Miniature endplate currents of muscle fibers from rat diaphragm during galantamine-induced acetylcholinesterase inhibition

Miniature endplate currents (MEPC) were recorded in muscle fibers of rat diaphragm using voltage clamp technique during acetylcholinesterase (AChE) inhibition induced by various concentrations of galantamine. Their amplitude and time course began to increase at a galantamine concentration of 3.16·10^–8 g/ml. Increased concentrations of galantamine produced a greater effect. Maximum amplitude and time course were reached at a concentration of 10^–6 g/ml. The input resistance of muscle fibers increased under the effects of galantamine. In all cases MEPC fell exponentially. At a concentration of 10^–5 g/ml galantamine produced a curarelike effect; amplitude and time course of decay increased to a lesser extent than at a concentration of 10^–6 and the decay in MEPC became biphasic. Following washout of galantamine (10^–5 g/ml) the time course of MEPC first rose, then fell, returning to the initial level in 3 h, and decay again became exponential. Changes in MEPC parameters under the effects of different concentrations of galantamine and washout were closely correlated. A positive correlation was found between the time course of decay and MEPC amplitude both in the presence and absence of AChE inhibition. It is postulated that the functional importance of synaptic AChE in repressing the postsynaptic action of acetylcholine is limited and that parameters of postsynaptic response may therefore be used to evaluate its action.A. A. Ukhtomskii Institute of Physiology, A. A. Zhdanov State University, Leningrad. Translated from Neirofiziologiya, Vol. 17, No. 5, pp. 607–614, September–October, 1985.     

Effects of Etomidate on GABAergic and Glutamatergic Transmission in Rat Thalamocortical Slices

Although accumulative evidence indicates that the thalamocortical system is an important target for general anesthetics, the underlying mechanisms of anesthetic action on thalamocortical neurotransmission are not fully understood. The aim of the study is to explore the action of etomidate on glutamatergic and GABAergic transmission in rat thalamocortical slices by using whole cell patch-clamp recording. We found that etomidate mainly prolonged the decay time of spontaneous GABAergic inhibitory postsynaptic currents (sIPSCs), without changing the frequency. Furthermore, etomidate not only prolonged the decay time of miniature inhibitory postsynaptic currents (mIPSCs) but also increased the amplitude. On the other hand, etomidate significantly decreased the frequency of spontaneous glutamatergic excitatory postsynaptic currents (sEPSCs), without altering the amplitude or decay time in the absence of bicuculline. When GABA_A receptors were blocked using bicuculline, the effects of etomidate on sEPSCs were mostly eliminated. These results suggest that etomidate enhances GABAergic transmission mainly through postsynaptic mechanism in thalamocortical neuronal network. Etomidate attenuates glutamatergic transmission predominantly through presynaptic action and requires presynaptic GABA_A receptors involvement.     

Quantum Properties of GABA Release in Cultured Neurons from the Rat Cortex

We analyzed the properties of inhibitory synaptic transmission between neurons in low-density cultures of cortical cells. Miniature, spontaneous, and evoked inhibitory postsynaptic currents were studied using a whole-cell path-clamp technique at a holding potential of -80 mV. These postsynaptic currents were identified as GABA release-activated Cl^- currents mediated by GABA_A receptors. Fitting amplitude histograms of these currents with Gaussian curves and an autocorrelation technique revealed the presence of equidistant peaks corresponding to a mean quantum size of 10 pA.     

Changes in synaptic transmission produced by hydrogen peroxide

The effect of hydrogen peroxide (H₂O₂) on excitatory and inhibitory synaptic transmission was studied at the lobster neuromuscular junction. H₂O₂ produced a dose dependent decrease in the amplitude of the junction potential (V_ejp). This decrease was due to changes in both presynaptic transmitter release and the postsynaptic response to the neurotransmitter. Observed presynaptic changes due to exposure to H₂O₂ were a decrease in the amount of transmitter released, that is, quantal content, as well as a decrease in the fast facilitation, that is, the amplitude increase of successive excitatory junction potentials at a rate of 3 Hz. To discern postsynaptic changes, glutamate, the putative excitatory neurotransmitter for this preparation was applied directly to the bathing medium in order to bypass the presynaptic release process. H₂O₂ produced a decreased response of the glutamate receptor/ ionophore. The action of H₂O₂ was not selective to excitatory (glutamate-mediated) transmission because inhibitory (GABA-mediated) transmission was also depressed by H₂O₂. This effect was primarily presynaptic since H₂O₂ produced no change in the postsynaptic response to applied GABA.     

