Role of Stored Calcium in the Regulation of Neurotransmitter Quantum Size

Release of stored calcium ions during activation of ryanodine receptors with ryanodine or caffeine elevates the mean amplitude of spontaneous miniature end-plate potentials. Blockade of these receptors with selective antagonists abolishes this effect. Preliminary loading of the motor nerve terminals with intracellular calcium buffer EGTA-AM, but not with BAPTA-AM, also completely prevented the ryanodine-induced increment of miniature end-plate potential amplitude probably induced by the release of stored calcium. Vesamicol, a selective blocker of acetylcholine transport into vesicles, prevented the ryanodine-induced increment of the mean amplitude of miniature end-plate potentials. This increment was 2-fold more pronounced after preliminary blockade of protein kinase C with chelerythrine and was completely abolished by blockade of protein kinase A with H-89.     

Kiss-and-Run Quantal Secretion in Frog Nerve-Muscle Synapse

Electrophysiological and optical methods were used to study exo- and endocytosis of synaptic vesicles underlying secretion of the neurotransmitter from motor nerve terminals in frog sternocutaneous muscle. Increase in extracellular concentration of K+ or sucrose produced similar increase in the frequency of miniature endplate currents recorded by extracellular microelectrode. Fluorescent microscopy revealed bright spots in nerve terminal during stimulation of secretion with high-potassium solutions in the presence of endocytosis marker FM1-43. These spots corresponded to clusters of synaptic vesicles that passed through the cycles of exo- and endocytosis. Subsequent high-potassium stimulation of exocytosis in normal Ringer solution led to disappearance of marker spots, while in hyperosmotic saline the spots were preserved. No spots were seen after stimulation of neurotransmitter secretion with sucrose in the presence of FM1-43. It is concluded that quantal secretion of the neurotransmitter in frog motor nerve endings can be realized via both complete exocytosis of synaptic vesicles with subsequent endocytosis and kiss-and-run mechanism with the formation of a temporary pore.     

The role of extracellular calcium in exo- and endocytosis of synaptic vesicles at the frog motor nerve terminals

In the present study we combined FM 1-43 imaging and electrophysiological recording of miniature end-plate currents (MEPCs) to determine the role of extracellular calcium in synaptic vesicle exo- and endocytosis at the frog motor nerve terminals. We replaced extracellular Ca2+ ions with other bivalent cations (Sr2+, Ba2+, Cd2+, Mg2+) or used a calcium-free solution and monitored fluorescent staining of the nerve terminals in the presence of caffeine, which promotes the release of Ca2+ from intracellular stores. Caffeine has induced FM1-43 internalization only in the presence of bivalent cations in the external solution. The exposure of the neuromuscular junction to caffeine in a calcium-free solution caused a reversible failure of FM 1-43 loading and an increase in the nerve terminal width. This effect of a calcium-free solution was not due to a decrease in exocytosis, because caffeine-induced FM1-43 unloading from the previously loaded nerve terminals, as well as a degree of the MEPCs frequency increase, was unchanged. We conclude that the presence of Ca2+ or other bivalent cations in extracellular space is necessary for endocytosis but not for exocytosis of synaptic vesicles, while transmitter release is promoted by efflux of Ca2+ from intracellular stores. The effect of extracellular Ca2+ on endocytosis might be driven by the non-specific interactions with membrane lipids.     

Empty synaptic vesicles recycle and undergo exocytosis at vesamicol-treated motor nerve terminals.

