Syogoro Nishi

Characterization and ionic basis of GABA-induced depolarizations recorded in vitro from cat primary afferent neurones.

1. Responses of single cells in the isolated cat spinal ganglion to GABA applied by superfusion or by iontophoresis were recorded using intracellular micro‐electrodes. 2. Of the twelve structurally related compounds investigated, GABA was the most effective in its ability to produce a depolarization of the cell membrane. 3. Studies determining concentration‐response relationships indicate that two to three molecules of GABA are required to combine with the GABA receptor for activation. 4. Bicuculline and picrotoxin, each act in a non‐competitive manner to antagonize the GABA‐induced membrane current. 5. The equilibrium potential for iontophoretically induced GABA depolarizations (EGABA) was found to be ‐23.5 plus or minys 6.1 mV. EGABA was independent upon [cl‐]o, but independent of [Na+]o, [K+], or [Ca2+]o. 6. Intracellular injection of twenty antions (Br‐, I‐, NO2‐, NO3‐, ClO4‐, SCN‐, Bf4‐, HS‐, OCN‐, ClO3‐, BrO3‐, F‐, HCO2‐, HSO3‐, HCO3‐, CH3CO2‐, SO42‐, C6H5O73‐) indicated that the activated GABA receptor membrane was permeable to those anions whose hydrated diameter is no larger than that of ClO‐3. 7. Restoration of the GABA depolarization to its control level after augmentation by Cl‐ injection had a mean time constant of 27.8 plus or minus 2.6 min. Picrotoxin did not alter this value. 8. When foreign anions were exchanged for Cl‐ in the perfusion solution, the ten anaions smaller or equal to ClO3‐, decreased the GABA depolarization by 50‐90% and increased its time course 1.5‐2.0 x control. The only exception having a small radius was Br‐ which augmented the amplitude 10‐30%. 9. The ten anions larger than ClO3‐ produced a biphasic effect, i.e. an initial augmentation followed by a marked (up to 100%) depression of the response. Experiments with CH3COO‐, CH3SO4‐, or HOCH2CH2SO3‐, indicated that this depression was non‐competitive.

Blind patch-clamp recordings from substantia gelatinosa neurons in adult rat spinal cord slices: Pharmacological properties of synaptic currents

Whole cell patch-clamp recordings were made from substantia gelatinosa neurons in the thick slice of the adult rat spinal cord, which retained an attached dorsal root to study the pharmacological properties of spontaneous and primary afferent fibre-evoked synaptic currents. The majority of substantia gelatinosa neurons tested exhibited miniature excitatory postsynaptic currents in the presence of tetrodotoxin (0.5 microM). Stimulation of primary afferent A delta fibres evoked monosynaptic and/or polysynaptic excitatory postsynaptic currents. In Mg(2+)-containing solution, 6-cyano-7-nitroquinoxaline-2,3-dione (10 microM) abolished the evoked excitatory postsynaptic currents and the miniature excitatory postsynaptic currents. 2-Amino-5-phosphonovaleric acid (50-100 microM) had little effect on the miniature excitatory postsynaptic current. In Mg(2+)-free solution, however, 6-cyano-7-nitroquinoxaline-2,3-dione reduced but did not abolish the miniature excitatory postsynaptic currents, leaving the miniature excitatory postsynaptic currents with a small amplitude and a slow time course, which were abolished by 2-amino-5-phosphonovaleric acid. At holding potentials more positive than -60 mV, stimulation of A delta fibres evoked outward postsynaptic currents in 11 out of 28 substantia gelatinosa neurons. The evoked inhibitory postsynaptic currents were abolished in seven out of 11 neurons by either strychnine (0.5 microM) or bicuculline (10 microM), and in the remaining four neurons by the combination of both antagonists.(ABSTRACT TRUNCATED AT 250 WORDS)

Primary afferent-evoked glycine- and GABA-mediated IPSPs in substantia gelatinosa neurones in the rat spinal cord in vitro.