Effects of blockers of voltage-operated potassium channels on an NMDA component of excitatory synaptic transmission in theCA1 subfield of the rat hippocampus

The effects of voltage-operated potassium channel blockers on evoked excitatory synaptic transmission were studied in theCA1 subfield of rat hippocampal slices. Incubation with 50 μM 4-aminopyridine (n=27), 300 nM α-dendrotoxin (n=3), or 5 to 25 mM tetraethylammonium (n=7) resulted in an enhancement of the peak amplitude of excitatory postsynaptic currents (EPSC) and significant prolongation of their decay at strong stimuli, due to an increased contribution of NMDA receptors into EPSC. In five experiments, the presence of an AMPA receptor antagonist, 4-aminopyridine, led to the appearance of NMDA receptor-mediated field excitatory postsynaptic potentials (fEPSP). It is suggested that various modulations increasing presynaptic Ca²⁺ entry and, consequently, glutamate release may increase an NMDA component of synaptic transmission via excitation of polysynaptic excitatory pathways and/or due to glutamate spillover to distant extrasynaptic NMDA receptors.     

Postsynaptic effects of substance P in frog neuromuscular junction

We have studied the effect of substance P on the end-plate currents (EPC) and the miniature EPC (MEPC) after acetylcholine esterase (ACE) inhibition in the cut neuromuscular preparation of the frog sartorius muscle using the voltage-clamp technique. At concentrations of 5·10^–7–1·10^–6 moles/liter substance P had no effect on the amplitude and the time characteristics of single EPC and MEPC but promoted prolongation of EPC decay on repetitive stimulation of the nerve with a frequency of 10/sec, indicating intensification of postsynaptic potentiation. Elevation of the concentration of the given peptide to 5·10^–6 moles/liter led to the shortening of the decay of single EPC and a more marked depression of the EPC amplitude in the trains as compared to the control, reflecting a decrease in the sensitivity of the postsynaptic membrane to the mediator, i.e., development of desensitization.S. V. Kurashov State Medical Institute, Kazan. I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Leningrad. Translated from Neirofiziologiya, Vol. 23, No. 4, pp. 436–441, July–August, 1991.     

GABA-Mediated Inhibition in Cultured Neurons of the Rat Brain Cortex

In cultured pyramidal neurons of the rat brain cortex, we recorded (in the whole-cell configuration) postsynaptic currents (PSC) evoked by direct electrical microstimulation of an axon of the interneuron adjacent to the pyramidal cell. Application of 5 M bicuculline rapidly, entirely, and reversibly blocked these currents. Linear changes in the holding potential on the membrane of the postsynaptic cell resulted in linear changes in the amplitude of averaged currents. The currents underwent reversion when the holding potential was –16 mV, which was close to the reversal potential for Cl^- ions at their respective concentrations in the extra- and intracellular solutions. We conclude that the recorded currents are inhibitory PSC (IPSC) mediated by GABA release. The amplitudes of the recorded currents varied from a measurable minimum (8 pA) to more than 150 pA at a holding potential on the postsynaptic cell membrane of –80 mV. Times to peak of the high- and low-amplitude currents showed no significant differences, being about 6.4 msec on average. Decays of the current could be satisfactorily approximated by a monoexponential function with a mean time constant of 17 msec. The time constants of IPSC decay were distributed accordingly to the Gaussian law. In some cases, the amplitude distributions of IPSC were unimodal ((with a rightward asymmetry), but in most cases they were clearly polymodal. The amplitude distribution can be described by the sum of several Gaussian distributions; the distance between modes of the Gaussians was 25 ± 6 pA, on average. The obtained estimates of the amplitude of monoquantal GABA-induced IPSC in neurons of the brain cortex allow us to conclude that in various CNS regions the dimension of the vesicles in GABA-ergic synapses formed by inhibitory interneurons is identical.     

