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1. In freshly isolated spinal dorsal horn (DH) neurons (laminae I-III) of the young rat, the effects of tachykinins (substance P, neurokinin A) on inward current induced by excitatory amino acids were studied under whole-cell voltage-clamp conditions. 2. When the cells were clamped to a holding potential of -60 mV, a simultaneous application of N-methyl-D-aspartate (NMDA) (10(-4) M) and substance P (SP) (2 x 10(-9)-10(-7) M) for 10 s reversibly enhanced (by 129.6 +/- 8.2%, mean +/- SE) the peak amplitude of the initial transient component of the NMDA-induced current in approximately 60% of the examined cells and reduced it (to 83.3 +/- 2.7%) in 27% of the cells. In addition, SP produced an increase (by 133.6 +/- 11.7%) or a small decrease (to 85.9 +/- 1.4%) in the steady-state component of the NMDA response. In difference to SP, a simultaneous application of NMDA (10(-4) M) and neurokinin A (NKA) (10(-10)-10(-7) M) reversibly suppressed (to 86.8 +/- 2.1%) the peak amplitude of the NMDA-induced current in 75% of the examined cells. 3. The NMDA-induced currents were modulated by tachykinins not only during the coadministration but up to 20 min after the removal of the peptide. SP potentiated the initial peak NMDA current by 147.9 +/- 8.1% in 78% of examined cells and decreased it (76.3 +/- 5.7%) in 11% of cells. The potentiating effect was concentration-dependent (range: 10(-11)-10(-8) M) and reversible, but it was reduced with repeated applications. In addition, SP increased (by 125.4 +/- 3.6%) or reduced (to 86.0 +/- 1.8%) the steady-state component of the NMDA response. 4. When the single DH neurons were exposed to SP or NKA for 30 s-7 min before the testing of the NMDA responses, tachykinins had two distinct effects on the peak amplitude of the transient component of the NMDA-induced current, consisting of an initial depression (SP: to 64.8 +/- 2.1%; NKA: to 76.3 +/- 4.4%) followed by a potentiation (SP: by 146.6 +/- 6.8%; NKA: by 178.4 +/- 35.2%). The enhancing effect in some cells lasted less than or equal to 1 h. 5. A claimed novel nonselective tachykinin antagonist, spantide II (10(-8) M) coadministered with NMDA (10(-4) M), slightly depressed the peak component of NMDA-induced current. In addition, it effectively blocked the SP-induced potentiation of the responses of DH neurons to NMDA.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of noradrenaline precursors on glycine and N-methyl-D-aspartate (NMDA) receptors in spinal cord neurons recently isolated from chick embryo were investigated using whole cell patch-clamp and concentration clamp techniques. Both L-alanine and L-DOPA were found to be glycine agonists capable of potentiating NMDA response, while L-tyrosine does not activate glycine but can potentiate NMDA response. Lastly, L-phenylalanine and dopamine do not interact with either glycine or NMDA receptors.

Glutamate-gated ion channels mediate excitatory synaptic transmission in the central nervous system and are involved in synaptic plasticity, neuronal development and excitotoxicity (5,24). These ionotropic glutamate receptors were classified according to their preferred agonists as AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid), KA (kainate), and NMDA (N-methyl-D-aspartate) receptors [Trends Pharmacol. Sci., 11 (1990) 25-33]. The present study of NMDA receptor channels expressed in acutely isolated spinal dorsal horn (DH) neurons of young rat reveals that they are subject to modulation through the adenylate cyclase cascade. Whole-cell voltage-clamp recording mode was used to examine the effect of adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase (PKA) on the responses of DH neurons to NMDA. Whole-cell current response to NMDA was enhanced by 8 Br-cAMP, a membrane permeant analog of cAMP or by intracellular application of cAMP or catalytic subunit of PKA.

Substance P and neurokinin A both potentiated N-methyl-d-aspartate (NMDA)-induced currents recorded in acutely isolated neurons from the dorsal horn of the rat. To elucidate the mechanism underlying this phenomenon, we measured the effects of tachykinins and glutamate receptor agonists on [Ca2+]i in these cells. Substance P, but not neurokinin A, increased [Ca2+]i in a subpopulation of neurons. The increase in [Ca2+]i was found to be due to Ca2+ influx through voltage-sensitive Ca2+ channels. Substance P and neurokinin A also potentiated the increase in [Ca2+]i produced by NMDA, but not by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, kainate, or 50 mM K+. Phorbol esters enhanced the effects of NMDA and staurosporine inhibited the potentiation of NMDA effects by tachykinins. It is concluded that activation of protein kinase C may mediate the enhancement of NMDA effects by tachykinins in these cells. However, the effects of tachykinins on [Ca2+]i can be dissociated from their effects on NMDA receptors.