1. The possible roles of glycine and gamma‐aminobutyric acid (GABA) as inhibitory transmitters in the spinal dorsal horn were studied by intracellular recordings from substantia gelatinosa (SG) neurones in transverse slices of the adult rat spinal cord which retained an attached dorsal root. 2. Stimulation of primary afferent A delta fibres evoked an initial excitatory postsynaptic potential (fast EPSP) followed by a short and/or long inhibitory postsynaptic potential (short and long IPSP). The short IPSP, observed in twenty‐nine SG neurones (37%) which received inhibitory inputs, had a mean latency of 3.6 ms and a half‐decay time of 11 ms, while the long IPSP had a mean latency of 3.7 ms and a half‐decay time of 42 ms and was observed in thirty‐seven SG neurones (47%). The remaining twelve neurones (16%) exhibited both short and long IPSPs. Both IPSPs reversed polarity at a membrane potential of ‐70 +/‐ 4 mV. The short IPSP was reversibly blocked by the glycine receptor antagonist strychnine (0.5‐2 microM), while the long IPSP was reversibly blocked by the GABAA receptor antagonist bicuculline (10‐20 microM). 3. In the majority of SG neurones, the short and long IPSPs appeared to be disynaptic and were blocked by the non‐N‐methyl‐D‐aspartic acid (non‐NMDA) receptor antagonist 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX; 5‐10 microM). Both IPSPs were less sensitive (depressed by less than 30%) to the NMDA receptor antagonist DL‐2‐amino‐5‐phosphonovaleric acid (APV; 50‐100 microM). 4. In ten SG neurones (13%), bath‐applied glutamate (0.5‐2 mM) increased the amplitude and frequency of IPSPs, which had a similar time course to that of the short IPSP evoked by afferent A delta fibres. The glutamate‐induced short IPSPs were blocked by tetrodotoxin (0.5 microM) or strychnine (0.5‐1 microM). In twelve neurones (16%), glutamate hyperpolarized the membrane or increased the amplitude and frequency of IPSPs that had a similar time course to that of the A delta fibre‐evoked long IPSPs. The glutamate‐induced membrane hyperpolarization and long IPSPs decreased in amplitude with membrane hyperpolarization and reversed polarity at ‐70 +/‐ 6 mV. These hyperpolarizing responses were blocked by tetrodotoxin (0.5 microM) or bicuculline (10 microM). 5. These observations suggest that primary afferent A delta fibres activate glycinergic and/or GABAergic interneurones primarily through the non‐NMDA receptor subclass and result in inhibition of nearby SG neurones in the dorsal horn of the spinal cord.(ABSTRACT TRUNCATED AT 400 WORDS)

Hyperpolarizing and depolarizing actions of dopamine via D-1 and D-2 receptors on nucleus accumbens neurons

The effect of dopamine (DA) on the nucleus accumbens neurons in guinea-pig brain slices was studied by intracellular recordings. DA caused a hyperpolarization in 28% of the neurons tested, a depolarization in 11%, and a hyperpolarization followed by a depolarization in 53%. The remaining neurons were unaffected. Analyses of the responses revealed that the DA hyperpolarization was produced by activation of the D-1 receptor and associated with an increase in potassium conductance, whereas the DA depolarization was generated by activation of the D-2 receptor and accompanied by a decrease in potassium conductance. DA uptake inhibitors augmented both the hyperpolarizing and depolarizing responses, while cyclic adenosine monophosphate selectively enhanced the former.

5-Hydroxytryptamine receptors of visceral primary afferent neurones on rabbit nodose ganglia

1. The electrophysiological characteristics of 5‐hydroxytryptamine (5‐HT) receptors distributed on visceral primary afferent neurones (the nodose ganglion cells of the vagus) in rabbits were investigated with intracellular recording and voltage‐clamp techniques.

Intracellular recordings from lateral horn cells of the spinal cord in vitro

Intracellular recording was used in studies of the preganglionic neurons of the autonomic nervous system. These were carried out on isolated segments of the cat spinal cord. It was found that the lateral horn cells have electrical membrane characteristics similar to postganglionic neurons, but many of them have a much longer afterhyperpolarization. 5-Hydroxytryptamine, noradrenaline and aspartate induce depolarizations in lateral horn cells which are characteristically associated with an increased membrane resistance. EPSPs in lateral horn cells are not cholinergic in nature, though many cells are endowed with excitatory nicotinic receptors. Some interneurons also appear to have excitatory muscarinic receptors. Glutamate can depolarize many lateral horn cells. This excitatory amine does not seem to be responsible for the production of an EPSP, since the EPSP persisted during the continued presence of glutamate in the superfusing medium. All the neurons examined in the lateral horn are susceptible to the hyperpolarizing and shunting actions of GABA and glycine. In a small group of neurons, noradrenaline caused a hyperpolarization.

Changes in spontaneous firing patterns of rat hippocampal neurones induced by volatile anaesthetics.

1. The effects of the volatile anaesthetics, halothane, isoflurane and enflurane, on rat hippocampal CA1 and CA3 neurones in in vitro preparations were studied by intracellular recording methods. 2. The three anaesthetics, at concentrations similar to those used clinically (0.2‐1.2 mM), initially increased and then subsequently decreased the spontaneous firing of CA1 neurones without affecting the resting membrane properties or the EPSPs evoked by focal stimuli. 3. The anaesthetics at these concentrations depressed both the fast after‐hyperpolarization of the soma spike and the post‐tetanic hyperpolarization induced by repetitive stimulation. They also decreased the IPSPs evoked by focal stimuli. 4. The threshold for spike generation was gradually elevated by as much as 4‐6 mV during application of the anaesthetics at these concentrations. The subthreshold potential oscillations (which are likely to be associated with periodic alterations in non‐inactivating Ca2+ and Na+ currents) were enhanced in the low concentrations (0.2‐0.5 mM), but were depressed in the high concentrations (0.8‐1.2 mM). 5. The results suggest that the transient increase in the firing frequency was caused by a depression of both the spike after‐hyperpolarization and the post‐tetanic hyperpolarization, and that the reduction of spontaneous firing was mainly due to an elevated threshold for spike generation. 6. The three anaesthetics altered the pattern of spontaneous spike‐firing in CA3 neurones from solitary spiking to burst firing without affecting the resting membrane properties. 7. The effects of the anaesthetics on the active membrane properties and the postsynaptic potentials in CA3 neurones were similar to the effects in CA1 neurones. 8. In the majority of CA3 neurones, soma spikes elicited by depolarizing current pulses were followed by a Ca2+‐dependent after‐depolarization, which was in turn followed by a prolonged after‐hyperpolarization (post‐burst hyperpolarization). The anaesthetics facilitated the after‐depolarizing potential, while they depressed the post‐burst hyperpolarization. Combination of the two effects would give rise to the highly stereotyped burst (about 1 Hz in frequency) in the presence of the volatile anaesthetics.