Mechanisms of the Effect of Dimephosphone on Synaptic Transmission in the Frog Neuromuscular Junction

We studied the influence of dimephosphone, an organophosphorus drug with a broad spectrum of therapeutic effects on the peripheral and central nervous systems, on postsynaptic end-plate currents (EPC) in the frog neuromuscular junction. Dimephosphone was demonstrated to decrease in a voltage-independent manner the EPC amplitude and to prolong the EPC decay. These effects are not related to inhibition of acetylcholinesterase. We propose a theoretical interpretation of the observed phenomena based on the model of blockade of an open ion channel of the acetylcholine receptor and conclude that postsynaptic receptors are one of the most probable targets for the action of dimephosphone.     

Phencyclidine (PCP) blocks glutamate-activated postsynaptic currents

Phencyclidine (PCP) was tested on the metathoracic tibialis muscles of Locusta migratoria. In physiological solution, the peak amplitude of the excitatory postsynaptic currents (EPSCs) evoked by nerve stimulation was linearly related to membrane potential between -50 and -150 mV. The decay time constant of the EPSC (tau EPSC) was exponentially dependent on voltage and decreased with hyperpolarization. The membrane potential change required to produce an e-fold change in tau EPSC was 315 mV. PCP (5-40 microM) produced a concentration-dependent depression of both EPSC peak amplitude and tau EPSC. A slight nonlinearity in the current-voltage relationship could be discerned at high concentrations of PCP. The shortening of the decay time constant of EPSC (tau EPSC) occurred without significant change in the voltage sensitivity observed under control conditions. Under all experimental conditions, the decay of the EPSCs remained a single exponential of time. Fluctuation analysis indicated that 5 microM PCP shortens the lifetime of the glutamate-activated channels by 25.7 +/- 3%. PCP (10-80 microM) did not induced desensitization of the glutamate receptors. These results suggest that PCP interacts with the open conformation of ion channels activated by the glutamate receptor.     

The signaling pathways mediated by P2Y nucleotide receptors in the formation and maintenance of the skeletal neuromuscular junction

The motor neuron, the Schwann cell and the muscle cell are highly specialized at the vertebrate skeletal neuromuscular junction (NMJ). The muscle cell surface contains a high local density of acetylcholine (ACh) receptors (AChRs), acetylcholinesterase (AChE) and their interacting macromolecules at the NMJ, forming the postsynaptic specializations. During the early stages of development, the incoming nerve terminal induces the formation of these postsynaptic specializations; the nerve secretes agrin and neuregulin (NRG), which are known to aggregate existing AChRs and to increase the expression of AChR at the synaptic region, respectively. In addition, adenosine 5'-triphosphate (ATP) is stored at the motor nerve terminals and is coreleased with ACh during muscle contraction. Recent evidence suggests that ATP can play a role in forming and maintaining the postsynaptic specializations by activating its corresponding receptors. In particular, one of the nucleotide receptor subtypes, the P2Y(1) receptor, is specifically localized at the NMJs. The gene expression of AChR and AChE is upregulated after the activation of P2Y(1) receptors. Thus, the synaptic ATP together with agrin and NRG can act as a synapse-organizing factor to induce the expression of postsynaptic functional effectors.     

Differential Modulation of GABAA Receptors Underlies Postsynaptic Depolarization- and Purinoceptor-Mediated Enhancement of Cerebellar Inhibitory Transmission: A Non-Stationary Fluctuation Analysis Study

