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R Y Pun

Publications and source records attributed to R Y Pun.

At least 37 records · Page 2Linked to original sources
PubMed1986

Synaptic excitation in cultures of mouse spinal cord neurones: receptor pharmacology and behaviour of synaptic currents.

Fast monosynaptic excitatory post-synaptic potentials between spinal cord neurones in cell culture (s.c.-s.c. e.p.s.p.s) were studied with current-clamp and two-electrode voltage-clamp methods. The reversal potential, response to acidic amino acid antagonists, and behaviour of the synaptic current were examined. The amplitude of the e.p.s.p. increased with membrane potential hyperpolarization and decreased with depolarization. The reversal potential of the e.p.s.p. was +3.8 +/- 2.5 mV (mean +/- S.E. of mean). The reversal potential for responses to ionophoretically applied L-glutamate and L-aspartate was also near 0 mV. The acidic amino acid antagonist, cis-2,3-piperidine dicarboxylic acid (PDA, 0.25-1.0 mM) reversibly antagonized the monosynaptic e.p.s.p.s as well as responses to kainate (KA) or quisqualate (QA). The selective N-methyl-D-aspartate antagonist, (+/-) 2-amino-5-phosphonovaleric acid (APV), had little effect on either the monosynaptic e.p.s.p.s or responses to QA or KA at concentrations that abolished responses to L-aspartate. Under voltage clamp, the peak synaptic current (e.p.s.c.) was linearly related to the membrane potential, increasing in amplitude with hyperpolarization and decreasing with depolarization from the resting potential. The decay of a somatic e.p.s.c. was well fitted by a single exponential function with a time constant of 0.6 ms at 25 degrees C. E.p.s.c.s which had proximal dendritic locations had decay time constants of 1-2 ms. The decay time constant was voltage-insensitive between -80 and +10 mV. We suggest that an acidic amino acid receptor other than that for NMDA mediates excitatory transmission at the s.c.-s.c. synapse; and that the underlying conductance mechanism is voltage insensitive with an estimated mean channel lifetime of less than 1 ms.

2-Amino-5-phosphonovalerate↗
PubMed1986

Active and inactive central synapses in cell culture.

Synaptic interactions between pairs of spinal cord (SC) neurons and between dorsal root ganglion neurons and SC neurons were studied in dissociated cell cultures prepared from fetal mouse. Combined injection of horseradish peroxidase into presynaptic neurons and Lucifer yellow into postsynaptic neurons allowed detailed correlation of morphological-physiological analyses of synaptically linked cells. Statistical analysis of trains of evoked EPSPs under conditions of high and of low transmitter output was used to determine the number of physiological release elements, n, involved in a given synaptic connection. When n was compared with the number of boutons subserving a synaptic connection, it was found that in 80% of cases the number of boutons was equal to or greater than the number of release elements. In some cases, the bouton count was more than fivefold greater than n. The simplest explanation is that, in general, one bouton can release no more than one quantum of transmitter and, in a significant proportion of synaptic connections, a large fraction of boutons do not participate in the release process. Theoretical consideration and analysis of the electrotonic structure of some of the neurons studied indicate that the dendritic location of synaptic inputs does not affect our results. Variations in the probability of release, p, may contribute to the apparent disparity between n and bouton number. If so, this variation must be large with many boutons having a very low p, difficult to distinguish experimentally from zero.

Animals↗
PubMed1985

Effects of nitrendipine on the voltage-sensitive calcium channel in mammalian sensory neurons.

