stages Ca2+ current could be blocked almost completely by combined application of omega-CgTX GVIA and nitrendipine. At young embryonic ages there was significant overlap in sensitivity, with approximately 75% of the current sensitive to either blocker applied independently. By stage 40, there was very little or no overlap in sensitivity, with approximately 75% of the current blocked by omega-CgTX GVIA (N-type) and 30% blocked by nitrendipine (L-type). These data are consistent with earlier findings that the pharmacology of acetylcholine release from ciliary ganglion nerve terminals changes during development from sensit...
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To assess physiological accommodative function of intact intraocular lenses, as measured by focal length changes, in chickens of various ages. Eyes of white leghorn chickens, aged 0 days (hatchlings), 7 days, 14 days, 6 weeks, 1 year, and... more
To assess physiological accommodative function of intact intraocular lenses, as measured by focal length changes, in chickens of various ages. Eyes of white leghorn chickens, aged 0 days (hatchlings), 7 days, 14 days, 6 weeks, 1 year, and 2 years, were enucleated and the backs of the globes, except for the ciliary nerve and ganglion, were removed. The ciliary nerve and ganglion were suctioned into the tip of a suction electrode, and lenses were optically scanned before, during, and after accommodation. Accommodation was elicited by delivery of 30-Hz electrical pulses. For all age groups, lenticular focal lengths for stimulated eyes were significantly shorter than for relaxed eyes, indicating that accommodation had been induced. Lenticular accommodative amplitudes decreased significantly with age, with reduced function occurring between 0 and 7 days and again between 14 days and 1 year. Although accommodation was associated with an increased spherical aberration in all age groups, th...
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The understanding of neurotransmitter release at vertebrate synapses has been hampered by the paucity of preparations in which presynaptic ionic currents and postsynaptic responses can be monitored directly. We used cultured embryonic... more
The understanding of neurotransmitter release at vertebrate synapses has been hampered by the paucity of preparations in which presynaptic ionic currents and postsynaptic responses can be monitored directly. We used cultured embryonic Xenopus neuromuscular junctions and simultaneous pre- and postsynaptic patch-clamp current-recording procedures to identify the major presynaptic conductances underlying the initiation of neurotransmitter release. Step depolarizations and action potential waveforms elicited Na and K currents along with Ca and Ca-activated K (KCa) currents. The onset of KCa current preceded the peak of the action potential. The predominantly omega-CgTX GVIA-sensitive Ca current occurred primarily during the falling phase, but there was also significant Ca2+ entry during the rising phase of the action potential. The postsynaptic current began a mean of 0.7 msec after the time of maximum rate of rise of the Ca current. omega-CgTX also blocked KCa currents and transmitter release during an action potential, suggesting that Ca and KCa channels are colocalized at presynaptic active zones. In double-ramp voltage-clamp experiments, KCa channel activation is enhanced during the second ramp. The 1 msec time constant of decay of enhancement with increasing interpulse interval may reflect the time course of either the deactivation of KCa channels or the diffusion/removal of Ca2+ from sites of neurotransmitter release after an action potential.
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The mechanical and pharmacological characteristics of the cholinergic activation of the smooth muscle in the choroidal coat of the chick eye have been assessed in tissues isolated from birds 1 d posthatching using histological,... more
The mechanical and pharmacological characteristics of the cholinergic activation of the smooth muscle in the choroidal coat of the chick eye have been assessed in tissues isolated from birds 1 d posthatching using histological, electrophysiological, and immunological techniques. The choroidal coat is innervated by a dense network of cholinergic nerves that make en passant synapses with smooth muscle. Thirty-hertz stimulation of these nerves initiates red blood cell (RBC) movement in the vessels of the choroidal coat, and this activation is blocked by muscarinic ACh receptor (AChR) antagonists. Force-transducer recordings of nerve-induced contractions of this tissue have a slow onset and relaxation time course similar to those of smooth muscle contractions. Furthermore, since nearly half the cholinergic neurons innervating the choroid die within a defined period during development, the onset and pharmacology of this innervation were studied during embryogenesis. With a neural cytoske...