Cerebellar GABAergic inhibitory transmission between interneurons and Purkinje cells (PCs) undergoes a long-lasting enhancement following different stimulations, such as brief depolarization or activation of purinergic receptors of postsynaptic PCs. The underlying mechanisms, however, are not completely understood. Using a peak-scaled non-stationary fluctuation analysis, we therefore aimed at characterizing changes in the electrophysiological properties of GABA_A receptors in PCs of rat cerebellar cortex during depolarization-induced “rebound potentiation (RP)” and purinoceptor-mediated long-term potentiation (PM-LTP), because both RP and PM-LTP likely depend on postsynaptic mechanisms. Stimulation-evoked inhibitory postsynaptic currents (eIPSCs) were recorded from PCs in neonatal rat cerebellar slices. Our analysis showed that postsynaptic membrane depolarization induced RP of eIPSCs in association with significant increase in the number of synaptic GABA_A receptors without changing the channel conductance. By contrast, bath application of ATP induced PM-LTP of eIPSCs with a significant increase of the channel conductance of GABA_A receptors without affecting the receptor number. Pretreatment with protein kinase A (PKA) inhibitors, H-89 and cAMPS-Rp, completely abolished the PM-LTP. The CaMKII inhibitor KN-62 reported to abolish RP did not alter PM-LTP. These results suggest that the signaling mechanism underlying PM-LTP could involve ATP-induced phosphorylation of synaptic GABA_A receptors, thereby resulting in upregulation of the channel conductance by stimulating adenylyl cyclase-PKA signaling cascade, possibly via activation of P2Y₁₁ purinoceptor. Thus, our findings reveal that postsynaptic GABA_A receptors at the interneuron-PC inhibitory synapses are under the control of two distinct forms of long-term potentiation linked with different second messenger cascades.     

Depression of glutamate-mediated synaptic transmission by benzyl alcohol.

The data obtained from this study suggest that the nonionizable anesthetic benzyl alcohol has two prominent actions on GABA- and glutamate-mediated synaptic transmission at the lobster neuromuscular junction. They are as follows: (1) depression of the excitatory end-plate potential and the postsynaptic membrane response to applied glutamate, and (2) a hyperpolarization of the postsynaptic resting membrane potential associated with a decrease in effective membrane resistance. No change in amplitude of the inhibitory end-plate potential or inhibitory reversal potential was seen. Excitatory miniature end-plate potential frequency was also unaffected. The depression of excitatory synaptic transmission appears to be due to a decreased responsiveness of the postsynaptic receptor-ionophore complex.     

Actions of histamine on muscle and ganglia of the guinea pig gallbladder

Histamine is an inflammatory mediator present in mast cells, which are abundant in the wall of the gallbladder. We examined the electrical properties of gallbladder smooth muscle and nerve associated with histamine-induced changes in gallbladder tone. Recordings were made from gallbladder smooth muscle and neurons, and responses to histamine and receptor subtype-specific compounds were tested. Histamine application to intact smooth muscle produced a concentration-dependent membrane depolarization and increased excitability. In the presence of the H(2) antagonist ranitidine, the response to histamine was potentiated. Activation of H(2) receptors caused membrane hyperpolarization and elimination of spontaneous action potentials. The H(2) response was attenuated by the ATP-sensitive K(+) (K(ATP)) channel blocker glibenclamide in intact and isolated smooth muscle. Histamine had no effect on the resting membrane potential or excitability of gallbladder neurons. Furthermore, neither histamine nor the H(3) agonist R-alpha-methylhistamine altered the amplitude of the fast excitatory postsynaptic potential in gallbladder ganglia. The mast cell degranulator compound 48/80 caused a smooth muscle depolarization that was inhibited by the H(1) antagonist mepyramine, indicating that histamine released from mast cells can activate gallbladder smooth muscle. In conclusion, histamine released from mast cells can act on gallbladder smooth muscle, but not in ganglia. The depolarization and associated contraction of gallbladder smooth muscle represent the net effect of activation of both H(1) (excitatory) and H(2) (inhibitory) receptors, with the H(2) receptor-mediated response involving the activation of K(ATP) channels.     