We examined the ability of nitrendipine, a calcium antagonist believed to block the voltage-sensitive calcium channels in a number of excitable membranes, to affect the calcium spike duration and rate of rise (Dv/Dt) in 4-week-old cultured mouse dorsal root ganglion neurons. Concentrations of 10 nM-1.0 microM nitrendipine reduced but did not eliminate calcium spike as measured by the rate of rise. These results were obtained at either 22 degrees or 37 degrees C in solutions containing 5 mM calcium. A similar lack of total block was seen with prolonged incubation in sodium-free recording media containing 1 microM tetrodotoxin (TTX), 1.8 mM calcium, and 1 microM nitrendipine. Calcium spike duration was affected in an inconsistent manner by nitrendipine and other 1,4 dihydropyridines. Cobalt (10 mM) totally blocked the voltage-dependent calcium mechanism as measured by either the spike duration or the rate of rise. We conclude that calcium influx through the voltage-sensitive calcium channel in mouse dorsal root ganglion neurons is not blocked by even high concentrations of nitrendipine.

Action Potentials↗
PubMed1985

Noradrenergic responses of spinal neurons in locus coeruleus-spinal cord co-cultures.

Locus coeruleus (LC) explants were co-cultured with dissociated spinal neurons of mice. Nerve fibers exhibiting catecholamine fluorescence radiated from the explants and frequently invested spinal cord (SC) neurons close to the explants. Electrical stimulation of the explant and iontophoretic application of norepinephrine evoked a spectrum of slow depolarizing, hyperpolarizing, and biphasic responses in the SC cells. The responses to LC stimulation and to application of norepinephrine were usually similar in a given cell. The depolarizing responses were associated with an increase in apparent input resistance and pharmacologic tests indicated that the responses were mediated by alpha-receptors. Neurons in regions innervated by catecholamine-containing fibers usually gave depolarizing responses to LC stimulation and such neurons had a very high probability of exhibiting depolarizing responses to applied norepinephrine. It would appear that either locus coeruleus explants favored the survival of cells with alpha-receptors or expression of these receptors in SC neurons was induced by innervation of the neurons by locus coeruleus axons.

Action Potentials↗
PubMed1983

Synaptic interactions between mammalian central neurons in cell culture. I. Reversal potential for excitatory postsynaptic potentials.

Intracellular recording and stimulation techniques were used to study the electrical properties of neurons in cell cultures from fetal mouse spinal cord (SC). The morphology of SC neurons and the distribution on SC neurons of boutons formed by synaptically connected SC or dorsal root ganglion (DRG) neurons were demonstrated with horseradish peroxidase (HRP) injection. Postsynaptic polarization in conjunction with synaptic activation of SC neurons was used to determine the reversal potential for excitatory postsynaptic potentials (EPSPs). Tetraethylammonium ions were injected postsynaptically in order to obtain reversal of the EPSPs. Both SC-SC and DRG-SC excitatory connections could be reversed by postsynaptic depolarization. The average reversal potential for the SC-SC EPSP was -4 +/- 12.2 (SD) mV and that for the DRG-SC EPSP was +8 +/- 7.9 (SD) mV, a statistically significant difference (Wilcoxon two-sample rank; P less than 0.05). Scatter was quite large, particularly for the SC-SC connection. While some neurons gave clear electrophysiological evidence of significant dendritic effects, the average total electrotonic length was small (0.58 +/- 0.65 (SD) of a length constant). The morphological extent of the dendrites of SC neurons was substantially less than that of mature motoneurons in vivo. We concluded that both SC-SC and DRG-SC EPSPs were mediated by a conventional conductance increase and that most synaptic input was not far removed electrically from the recording site in the neuron cell body.

Animals↗
PubMed1983

Synaptic interactions between mammalian central neurons in cell culture. II. Quantal Analysis of EPSPs.

The presynaptic release mechanism involved in excitatory synaptic connections between neurons in cell cultures of fetal mouse spinal cord were studied by statistical analysis of intracellularly recorded postsynaptic responses. Quantal parameters were determined for the EPSPs evoked in spinal cord (SC) neurons by stimulation of either other SC or dorsal root ganglion (DRG) neurons. Transmitter release was manipulated by varying the Ca2+ and Mg2+ content of the culture medium. The release process was represented better by binomial than by Poisson statistics. A method was derived for obtaining the probability of release and the number of release elements. The quantal content and the number of release elements were substantially higher for the SC-SC connection than for the DRG-SC connection. This was partially compensated for by a larger quantal amplitude for the DRG-SC connection. There was some indication that the probability of release was higher for the SC-SC connection. The relationship between transmitter output and effective external Ca2+ ion concentration was approximately linear.