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We have described in the preceding 2 papers the development of the pharmacological and contractile properties of all targets of the ciliary ganglion: the iris and ciliary body (Pilar et al., 1987), and the choroidal coat (Meriney and... more
We have described in the preceding 2 papers the development of the pharmacological and contractile properties of all targets of the ciliary ganglion: the iris and ciliary body (Pilar et al., 1987), and the choroidal coat (Meriney and Pilar, 1987). In this paper, we examine the chronic effects of ACh receptor (AChR) blockade on ciliary ganglion neuron survival. Nicotinic or muscarinic AChR blockers were administered daily to developing chicken embryos during the normal neuronal death period in the ciliary ganglion. The effects of the blockers on ganglionic and neuromuscular transmission were assessed, and neuronal survival was assayed by counting both the total number of ganglion neurons and the selectively HRP-labeled ciliary neurons after the normal neuronal death period. Blockade of ganglionic transmission decreases survival in both populations of neurons. Blockade of neuromuscular muscular transmission increases survival in the ciliary population, which innervates the striated ir...
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The development of the mechanical characteristics of contraction and the pharmacology of synaptic activation in chick iris and ciliary body were examined from embryonic day 9 through posthatching. The ciliary ganglion-target muscle system... more
The development of the mechanical characteristics of contraction and the pharmacology of synaptic activation in chick iris and ciliary body were examined from embryonic day 9 through posthatching. The ciliary ganglion-target muscle system has proven to be a useful model for both in vivo and in vitro studies of neuron-target interactions; one such interaction is involved in neuronal cell death, which in the ciliary ganglion occurs from Stage (St) 34 to 40. To understand the mechanism by which cholinergic blocking agents prevent naturally occurring neuronal death in the chick ciliary ganglion (see the following paper, Meriney et al., 1987), it was necessary to determine the effect of these agents on synaptic transmission at target structures during the cell death period. Initially (St 34-36), iris muscle contraction are synaptically mediated via muscarinic ACh receptors (AChRs) on myoepithelial cells, which have the contractile and structural characteristics of smooth muscle. Close ap...
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Most studies on the trophic regulation of the normal neuronal competition for survival have focused on interactions between neurons and their target environment. However, it is also likely that trophic modulators are released from... more
Most studies on the trophic regulation of the normal neuronal competition for survival have focused on interactions between neurons and their target environment. However, it is also likely that trophic modulators are released from premotor inputs onto motoneurons. We have examined the developmental distribution of endogenous enkephalin-like immunoreactivity and the role that these endogenous opioid peptides play in normal neuronal degeneration. During the early portion of the normal cell death period, enkephalin-like immunoreactivity is highest within preganglionic cell bodies in the midbrain and their nerve terminals in the ciliary ganglion. Exogenous daily morphine administration to the chick embryo has previously been shown to delay most of the normal neuronal death in the ciliary ganglion (see Meriney et al., 1985). We hypothesized that opiate receptor activation increases the probability that ciliary ganglion neurons will survive their developmental competition and, further, th...
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The quantitative relationship between presynaptic calcium influx and transmitter release critically depends on the spatial coupling of presynaptic calcium channels to synaptic vesicles. When there is a close association between calcium... more
The quantitative relationship between presynaptic calcium influx and transmitter release critically depends on the spatial coupling of presynaptic calcium channels to synaptic vesicles. When there is a close association between calcium channels and synaptic vesicles, the flux through a single open calcium channel may be sufficient to trigger transmitter release. With increasing spatial distance, however, a larger number of open calcium channels might be required to contribute sufficient calcium ions to trigger vesicle fusion. Here we used a combination of pharmacological calcium channel block, high-resolution calcium imaging, postsynaptic recording, and 3D Monte Carlo reaction-diffusion simulations in the adult frog neuromuscular junction, to show that release of individual synaptic vesicles is predominately triggered by calcium ions entering the nerve terminal through the nearest open calcium channel. Furthermore, calcium ion flux through this channel has a low probability of triggering synaptic vesicle fusion (~6%), even when multiple channels open in a single active zone. These mechanisms work to control the rare triggering of vesicle fusion in the frog neuromuscular junction from each of the tens of thousands of individual release sites at this large model synapse.