We investigated whether recycled cholinergic synaptic vesicles, which were not refilled with ACh, would join other synaptic vesicles in the readily releasable store near active zones, dock, and continue to undergo exocytosis during prolonged stimulation. Snake nerve-muscle preparations were treated with 5 microM vesamicol to inhibit the vesicular ACh transporter and then were exposed to an elevated potassium solution, 35 mM potassium propionate (35 KP), to release all preformed quanta of ACh. At vesamicol-treated endplates, miniature endplate current (MEPC) frequency increased initially from 0.4 to >300 s-1 in 35 KP but then declined to <1 s-1 by 90 min. The decrease in frequency was not accompanied by a decrease in MEPC average amplitude. Nerve terminals accumulated the activity-dependent dye FM1-43 when exposed to the dye for the final 6 min of a 120-min exposure to 35 KP. Thus synaptic membrane endocytosis continued at a high rate, although MEPCs occurred infrequently. After a 120-min exposure in 35 KP, nerve terminals accumulated FM1-43 and then destained, confirming that exocytosis also still occurred at a high rate. These results demonstrate that recycled cholinergic synaptic vesicles that were not refilled with ACh continued to dock and undergo exocytosis after membrane retrieval. Thus transport of ACh into recycled cholinergic vesicles is not a requirement for repeated cycles of exocytosis and retrieval of synaptic vesicle membrane during prolonged stimulation of motor nerve terminals.     

Effects of high-potassium solutions and caffeine on synaptic vesicle exoendocytosis processes in the frog neuromuscular junction

Studies on frog skin-pectoris muscle preparations using vital fluorescent microscopy showed that stimulation of transmitter secretion using high-potassium solutions with the endocytosis marker FM 1–43 induced bright spots in all motor nerve terminals, these representing accumulations of vesicles undergoing the exoendocytic cycle in the active zones of nerve endings. Stimulation of transmitter secretion with caffeine evoked bright spots only in some nerve terminals and only in some parts of the terminals. In summer, the number of bright spots on stimulation of transmitter secretion by caffeine increased sharply. Extracellular recording of spontaneous synaptic signals showed that high-potassium solutions, like caffeine, produced dose-dependent increases in the frequency of miniature endplate currents. However, while high-potassium solutions always increased the frequency, this occurred with caffeine in only a proportion of experiments. This leads to the conclusion that exoendocytosis processes can occur both because of the influx of Ca²⁺ ions into nerve endings as a result of depolarization (high-potassium solutions) and because of the release of Ca²⁺ ions from the endoplasmic reticulum (caffeine). The possible spatial localization of the endoplasmic reticulum in nerve endings is discussed. The endoplasmic reticulum is suggested to have a role in synapse remodeling processes. __________ Translated from Rossiiskii Fiziologicheskii Zhurnal imeni I. M. Sechenova, Vol. 91, No. 7, pp. 821–831, July, 2005.     

The effects of taipoxin and notexin on the function and fine structure of the murine neuromuscular junction

The isolated neurotoxins taipoxin and notexin from the venoms of the Elapidae, Oxyuranus scutellatus and Notechis scutatus scutatus respectively cause a neuromuscular block when administered to the mouse in vivo or to the phrenic nerve-hemidiaphragm preparation in vitro. The block is preceded by a latency period during which the toxins bind irreversibly to the nerve. The period is shortened by nerve activity. The frequency of the miniature end-plate potentials is gradually reduced, almost to zero, and their amplitude distribution is altered; small and very large miniature endplate potentials appearing. Ultrastructurally the endplates are altered in the presynaptic portion but not in the postsynaptic part. In an early stage of poisoning the axolemma has an increased number of omega-shaped indentations similar in size to synaptic vesicles. At a later stage, when the animals die of respiratory paralysis, the axolemmal indentations are more numerous and the synaptic vesicles greatly reduced in number, the remaining vesicles having a variable and frequently larger than normal size. When impulse activity in the phrenic nerve is stopped by cutting the nerve before the administration of toxin there is no reduction in the number of synaptic vesicles, only the appearance of an increased number of axolemmal indentations. It is suggested that taipoxin and notexin irreversibly interfere with the formation of synaptic vesicles by arresting vesicle membrane recycling at the level of the axolemma. When the pre-existing store of vesicles is depleted, by nerve activity, a neuromuscular block results.     