Animals↗
PubMed1981

A coeruleo-spinal system in culture.

In combined cultures of dissociated spinal neurons and explants from the region of locus coeruleus, rich catecholamine-containing fiber projections from the explant to the surrounding regions of spinal neurons were demonstrated by fluorescence histochemistry. Electrical stimulation of the explant resulted in slow depolarizing responses in many of the spinal neurons. Cells exhibiting this type of response were also usually depolarized by local application of noradrenaline, whereas other, unresponsive neurons usually were not. The depolarizing responses to electrical stimulation and to noradrenaline were both increased by depolarizing current injection and decreased by hyperpolarizing current. These and other data suggest that the depolarizing responses of the spinal neurons to explant stimulation are mediated by noradrenaline released from axons of locus coeruleus neurons.

Animals↗
PubMed1981

A procedure for comparing the mobilities of unlabeled drugs used in microelectrophoresis experiments.

A novel method for comparing the absolute mobilities of unlabeled compounds released from micropipettes in microelectrophoresis experiments is described. The method is based on the principle that the introduction of a "foreign" ion into an electrolyte reduces the transport number of a "reference" ion present in the electrolyte. Using [14C]-noradrenaline as the "reference" ion, the mobilities of two "foreign" ions, methoxamine and phenylephrine, were compared. No significant difference was found between the mobilities of the two drugs. It was concluded that the two drugs probably have similar transport numbers when released from solutions of equal molarity in microelectrophoresis experiments in vivo, and thus the previously reported difference between the apparent potencies of the two drugs is presumably of biological origin. The method described here may be of use in comparing the mobilities of other compounds, the radiolabeled forms of which are either unavailable or prohibitively expensive.

Biological Transport↗
PubMed1981

Comparison of the effects of methoxamine with those of noradrenaline and phenylephrine on single cerebral cortical neurones.

1 The technique of microelectrophoresis was used to compare the actions of methoxamine, noradrenaline and phenylephrine on single neurones in the somatosensory cerebral cortex of the rat.2 Methoxamine evoked only excitatory responses on cortical neurones. The methoxamine-sensitive cells were also excited by phenylephrine; cells excited by methoxamine could either be excited or depressed by noradrenaline.3 Methoxamine appeared to be less potent than either noradrenaline or phenylephrine in evoking excitatory responses.4 Responses to methoxamine had a slower time course than responses to either noradrenaline or phenylephrine, both the latencies to onset and the recovery times being longer for responses to methoxamine than for responses to noradrenaline or phenylephrine.5 When the absolute mobilities of methoxamine, noradrenaline and phenylephrine were compared using an in vitro method, no significant differences were found between the mobilities of the three ionic species, suggesting that the three drugs have similar transport numbers. Thus the differences in potency between methoxamine and the other two drugs, and the difference between the time courses of responses to methoxamine and the other two drugs, are presumably of biological origin.6 The alpha-adrenoceptor antagonist, phenoxybenzamine, antagonized equally excitatory responses to methoxamine and noradrenaline, and responses to methoxamine and phenylephrine, without affecting responses to acetylcholine.7 When responses to methoxamine and noradrenaline and responses to methoxamine and acetylcholine were summated on the same cells, the net responses were smaller than those expected on the basis of additive effects; the deviation from additivity was greater in the case of the summation of responses to methoxamine and noradrenaline than in the case of summation of responses to methoxamine and acetylcholine. This observation is consistent with the hypothesis that the interaction between methoxamine and noradrenaline follows the model of competitive dualism, whereas the interaction between methoxamine and acetylcholine follows the model of functional synergism.8 The results suggest that methoxamine may act as a partial agonist at excitatory alpha-adrenoceptors on cerebral cortical neurones.