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Research Interests: Cognitive Science, Calcium, Humans, Animals, Membrane Fusion, and 8 morePKC, Snap, Exocytosis, SNARE proteins, Rab, GFP, Neurosciences, and TIRF
Research Interests: Neuroscience, Psychology, Monte Carlo Simulation, Kinetics, Patch-clamp and imaging techniques, and 15 moreCell line, Computer Simulation, Animals, Calcium Signaling, Calcium Channel, Synaptic Transmission, Neurons, Rats, Time Factors, Lumping Kinetic Model, Neurosciences, Monte Carlo Method, Protein Kinase Inhibitors, Patch Clamp, and Cell Membrane
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Lambert-Eaton myasthenic syndrome (LEMS) is an autoimmune disease that disrupts the normally reliable neurotransmission at the neuromuscular junction (NMJ). This disruption is thought to result from an autoantibody-mediated removal of a... more
Lambert-Eaton myasthenic syndrome (LEMS) is an autoimmune disease that disrupts the normally reliable neurotransmission at the neuromuscular junction (NMJ). This disruption is thought to result from an autoantibody-mediated removal of a subset of the P/Q-type Ca(2+) channels involved with neurotransmitter release. With less neurotransmitter release at the NMJ, LEMS patients experience debilitating muscle weakness. The underlying cause of LEMS in slightly more than half of all patients is small cell lung cancer, and cancer therapy is the priority for these patients. In the remaining cases, the cause of LEMS is unknown, and these patients often rely on symptomatic treatment options, as there is no cure. However, current symptomatic treatment options, such as 3,4-diaminopyridine (3,4-DAP), can have significant dose-limiting side effects; thus, additional treatment approaches would benefit LEMS patients. Recent studies introduced a novel Ca(2+) channel agonist (GV-58) as a potential therapeutic alternative for LEMS. Additionally, this work has shown that GV-58 and 3,4-DAP interact in a supra-additive manner to completely restore the magnitude of neurotransmitter release at the NMJs of a LEMS mouse model. In this review, we discuss synaptic mechanisms for reliability at the NMJ and how these mechanisms are disrupted in LEMS. We then discuss the current treatment options for LEMS patients, while also considering recent work demonstrating the therapeutic potential of GV-58 alone and in combination with 3,4-DAP.
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Lambert-Eaton myasthenic syndrome (LEMS) is an autoimmune disorder in which a significant fraction of the presynaptic P/Q-type Ca(2+) channels critical to the triggering of neurotransmitter release at the neuromuscular junction (NMJ) are... more
Lambert-Eaton myasthenic syndrome (LEMS) is an autoimmune disorder in which a significant fraction of the presynaptic P/Q-type Ca(2+) channels critical to the triggering of neurotransmitter release at the neuromuscular junction (NMJ) are thought to be removed. There is no cure for LEMS, and the current most commonly used symptomatic treatment option is a potassium channel blocker [3,4-diaminopyridine (3,4-DAP)] that does not completely reverse symptoms and can have dose-limiting side-effects. We previously reported the development of a novel Ca(2+) channel agonist, GV-58, as a possible alternative treatment strategy for LEMS. In this study, we tested the hypothesis that the combination of GV-58 and 3,4-DAP will elicit a supra-additive increase in neurotransmitter release at LEMS model NMJs. First, we tested GV-58 in a cell survival assay to assess potential effects on cyclin-dependent kinases (Cdks) and showed that GV-58 did not affect cell survival at the relevant concentrations fo...