Modulation of transmitter release from the locust forewing stretch receptor neuron by GABAergic interneurons activated via muscarinic receptors

The role of muscarinic receptors in the down‐regulation of acetylcholine (ACh) release from the locust forewing stretch receptor neuron (fSR) terminals has been investigated. Electrical stimulation of the fSR evokes monosynaptic excitatory postsynaptic potentials (EPSPs) in the first basalar motoneuron (BA1), produced mainly by the activation of postsynaptic nicotinic cholinergic receptors. The general muscarinic antagonists scopolamine (10⁻⁶ M) and atropine (10⁻⁸ to 10⁻⁶ M) caused a reversible increase in the amplitude of electrically evoked EPSPs. However, scopolamine (10⁻⁶ M) caused a slight depression in the amplitude of responses to ACh pressure‐applied to the soma of BA1. These observations indicate that the EPSP amplitude enhancement is due to the blockade of muscarinic receptors on neurons presynaptic to BA1. The muscarinic receptors may be located on the fSR itself and act as autoreceptors, and/or they may be located on GABAergic interneurons which inhibit ACh release from the fSR. Electron microscopical immunocytochemistry has revealed that GABA‐immunoreactive neurons make presynaptic inputs to the fSR. The GABA antagonist picrotoxin (10⁻⁶ M) caused a reversible increase in the EPSP amplitude, which does not appear to be due to an increase in sensitivity of BA1 to ACh, as picrotoxin (10⁻⁶ M) slightly decreased ACh responses recorded from BA1. Application of scopolamine (10⁻⁶ M) to a preparation preincubated with picrotoxin did not cause the EPSP amplitude enhancement normally seen in control experiments; in fact, it caused a slight depression. This indicates that at least some of the presynaptic muscarinic receptors are located on GABAergic interneurons that modulate transmission at the fSR/BA1 synapse. © 1999 John Wiley & Sons, Inc. J Neurobiol 40: 420–431, 1999     

Presynaptic receptors regulating the time course of neurotransmitter release from vertebrate nerve endings

A number of different types of presynaptic receptors was revealed in central and peripheral chemical synapses activated both by main mediator and co-mediators released simultaneously. Physiological significance and mechanisms of functioning of these receptors are not clear yet. They are assumed to provide negative or positive feedback decreasing or increasing the number of neurotransmitter quanta released in response to nerve impulse and thus regulating synaptic transmission. At the same time, there is one more way of secretion process modulation associated with the changes of timing of transmitter release. This mechanism was shown to contribute to the efficiency of synaptic transmission. The role of presynaptic receptors in regulation of the kinetics of quanta release is one of the interesting questions of modern neurophysiology. This paper overviews the results obtained by the authors that demonstrate the contribution of presynaptic receptors of different types into the regulation of temporal parameters of quantal secretion at the vertebrates neuromuscular junction. It was shown that activation of the cholinergic nicotinic receptors leads to a decrease of the amplitude of postsynaptic response not only due to reduction of the quantity of released quanta but also due to increased the level of asynchronous release. On the contrary, the facilitating effect of catecholamines on the neuromuscular synapse is the result of activation of presynaptic β₁-adrenoreceptors which leads to greater synchronization of release process and, consequently, to the increase of the amplitude of the postsynaptic response. Presynaptic purine receptors, involved in the modulation the intensity of secretion, are also capable of alteration of the time course of secretion. Activation of ryanodine receptors results in the increase of the number of quanta released with prolonged latencies leading to appearance of the phase of delayed asynchronous neurotransmitter release.     

Agonistic action of synthetic analogues of quisqualic acid at the insect neuromuscular junction

One hundred twenty analogues of quisqualic acid were synthesized and assayed on the neuromuscular junction of larva of the mealworm, Tenebrio molitor. Two new agonists for amino acid receptors, L-glutamic acid N-thiocarboxyanhydride (L-GANTA) and DL-hydantoinpropionic acid (DL-HPA), were discovered in this study. L-GANTA and DL-HPA produced muscle membrane depolarization, accompanied by a reduction of the muscle input resistance. The amplitude of excitatory postsynaptic potentials was decreased in the presence of L-GANTA and DL-HPA. The apparent dissociation constants obtained from dose-depolarization plots were 7 x 10^?4 M for L-GANTA and 9 x 10^?4 M for DL-HPA. Some structural constraints imposed on agonists at amino acid receptors on insect muscle were discussed.     