The pool of fast releasing vesicles is augmented by myosin light chain kinase inhibition at the calyx of Held synapse

Synaptic strength is determined by release probability and the size of the readily releasable pool of docked vesicles. Here we describe the effects of blocking myosin light chain kinase (MLCK), a cytoskeletal regulatory protein thought to be involved in myosin-mediated vesicle transport, on synaptic transmission at the mouse calyx of Held synapse. Application of three different MLCK inhibitors increased the amplitude of the early excitatory postsynaptic currents (EPSCs) in a stimulus train, without affecting the late steady-state EPSCs. A presynaptic locus of action for MLCK inhibitors was confirmed by an increase in the frequency of miniature EPSCs that left their average amplitude unchanged. MLCK inhibition did not affect presynaptic Ca²⁺ currents or action potential waveform. Moreover, Ca²⁺ imaging experiments showed that [Ca²⁺]_i transients elicited by 100-Hz stimulus trains were not altered by MLCK inhibition. Studies using high-frequency stimulus trains indicated that MLCK inhibitors increase vesicle pool size, but do not significantly alter release probability. Accordingly, when AMPA-receptor desensitization was minimized, EPSC paired-pulse ratios were unaltered by MLCK inhibition, suggesting that release probability remains unaltered. MLCK inhibition potentiated EPSCs even when presynaptic Ca²⁺ buffering was greatly enhanced by treating slices with EGTA-AM. In addition, MLCK inhibition did not affect the rate of recovery from short-term depression. Finally, developmental studies revealed that EPSC potentiation by MLCK inhibition starts at postnatal day 5 (P5) and remains strong during synaptic maturation up to P18. Overall, our data suggest that MLCK plays a crucial role in determining the size of the pool of synaptic vesicles that undergo fast release at a CNS synapse.     

Long-term use-dependent enhancement of impulse-induced exocytosis by adrenaline at frog motor nerve terminals

Adrenaline (5-20 microM) use-dependently increased end-plate potentials (EPPs) in normal Ringer solution (containing d-tubocurarine to partially block acetylcholine receptors) and a low Ca2+, high Mg2+ solution for more than several hours and decreased the coefficient of variation of EPP amplitude in the latter solution in frog neuromuscular junctions. The amplitude and frequency of miniature EPPs and impulse-induced increases in intraterminal Ca2+ concentration were unaffected. Adrenaline thus causes sustained enhancement of impulse-induced exocytosis by acting at a mechanism of exocytosis downstream to Ca2+ entry.     

Conductance of end-plate channels is voltage dependent.

The amplitude of miniature end-plate currents (MEPCs) and end-plate currents generated by iontophoresis of acetylcholine (ACh) were recorded in voltage-clamped toad sartorius fibres. Single channel conductance was determined from analysis of current fluctuations. In normal solution, but even more so in solutions containing lithium instead of sodium, both the peak conductance during a miniature end-plate current and the conductance of individual end-plate channels varied with membrane potential, becoming less at more hyperpolarized potentials. Single channel conductance was not influenced by membrane potential in solutions containing potassium or caesium instead of sodium. It was concluded that the nature of the ions passing through an end-plate channel influences its conductance.     

安氟醚对海马CA1区锥体神经元的自发性微小抑制性突触后电流的调控

为探讨吸入性麻醉剂安氟醚对海马CA1区锥体神经元的γ-氨基丁酸(GABA)能自发性微小抑制性突触后电流(mIP-SCs)的调控作用,本研究采用酶消化和机械分离的单细胞模型,应用制霉菌素穿孔膜片钳技术,记录安氟醚对海马CA1区锥体神经元的GABA能突触后电流的影响。结果显示:(1)安氟醚可使GABA的浓度-效应曲线平行左移,但不影响GABA引起的最大反应;(2)安氟醚能够可逆性地增大GABA能自发性mIPSCs的发放频率而不影响其幅度;(3)在无钙细胞外液条件下,仍能观察到安氟醚对GABA能自发性mIPSCs发放频率的增强作用;膜通透性胞内钙库Ca2+的螯合剂BAPTA-AM可抑制安氟醚的增强作用。以上结果提示在海马CA1区安氟醚可能通过释放胞内钙库内的Ca2+使神经终末内Ca2+浓度升高而增加GABA的释放,从而达到中枢抑制作用。     