Effect of barbiturates on the GABA receptor of cat primary afferent neurones

1. The effects of the barbiturate anaesthetics, pentobarbitone and thiopentone, on the membrane properties and the γ‐aminobutyric acid (GABA)‐induced responses of cat primary afferent neurones were studied with intracellular recording and voltageclamp techniques.

Fast excitatory postsynaptic potentials and the responses to excitant amino acids of sympathetic preganglionic neurons in the slice of the cat spinal cord

The properties of the excitatory postsynaptic potential evoked by focal stimulation and of the responses to excitatory amino acids were examined by intracellular recording from sympathetic preganglionic neurons in upper thoracic spinal cord slices of the adult cat. Single stimuli to the region dorsal to the intermedio-lateral nucleus evoked short-latency, presumably monosynaptic, excitatory postsynaptic potentials. The reversal potential of this response was -2.2 mV and became more negative when external Na+ or K+ concentration was decreased. The excitatory postsynaptic potential was depressed by the non-selective excitatory amino acid receptor antagonist cis-2,3-piperidine dicarboxylic acid and enhanced by a glutamate uptake inhibitor. The non-N-methyl-D-aspartate receptor antagonist 6-cyano-7-nitroquinoxaline-2.3-dione abolished the excitatory postsynaptic potential in 72% of neurons. In the remaining neurons, this antagonist only depressed the potential and unmasked a slower component which was abolished by the N-methyl-D-aspartate receptor antagonist D,L-2-amino-5-phosphonovaleric acid. In the presence of tetrodotoxin all neurons tested were depolarized by glutamate or aspartate, as well as by the selective agonists quisqualate, alpha-amino-3-hydroxy-5-methylisoxazole propionic acid, kainate and N-methyl-D-aspartate. The glutamate-evoked depolarization reversed at a membrane potential of -2.0 mV and at a more negative value when external Na+ or K+ concentration was decreased. The response to alpha-amino-3-hydroxy-5-methylisoxazole propionic acid was abolished by 6-cyano-7-nitroquinoxaline-2,3-dione in all neurons tested and that to kainate in only one-third of the cells. In the remainder the response to kainate was only slightly depressed by this antagonist. The responses to glutamate and aspartate were only slightly depressed by the combined action of the various amino acid receptor antagonists used. The responses to N-methyl-D-aspartate were abolished by D,L-2-amino-5-phosphonovaleric acid. The punched-out region of the intermedio-lateral nucleus, maintained in vitro, released glutamate and aspartate in the absence of stimulation. Field stimulation (20 Hz) enhanced release by between 40 and 100%. The increase was prevented by superfusion with calcium-free Krebs. It is concluded that excitatory amino acids, acting on both N-methyl-D-aspartate and non-N-methyl-D-aspartate receptors, but mainly on the latter, are likely mediators of the monosynaptic excitatory postsynaptic potential evoked in sympathetic preganglionic neurons by the stimulated region. The efflux data suggest that glutamate and aspartate are among the mediators.

Slow EPSP and the depolarizing action of noradrenaline on sympathetic preganglionic neurons

Intracellular recordings were made from sympathetic preganglionic neurons of the lateral horn in slices of cat thoracic cord maintained in vitro. Focal electrical stimulation of the slice evoked, in addition to the already described fast EPSPs, EPSPs of several seconds duration. The slow EPSPs, like the fast EPSPs, were graded with stimulus intensity and were abolished by TTX or low Ca and high Mg superfusion. The slow EPSP decreased in amplitude with membrane hyperpolarization and was nullified at -90 mV but did not reverse with further hyperpolarization. The slow EPSP was abolished by phentolamine or prazosin but not by yohimbine. Noradrenaline NA, 10-50 microM) caused in 30% of neurons a TTX-resistant depolarization. The NA-evoked depolarization had the same characteristics as the slow EPSP with respect to sensitivity to membrane potential and to adrenergic blockers. These results suggest that NA, acting on an alpha 1-receptor, may be the mediator of the slow EPSP evoked in this neuron by focal stimulation.