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Short-term synaptic facilitation occurs during high-frequency stimulation, is known to be dependent on presynaptic calcium ions, and persists for tens of milliseconds after a presynaptic action potential. We have used the frog... more
Short-term synaptic facilitation occurs during high-frequency stimulation, is known to be dependent on presynaptic calcium ions, and persists for tens of milliseconds after a presynaptic action potential. We have used the frog neuromuscular junction as a model synapse for both experimental and computer simulation studies aimed at testing various mechanistic hypotheses proposed to underlie short-term synaptic facilitation. Building off our recently reported excess-calcium-binding-site model of synaptic vesicle release at the frog neuromuscular junction (Dittrich M, Pattillo JM, King JD, Cho S, Stiles JR, Meriney SD. Biophys J 104: 2751-2763, 2013), we have investigated several mechanisms of short-term facilitation at the frog neuromuscular junction. Our studies place constraints on previously proposed facilitation mechanisms and conclude that the presence of a second class of calcium sensor proteins distinct from synaptotagmin can explain known properties of facilitation observed at the frog neuromuscular junction. We were further able to identify a novel facilitation mechanism, which relied on the persistent binding of calcium-bound synaptotagmin molecules to lipids of the presynaptic membrane. In a real physiological context, both mechanisms identified in our study (and perhaps others) may act simultaneously to cause the experimentally observed facilitation. In summary, using a combination of computer simulations and physiological recordings, we have developed a stochastic computer model of synaptic transmission at the frog neuromuscular junction, which sheds light on the facilitation mechanisms in this model synapse.
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1. The voltage dependence and kinetic properties of stage 40 ciliary ganglion calcium currents were determined using short (10 ms) voltage steps. These properties aided the interpretation of the action potential-evoked calcium current... more
1. The voltage dependence and kinetic properties of stage 40 ciliary ganglion calcium currents were determined using short (10 ms) voltage steps. These properties aided the interpretation of the action potential-evoked calcium current described below, and the comparison of our data with those observed in other preparations. 2. Three different natural action potential waveforms were modelled by a series of ramps to generate voltage clamp commands. Calcium currents evoked by these model action potentials were compared before and after alterations in the repolarization phase of each action potential. 3. Abrupt step repolarizations from various time points were used to estimate the time course of calcium current activation during each action potential. Calcium current evoked by fast action potentials (duration at half-amplitude, 0.5 or 1.0 ms) did not reach maximal activation until the action potential had repolarized by 40-50 %. In contrast, calcium current evoked by a slow action potential (duration at half-amplitude, 2.2 ms) was maximally activated near the peak of the action potential. 4. Slowing the rate of repolarization of the action potential (broadening) from different times was used to examine effects on peak and total calcium influx. With all three waveforms tested, broadening consistently increased total calcium influx (integral). However, peak calcium current was either increased or decreased depending on the duration of the control action potential tested and the specific timing of the initiation of broadening the repolarization phase. 5. The opposite effects on peak calcium current observed with action potential broadening beginning at different time points in repolarization may provide a mechanism for the variable effects of potassium channel blockers on transmitter release magnitude.
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Repeated administration of morphine in increasing doses delayed normal cell death in the ciliary ganglion of the chick embryo; the effect was completely blocked by naloxone. Survival of spinal motoneurons was not affected. Morphine also... more
Repeated administration of morphine in increasing doses delayed normal cell death in the ciliary ganglion of the chick embryo; the effect was completely blocked by naloxone. Survival of spinal motoneurons was not affected. Morphine also inhibited potassium-stimulated synthesis of acetylcholine in ganglion cells cultured with muscle, suggesting that morphine can influence neurotransmission. Morphine's effect on cell death may be due to an inhibition of transmission at the neuromuscular junction, but opiates may also directly affect cell death. Although it is now known whether the endogenous opiates in the ciliary ganglion influence neuronal survival during embryogenesis, exogenous opiates can affect normal cell death in the autonomic nervous system.