Effect of Fast and Slow Calcium Buffers on Induced Secretion of Neurotransmitter

Loading of mouse motor nerve terminals with EGTA-AM, but not with BAPTA-AM, inhibited the release of the neurotransmitter in response to stimulation of the nerve with rare (0.3 Hz) “single” pulses. During rhythmic stimulation with short (50 EPP) high-frequency (20 Hz) series, BAPTA-AM buffer modified burst pattern in a dose-dependent manner: it replaced the phase of initial facilitation by persistent depression of secretion and decreased its plateau level at the end of the burst. In contrast, loading of the nerve terminals with EGTA-AM buffer produced no effect on the phase of initial facilitation, but decreased the plateau level to the same degree as BAPTA-AM did. Probably, the different effects of both buffers on secretion of neurotransmitter reflect peculiarities of involvement of fast and slow Ca²⁺ signals of motor terminals in single and rhythmic release of the neurotransmitter.     

Ca(2+)-induced Ca(2+) release activates spontaneous miniature outward currents (SMOCs) in parasympathetic cardiac neurons.

Mudpuppy parasympathetic cardiac neurons exhibit spontaneous miniature outward currents (SMOCs) that are thought to be due to the activation of clusters of large conductance Ca(2+)-activated K(+) channels (BK channels) by localized release of Ca(2+) from internal stores close to the plasma membrane. Perforated-patch whole cell recordings were used to determine whether Ca(2+)-induced Ca(2+) release (CICR) is involved in SMOC generation. We confirmed that BK channels are involved by showing that SMOCs are inhibited by 100 nM iberiotoxin or 500 microM tetraethylammonium (TEA), but not by 100 nM apamin. SMOC frequency is decreased in solutions that contain 0 Ca(2+)/3.6 mM Mg(2+), and also in the presence of 1 microM nifedipine and 3 microM omega-conotoxin GVIA, suggesting that SMOC activation is dependent on calcium influx. However, Ca(2+) influx alone is not sufficient; SMOC activation is also dependent on Ca(2+) release from the caffeine- and ryanodine-sensitive Ca(2+) store, because exposure to 2 mM caffeine consistently caused an increase in SMOC frequency, and 10-100 microM ryanodine altered the configuration of SMOCs and eventually inhibited SMOC activity. Depletion of intracellular Ca(2+) stores by the Ca-ATPase inhibitor cyclopiazonic acid (10 microM) inhibited SMOC activity, even when Ca(2+) influx was not compromised. We also tested the effects of the membrane-permeable Ca(2+) chelators, bis-(o-aminophenoxy)-N,N,N', N'-tetraacetic acid-AM (BAPTA-AM) and EGTA-AM. EGTA-AM (10 microM) caused no inhibition of SMOC activation, whereas 10 microM BAPTA-AM consistently inhibited SMOCs. After SMOCs were completely inhibited by BAPTA, 3 mM caffeine caused SMOC activity to resume. This effect was reversible on removal of caffeine and suggests that the source of Ca(2+) that triggers the internal Ca(2+) release channel is different from the source of Ca(2+) that activates clusters of BK channels. We propose that influx of Ca(2+) through voltage-dependent Ca(2+) channels is required for SMOC generation, but that the influx of Ca(2+) triggers CICR from intracellular stores, which then activates the BK channels responsible for SMOC generation.     

Factors affecting the time course of decay of end-plate currents: a possible cooperative action of acetylcholine on receptors at the frog neuromuscular junction.