Animals↗
PubMed1979

The action of microelectrophoretically applied (3,4-dihydroxy-phenylamino)-2-imidazoline (DPI) on single cortical neurones.

1. The technique of microelectrophoresis was used in order to compare the actions of the imidazoline derivative, (3,4-dihydroxy-phenylamino)-2-imidazoline (DPI), with those of dopamine and phenylephrine on single neurones in the cerebral cortex of the rat anaesthetized with halothane. 2. DPI and phenylephrine were almost exclusively excitatory, whereas dopamine could evoke both excitatory and depressant responses. 3. In the case of excitatory responses, DPI appeared to be more potent than dopamine, and was approximately equipotent with phenylephrine. 4. The dopamine antagonist, haloperidol, could discriminate between excitatory responses to DPI and dopamine: responses to dopamine were abolished, whereas responses to DPI, and to a control agonist, acetylcholine, were unaffected. 5. The alpha-adrenoceptor antagonist, phenoxybenzamine, antagonized equally excitatory responses to DPI and phenylephrine. Responses to acetylcholine were not affected. 6. It is concluded that DPI does not stimulate dopamine receptors on cortical neurones; the excitatory responses of these cells to DPI may be mediated by alpha-adrenoceptors.

Animals↗
PubMed1978

Comparison of the responses of single cortical neurones to tyramine and noradrenaline: effects of desipramine.

1 The technique of microelectrophoresis was used in order to compare the actions of tyramine and noradrenaline on single neurones in the cerebral cortex of the rat.2 Tyramine could both excite and depress cortical neurones. Each tyramine-sensitive cell was also sensitive to noradrenaline. There was a high correlation between the directions of responses to tyramine and noradrenaline, most cells excited by tyramine being excited by noradrenaline, and most cells depressed by tyramine being depressed by noradrenaline.3 In the case of both excitatory and depressant responses, tyramine appeared to be less potent than noradrenaline.4 Tyramine evoked ;slower' responses than noradrenaline, both the latencies to onset and the recovery times being longer for responses to tyramine than for responses to noradrenaline.5 When the rates of release of tyramine and noradrenaline from micropipettes were measured in vitro, no significant difference could be observed between the transport numbers of the two drugs. Thus the difference in potency between the two drugs, and the difference in the time courses of responses to the two drugs, are presumably of biological origin.6 Desipramine could discriminate between neuronal responses to tyramine and noradrenaline: responses to tyramine were antagonized, while responses to noradrenaline were either potentiated or unaffected. Responses to DL-homocysteic acid were not affected by desipramine.7 The results are consistent with the hypothesis that tyramine is an indirectly acting sympathomimetic amine in the brain, and desipramine acts by blocking the uptake of both tyramine and noradrenaline into presynaptic noradrenergic nerve terminals.

Animals↗
PubMedDate not supplied

Voltage clamping with single microelectrodes: comparison of the discontinuous mode and continuous mode using the Axoclamp 2A amplifier.

The voltage clamp technique is a powerful method for studying the physiology of excitable membrane. This technique has made possible the determination of ionic responses generated by activation of either receptor-mediated or voltage-dependent processes. The development of the whole-cell, 'tight-seal' voltage clamp method has allowed the analysis and examination of membrane physiology at the single cell level. The method allows the characterization of voltage-dependent ionic conductances both at the macroscopic (whole-cell) and at the microscopic (unitary conductance or single channel) level in cells less than 10 micron in diameter, a feat difficult to achieve with 'conventional' fine-tipped micropipettes. In this paper, several methologies used for culturing neuronal and non-neuronal cells in the laboratory are described. A comparison between the two modes of voltage clamp using blunt-tipped 'patch'-microelectrodes, the switching (discontinuous) and the non-switching (continuous) modes, of the Axoclamp-2A amplifier is made. Some results on membrane currents obtained from neuronal and non-neuronal cells using the single electrode whole-cell 'tight-seal' voltage clamp is illustrated. The possible existence of two inactivating K+ currents, one dependent on Ca++ the other is not, is discussed.

Amplifiers, Electronic↗