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Research Interests: Electrophysiology, Calcium, Motor neuron, Cell Culture, Biological Sciences, and 17 moreSynapse, Calcium Imaging, Humans, Animals, Calcium Signaling, Calcium Channel, Synaptic Transmission, Neuromuscular Disease, Transmitter release, Neuromuscular diseases, Muscle contraction, Action potential, Neuromuscular Junction, Nervous System Diseases, Patch Clamp, Electron Microscope, and Motor Neurons(Synapse, Calcium Imaging, Humans, Animals, Calcium Signaling, Calcium Channel, Synaptic Transmission, Neuromuscular Disease, Transmitter release, Neuromuscular diseases, Muscle contraction, Action potential, Neuromuscular Junction, Nervous System Diseases, Patch Clamp, Electron Microscope, and Motor Neurons)
(Synapse, Calcium Imaging, Humans, Animals, Calcium Signaling, Calcium Channel, Synaptic Transmission, Neuromuscular Disease, Transmitter release, Neuromuscular diseases, Muscle contraction, Action potential, Neuromuscular Junction, Nervous System Diseases, Patch Clamp, Electron Microscope, and Motor Neurons)
Dynasore was recently developed as a small molecule, selective non-competitive inhibitor of the protein dynamin. This inhibitor has been shown to block dynamin-dependent endocytosis and is now used commonly to study vesicular recycling at... more
Dynasore was recently developed as a small molecule, selective non-competitive inhibitor of the protein dynamin. This inhibitor has been shown to block dynamin-dependent endocytosis and is now used commonly to study vesicular recycling at synapses. We have measured the effects of dynasore on spontaneous and evoked transmitter release at the frog neuromuscular junction and shown that, in addition to inhibiting endocytosis, dynasore increases the probability of transmitter release. Furthermore, we have shown that dynasore exposure leads to an increase in resting intra-terminal calcium, but this effect does not completely account for the dynasore-mediated increase in the probability of transmitter release. Therefore, in interpreting effects of the dynamin inhibitor dynasore at synapses, one must be alert to potential increases in presynaptic calcium concentration and transmitter release probability.
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Research Interests: Neuroscience, Psychology, Calcium, Animals, Calcium Signaling, and 14 moreCalcium Channel, Peptides, Acetylcholine, Potassium, Transmitter release, Xenopus laevis, Potassium Channels, Action potential, Action Potentials, Voltage Clamp, Neuromuscular Junction, Neurosciences, Patch Clamp, and Driving force
Neuroligins are a family of cell adhesion molecules critical in establishing proper central nervous system connectivity; disruption of neuroligin signaling in vivo precipitates a broad range of cognitive deficits. Despite considerable... more
Neuroligins are a family of cell adhesion molecules critical in establishing proper central nervous system connectivity; disruption of neuroligin signaling in vivo precipitates a broad range of cognitive deficits. Despite considerable recent progress, the specific synaptic function of neuroligin-1 (NL1) remains unclear. A current model proposes that NL1 acts exclusively to mature pre-existent synaptic connections in an activity-dependent manner. A second element of this activity-dependent maturation model is that an alternate molecule acts upstream of NL1 to initiate synaptic connections. SynCAM1 (SC1) is hypothesized to function in this capacity, though several uncertainties remain regarding SC1 function. Using overexpression and chronic pharmacological blockade of synaptic activity, we now demonstrate that NL1 is capable of robustly recruiting synapsin-positive terminals independent of synaptic maturation and activity in 2-week old primary hippocampal neuronal cultures. We further report that neither SC1 overexpression nor knockdown of endogenous SC1 impacts synapsin punctum densities, suggesting that SC1 is not a limiting factor of synapse initiation in maturing hippocampal neurons in vitro. Consistent with these findings, we observed profoundly greater recruitment of synapsin-positive presynaptic terminals by NL1 than SC1 in a mixed-culture assay of artificial synaptogenesis between primary neurons and heterologous cells. Collectively, our results contend multiple aspects of the proposed model of NL1 and SC1 function and motivate an alternative model whereby SC1 may mature synaptic connections forged by NL1. Supporting this model, we present evidence that combined NL1 and SC1 overexpression triggers excitotoxic neurodegeneration through SC1 signaling at synaptic connections initiated by NL1.
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The signal transduction cascade between the activation of the somatostatin (SOM) receptor and modulation of transmitter release was study using Acetylcholine (Ach) release measurements and patch clamp recordings of Ca2+ current from... more
The signal transduction cascade between the activation of the somatostatin (SOM) receptor and modulation of transmitter release was study using Acetylcholine (Ach) release measurements and patch clamp recordings of Ca2+ current from acutely dissociated St 40 ciliary ganglion neurons. As in intact synapses, somal ACh release was blocked by 100 nM SOM or 100 microM dibutyril cGMP, and the SOM-mediated inhibition could be reversed by 10 microM 1-NAME (a selective inhibitor of nitric oxide synthase, NOS) or 100 microM Rp-8p-CPT-cGMPs (a selective inhibitor of a cGMP protein dependent kinase, PKG). In whole cell recordings, SOM inhibition of Ca2+ current rapidly relaxes to control levels but is sustained in perforated patch recordings which decreases cell dialysis. Inhibition of NOS or PKG in perforated patch recordings, however caused SOM effects to become transient again. We hypothesize that PKG alters the characteristics of the membrane-delimited G protein inhibition of Ca2+ current. Therefore SOM receptors trigger a membrane-delimited signal transduction cascade that is modulated by soluble messengers, converging on voltage activated Ca2+ channels. When both pathways are active together, SOM causes a sustained inhibition of neuronal Ca2+ current leading to a decrease in transmitter release.