1. End-plate currents have been studied in gylcerol-treated frog sartorius nerve-muscle preparations with the voltage-clamp technique. 2. Adding the anticholinesterase prostigmine (3 muM) to the solution bathing the muscle caused a 2-7 (mean 3-3) times increase in the time constant of decay of end-plate currents. The anticholinesterase edrophonium (15 muM) also prolonged the time course of end-plate currents. 3. Pre-treatment of the preparation with collagenase, which leads to the removal of acetylcholinesterase in the synaptic cleft, prolongs the time course of end-plate currents. 4. Curare (1-2 muM), cobratoxin (0-13 muM), or alpha-bungarotoxin (0-13-0-26 muM) decreased the time constant of decay of end-plate currents in the presence of prostigmine. 5. These observations are consistant with the suggestion that repeated binding of acetylcholine (ACh) molecules to receptors as the ACh escapes from the synaptic cleft can contribute to the prolongation of end-plate currents which occurrs when acetylcholinesterase activity is eliminated. 6. Increasing the amount of transmitter released from the presynaptic nerve terminal leads to a prolongation of end-plate currents in the presence of prostigmine. 7. In the presence of prostigmine, the second of two end-plate currents (interval 2-10 msec) decays more slowly than the first. 8. ACh (1-40 muM) or carbachol (40 muM) applied in the solution bathing the muscle prolongs end-plate currents in the presence of prostigmine. 9. It is suggested on the basis of the observations described in paragraphs 6 to 8 that the time constant of decay of end-plate currents in the presence of prostigmine increases with increasing concentrations of ACh in the synaptic cleft. In the absence of prostigmine, increasing the concentration of ACh in the synaptic cleft did not change the time constant for decay of end-plate currents. 10. We interpret these results to suggest that ACh can have a cooperative action on receptors such that the association of ACh with one receptor (defined as binding a single ACh molecule) favours the binding or retention of ACh at other receptors. This implies that receptors can interact.     

Coupling of L-type calcium channels to neurotransmitter release at mouse motor nerve terminals

Previously, we have presented evidence for the presence of L-type voltage-dependent Ca2+ channels (VDCC) in 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, (acetoxymethyl)ester (BAPTA-AM)-incubated motor nerve terminals (MNTs) of the levator auris muscle of mature mice. The aim of the present work was to study the coupling of these L-type VDCC to neurotransmitter release by inhibiting protein phosphatases. We thus studied the effects of the protein phosphatase inhibitors okadaic acid (OA) and pervanadate on quantal content (QC) of transmitter release with the P/Q-type channels fully blocked. The QC was not significantly different under the three experimental conditions tested: incubation with dimethylsulphoxide (DMSO), ethylene-glycol-bis(beta-aminoethylether)-N,N,N',N'-tetraacetic acid, (acetoxymethyl)ester (EGTA-AM) and BAPTA-AM. After preincubation with OA (1 microM), but not with pervanadate, QC increased substantially in the BAPTA-AM-incubated (up to 400%) MNT, but not in those incubated with DMSO or EGTA-AM. The OA-induced increment of QC was attenuated greatly (approximately 95% reduction) by preincubation with either nitrendipine (10 microM) or calciseptine (300 nM). The effect of OA (1 microM) and pervanadate (0.1 mM) on spontaneous neurotransmitter release was also studied. After preincubation with OA, but not per-vanadate, miniature end-plate potential (MEPP) frequency increased only in the BAPTA-AM-incubated MNT (up to 700% increment). This response was attenuated (by approximately 80%) by nitrendipine (10 microM) or calciseptine (300 nM). In contrast, neither omega-agatoxin IVA (120 nM) nor omega-conotoxin GVIA (1 microM) affected this OA-induced increment significantly. We also evaluated the relationship between QC and extracellular [Ca2+] ([Ca2+]o) in BAPTA-AM-incubated MNT. Under conditions in which only P/Q-type VDCC were available to participate in neurotransmitter release, QC increased as [Ca2+]o was raised from 0.5 to 2 mM. However, when only L-type VDCC were available, QC increased when [Ca2+]o increased from 0.5 to 1 mM, but decreased significantly at 2 mM. The mean latency for P/Q-type VDCC-mediated EPP was 1.7-1.9 ms; for L-type VDCC-mediated EPP, 1.9-2.5 ms. The rise time of the L-type VDCC mediated EPP was significantly slower than that mediated by P/Q-type VDCC. Preincubation with H-7 (100 microM), a potent inhibitor of protein kinase C (PKC) and adenosine 3',5'cyclic monophosphate (cAMP)-dependent protein kinase (PKA), attenuated the OA-induced increment of both QC and MEPP frequency (50% and 70% decrement, respectively), suggesting the participation of at least these two protein kinases in the coupling of L-type VDCC. In summary, our results show coupling of L-type VDCC to neurotransmitter release when protein phosphatases are inhibited and intracellular [Ca2+] is buffered by the fast chelator BAPTA.     