The extent of the stimulated electrical potential decay under phosphorylating conditions and the H+ATP ratio in Rhodopseudomonas sphaeroides chromatophores following short flash excitation

1. In chromatophores from Rps. sphaeroides, the stimulation by ADP and P_i of the electric potential decay indicated by the carotenoid shift is greater than the stimulation of the decay of the pH change indicated by the colour change of added cresol red under similar conditions. This difference is attributed to H⁺ consumption during the synthesis of ATP. The ratio of H⁺ translocated across the membrane to ATP synthesized was estimated to be approximately 1.7 $\text{H}{}^{\mathrm{+}}\text{ATP}$.2. The stimulation of the electrical potential decay by ADP and P_i was found to be a constant fraction (10%) of the total decay when the flash intensity was varied. No ‘critical’ or ‘threshold’ potential was observed.3. The stimulated electrical potential decay after a second flash, given within a few seconds of the first, was related to the amplitude of the electrical potential produced by the second flash (10%) but neither to the dark time between the flashes, nor to the total extent of the electrical potential above the dark level. These results are consistent with two hypotheses (a) the chromatophores are a mixed population of vesicles, only a small fraction (10%) of which possess an active ATP synthesizing system (b) the activity of the ATP synthesizing system, though driven by a proton motive force, is controlled by electron transport processess. If alternative (a) is correct then the overall single turnover flash yield of 1 ATP per 1470 bacteriochlorophyll measured in (1) would mean that the yield of the active vesicles is approximately 10 ATP per 1470 bacteriochlorophyll or 30 ATP per vesicle.4. The stimulation of the electrical potential decay by ADP and P_i is approximately 40% less in antimycin-treated chromatophores. It is shown that this is probably a consequence of antimycin-inhibited H⁺-release on the inside of the chromatophore vesicles following a flash.     

Non-Decaying postsynaptics potentials and delayed spikes in hippocampal pyramidal neurons generated by a zero slope conductance created by the persistent Na+ current

The negative slope conductance created by the persistent sodium current (I_NaP) prolongs the decay phase of excitatory postsynaptic potentials (EPSPs). In a recent study, we demonstrated that this effect was due to an increase of the membrane time constant. When the negative slope conductance opposes completely the positive slope conductances of the other currents it creates a zero slope conductance region. In this region the membrane time constant is infinite and the decay phase of the EPSPs is virtually absent. Here we show that non-decaying EPSPs are present in CA1 hippocampal pyramidal cells in the zero slope conductance region, in the suprathreshold range of membrane potential. Na⁺ channel block with tetrodotoxin abolishes the non-decaying EPSPs. Interestingly, the non-decaying EPSPs are observed only in response to artificial excitatory postsynaptic currents (aEPSCs) of small amplitude, and not in response to aEPSCs of big amplitude. We also observed concomitantly delayed spikes with long latencies and high variability only in response to small amplitude aEPSCs. Our results showed that in CA1 pyramidal neurons I_NaP creates non-decaying EPSPs and delayed spikes in the subthreshold range of membrane potentials, which could potentiate synaptic integration of synaptic potentials coming from distal regions of the dendritic tree.     

Synaptogenesis in Co-Culture of Neurons of Dorsal Root Ganglia and Spinal Cord

In co-culture of spinal cord and dorsal root ganglion (DRG) neurons, we studied at different terms of culturing postsynaptic currents in DRG neurons evoked by direct electrical stimulation of single spinal neurons using a voltage-clamp technique in the whole-cell configuration. According to the reversal potential and sensitivity to bicuculline, these currents were classified as inhibitory postsynaptic currents (IPSC) carried by Cl^- ions through GABA_A receptors. During neuronal development in dissociated co-culture, the amplitude of evoked IPSC and their time to peak significantly increased. The time to peak of spontaneous IPSC (sIPSC) in DRG neurons remained unchanged, while the frequency of these currents increased with increasing culturing time. It is concluded that under culturing conditions spinal neurons establish inhibitory synaptic contacts with the somata of DRG neurons, and the number of such functional contacts increases in the course of culturing. Our findings show that in dissociated co-culture the process of formation of inhibitory synapses on the axon terminals of primary afferent neurons is akin to that realized in vivo, but with dissimilar topography of distribution of such synapses.