Research Interests: Life Sciences, Calcium, Signal Transduction, Somatostatin, Nitric oxide, and 14 moreAnimals, Acetylcholine, Whole cell recording, Nitric Oxide Synthase, Transmitter release, Chickens, Perforation, Protein Kinase, G protein, Patch Clamp, cyclic GMP, Biochemistry and cell biology, Cell Membrane, and Choroid
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Calcium (Ca2+) influx through presynaptic calcium channels triggers transmitter release, and any alterations in the gating of these calcium channels results in changes in the magnitude of transmitter released. We used (R)-roscovitine, a... more
Calcium (Ca2+) influx through presynaptic calcium channels triggers transmitter release, and any alterations in the gating of these calcium channels results in changes in the magnitude of transmitter released. We used (R)-roscovitine, a cyclin-dependent kinase inhibitor that also appears to act directly on calcium channels, as a tool to modulate presynaptic calcium influx and study effects on transmitter release. We show that this compound increased the quantal content of acetylcholine released from the Rana frog motor nerve terminal (by 149%) without changing paired-pulse facilitation (under low calcium conditions). In contrast, exposure to 3,4-diaminopyridine (DAP; which similarly affects transmitter release by partially blocking potassium channels, altering the shape of the presynaptic action potential, and indirectly increasing calcium entry) increased paired-pulse facilitation (by 23%). In addition, we show that (R)-roscovitine predominately slowed deactivation kinetics of calcium current (by 427%) recorded from Xenopus frog motoneurons, and as a result, increased the integral of calcium channel current evoked by a physiological action potential waveform (by 44%). Because we did not observe any significant effects of structurally related cyclin-dependent kinase inhibitors [(S)-roscovitine or olomoucine] on evoked transmitter release or calcium current kinetics, it appears that these effects of (R)-roscovitine are independent of cyclin-dependent kinases (cdks). In summary, we hypothesize that (R)-roscovitine effects on transmitter release at the adult frog neuromuscular junction (NMJ) are mediated by its effects on calcium channel gating, and these effects increase our understanding of calcium triggered secretion at this synapse.
Research Interests: Psychology, Cognitive Science, Calcium, Patch-clamp and imaging techniques, Animals, and 11 moreCalcium channels, Calcium Channel, Synaptic Transmission, Transmitter release, Xenopus laevis, Aminopyridine, Neuromuscular Junction, Neurosciences, Protein Kinase Inhibitors, Kinetin, and Excitatory Postsynaptic Potentials
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Despite decades of intense experimental studies, we still lack a detailed understanding of synaptic function. Fortunately, using computational approaches, we can obtain important new insights into the inner workings of these important... more
Despite decades of intense experimental studies, we still lack a detailed understanding of synaptic function. Fortunately, using computational approaches, we can obtain important new insights into the inner workings of these important neural systems. Here, we report the development of a spatially realistic computational model of an entire frog active zone in which we constrained model parameters with experimental data, and then used Monte Carlo simulation methods to predict the Ca(2+)-binding stoichiometry and dynamics that underlie neurotransmitter release. Our model reveals that 20-40 independent Ca(2+)-binding sites on synaptic vesicles, only a fraction of which need to bind Ca(2+) to trigger fusion, are sufficient to predict physiological release. Our excess-Ca(2+)-binding-site model has many functional advantages, agrees with recent data on synaptotagmin copy number, and is the first (to our knowledge) to link detailed physiological observations with the molecular machinery of Ca(2+)-triggered exocytosis. In addition, our model provides detailed microscopic insight into the underlying Ca(2+) dynamics during synapse activation.