UNC-46 is required for trafficking of the vesicular GABA transporter

Mutations in unc-46 in Caenorhabditis elegans cause defects in all behaviors that are mediated by GABA. Here we show that UNC-46 is a sorting factor that localizes the vesicular GABA transporter to synaptic vesicles. The UNC-46 protein is related to the LAMP (lysosomal associated membrane protein) family of proteins and is localized at synapses. In unc-46 mutants, the vesicular transporter is not found specifically in synaptic vesicles but rather is diffusely spread along the axon. Mislocalization of the transporter severely reduces the frequency of miniature currents, but the remaining currents are normal in amplitude. Because the number of synaptic vesicles is not depleted, it is likely that only a fraction of vesicles harbor the transporter in unc-46 mutants. Our data indicate that the transporter and UNC-46 have mutual roles in sorting. The vesicular GABA transporter recruits UNC-46 to synaptic vesicle precursors in the cell body, and UNC-46 sorts the transporter at the cell body and during endocytosis at the synapse.     

Mechanisms underlying LTP of inhibitory synaptic transmission in the deep cerebellar nuclei

Whole-cell recordings were used to investigate long-term potentiation of inhibitory synaptic currents (IPSCs) in neurons of deep cerebellar nuclei (DCN) in slices. IPSCs were evoked by electrical stimulation of the white matter surrounding the DCN in the presence of non-N-methyl-D-aspartate (non-NMDA) glutamate receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (20 microM). High-frequency stimulation induced a long-term potentation (LTP) of the IPSC amplitude without changing its reversal potential, rise time, and decay-time constant. This LTP did not require the activation of postsynaptic gamma-aminobutyric acid-A (GABA(A)) receptors but depended on the activation of NMDA receptors. LTP of IPSCs in DCN neurons could also be induced by voltage-depolarizing pulses in postsynaptic neurons and appeared to depend on an increase in intracellular calcium as the LTP was blocked when the cells were loaded with a calcium chelator, 1,2-bis-(2-amino-phenoxy)-N,N,N', N'-tetraacetic acid (BAPTA, 10 mM). LTP of IPSCs was accompanied by an increase in the frequency of spontaneous IPSCs and miniature IPSCs (recorded in the presence of tetrodotoxin 1 microM), but there was no significant change in their amplitude. In addition, during the LTP, the amplitude of response to exogenously applied GABA(A) receptor agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol hydrochloride was increased. Intracellular application of tetanus toxin, a powerful blocker of exocytosis, in DCN neuron prevented the induction of LTP of IPSCs. Our results suggest that the induction of LTP of IPSCs in the DCN neurons likely involves a postsynaptic locus. Plasticity of inhibitory synaptic transmission in DCN neurons may play a crucial role in cerebellar control of motor coordination and learning.     

Variability of quantal events in control solution and after cholinesterase blockade in frog

Amplitudes of miniature end-plate currents (AMEPCs) as well as their time constants of decay (TMEPCs) contribute to the amplitudes of miniature end-plate potentials and hence determine their mean values and variability. Larger mean amplitudes of miniature end-plate potentials observed after cholinesterase blockade result from both greater mean AMEPCs and greater mean TMEPCs. However, enhanced variability of amplitudes of miniature end-plate potentials is a result mainly of greater variability of TMEPCs. This occurs almost exclusively because the amplitude dependence of TMEPCs becomes markedly enhanced. Although the scatter of TMEPCs about TMEPC vs AMEPC curve is also larger, it does not contribute much to the increased variability of TMEPCs. The marked amplitude dependence of TMEPCs indicates that repetitive binding strongly depends on the quantal size--the number of acetylcholine molecules released per quantum, and suggests that if a quantum is the result of a release of several vesicles, then they should be released very close to each other. Marked amplitude dependence of TMEPCs also shows that, with cholinesterase inhibited, the quantal size can be estimated not only from AMEPCs, but also from TMEPCs. Greater scatter of TMEPCs from the TMEPC vs AMEPC curve is of more obscure origin and probably results because repetitive binding occurs over enlarged "critical areas" with more variable density of acetylcholine receptors.     

Physiological role of desensitized cholinoceptors in skeletal muscle

The effect of desensitized cholinoceptors on the time course of end-plate currents was evaluated in frog skeletal muscle at a high (physiological) level of acetylcholine secretion in the presence of active acetylcholinesterase, with two-electrode recording of the membrane potential. When the number of cholinoceptors was small so that they did not appreciably affect the amplitude of end-plate currents or the parameters of onequantum responses (miniature currents), the decay of multiquantum currents was significantly accelerated. Moreover, the presence of cholinoceptors drastically reduced the ability of acetylcholinesterase inhibitors to prolong the decay of end-plate currents. It is suggested that desensitized cholinoceptors in a synapse with a physiological level of acetylcholine secretion and active acetylcholinesterase may bind free acetylcholine with high affinity and thus supplement the well-known physiological role of acetylcholinesterase in limiting the reactivation of postsynaptic membrane cholinoceptors Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 119, No. 6, pp. 578–581, June, 1995 Presented by A. D. Ado, Member of the Russian Academy of Medical Sciences     

Bilirubin potentiates inhibitory synaptic transmission in lateral superior olive neurons of the rat

Bilirubin is a well-known neurotoxin that can result in multiple neurologic deficits. Previous studies have suggested that bilirubin affects aspects of synaptic transmission; however the acute effects of bilirubin on synaptic transmission have not been examined in real-time. In this study, using whole-cell voltage-clamp recordings, we observed the effect of bilirubin on inhibitory postsynaptic currents (IPSC) in postnatal 13-15-day-old neurons dissociated from lateral superior olive nuclei (LSO), one of the brainstem auditory nucleus that are highly vulnerable to bilirubin. The results showed that 10(-5)M bilirubin increased the frequency of spontaneous IPSC without causing change in their amplitudes or in the response to bath applied glycine, suggesting a presynaptic locus for the action. In the presence of tetrodotoxin, the frequency of miniature IPSC was also potentiated by 10(-5)M bilirubin. The facilitation by bilirubin was concentration dependent and increased with an increase in exposure time. Bicuculline only partially reduced the action of bilirubin. The action of bilirubin was observed in extracellular Ca(2+)-free ([Ca(2+)](o) free) solution but was fully occluded by pretreatment with BAPTA-AM in [Ca(2+)](o) free solution. Thus, in LSO neurons, bilirubin facilitates inhibitory synaptic transmission, in a manner independent of voltage-activated Na(+) and Ca(2+) channels but dependent on presynaptic [Ca(2+)](i). The increase of inhibitory synaptic transmission in response to acute bilirubin is a novel effect of bilirubin on the central nervous system and may have implications for neurotoxicity and the impairment of auditory transduction seen in hyperbilirubinemia.     

Effects of fenamate on inhibitory postsynaptic currents in Purkinje’s cells

The effects of nonsteroid antiinflammatory drugs of the fenamate group (mefenamic and tolfenamic acids) on spontaneous miniature inhibitory postsynaptic currents in Purkinje’s cells were studied in mouse cerebellar slices by the whole cell patch-clamp method. Both drugs in concentrations of 3–30 μM significantly prolonged miniature inhibitory postsynaptic currents and reduced their amplitude. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 145, No. 5, pp. 500–504, May, 2008