The effects of the metabotropic glutamate receptor agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid [(1S,3R)-ACPD] were examined on responses mediated by the ionotropic glutamate receptor agonists N-methyl D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA), and kainic acid (KA), in neurons acutely isolated from the dorsal horn of the rat spinal cord. (1S,3R)-ACPD produced an increase in the intracellular Ca2+ concentration in 50% of acutely isolated dorsal horn neurons, which could be prevented by blockers of voltage-sensitive Ca2+ channels. (1S,3R)-ACPD markedly potentiated increases in the intracellular Ca2+ concentration induced by NMDA, AMPA, and KA but not by 10-50 mM KCl. This potentiation occurred in all cells, required the simultaneous presence of both agonists, and was rapidly reversible. In the spinal cord slice preparation, (1S,3R)-ACPD potentiated the inward currents evoked by pressure application of AMPA, NMDA, and KA, an effect that was also rapidly reversible. These short term effects of (1S,3R)-ACPD may play an important role in the regulation of ionotropic responses mediated by glutamate in the spinal cord.

The action of glycine and GABA on isolated lamprey spinal cord neurons was investigated by means of intracellular perfusion and concentration clamp techniques. These amino acids activated desensitizing chloride ionic conductances. The concentrations of agonists evoking half-maximum effects (ED50) were equal to 16 microM and 1.5 mM for glycine- and GABA-activated currents, respectively. Increase in the transmitter concentration led to a decrease in the time constant of desensitization. Current-voltage relationships of glycine- and GABA-activated currents were strongly dose-dependent. At low agonist concentrations the time constant of activation decreased with membrane hyperpolarization. Glycine- and GABA-activated currents exhibited complete cross-desensitization. The specific glycine antagonist, strychnine, suppressed both glycine- and GABA-activated currents to the same degree. Selective antagonists of GABA receptors, bicuculline and picrotoxin, produced equal blocking effects on glycine- as well as GABA-evoked responses. In the cells studied, taurine activated desensitizing ionic conductance. Responses evoked by taurine and glycine applications demonstrated complete cross-desensitization. Taurine-activated currents were sensitive to strychnine, bicuculline and picrotoxin. These results suggest the existence of one receptor-channel complex for the main inhibitory transmitters in lamprey spinal cord neurons. 3,4-Dioxy-L-beta-phenylalanine evoked desensitizing strychnine-sensitive ionic responses which exhibited cross-desensitization with glycine-activated currents.

1. The membrane currents elicited by L-glutamate and glycine applications in morphologically different neurone types were investigated in isolated spinal cord cells from the lumbar enlargement of 6 to 11-day-old chick embryos. The whole-cell patch-clamp technique and concentration clamp methods have been used. Isolated spinal cord neurones of four stages were investigated: 6, 7.5, 9 and 11 days of incubation (29th, 32nd, 35th and 37th stages of development, respectively). 2. The L-glutamate-activated conductance consisted of desensitizing and non-desensitizing components. The Hill coefficient for the first component was 1, and for the second was 2. The number of cells responding to L-glutamate application with only a desensitizing component decreased from 53.4% on the 6th day of incubation to 6.7% on the 11th day, whereas the number of cells responding with bicomponent responses increased during the same period from 13.3 to 87%. 3. From the 6th and 11th day of chick embryo development the characteristics of the desensitizing component of L-glutamate-activated conductance remained constant (half-maximal dose, (ED50 = 2.6 +/- 0.3 mM) whereas the ED50 for the non-desensitizing component decreased 10 times. 4. It was found that the density of the desensitizing L-glutamate-activated ionic current increased during morphological and age-dependent differentiation of spinal cord cells. 5. Of the investigated cells 88.7% were sensitive to glycine application. The smallest percentage of neurones responding to glycine application was observed at 7.5 days of incubation. Glycine-activated conductance did not change at the investigated stages (ED50, 71 +/- 2 microM; Hill coefficient, 2). 6. A significant decrease in the glycine-activated current density was observed on the 9th day of incubation in multipolar neurones with three neurites, against a tendency to an increase of glycine-activated ionic currents during morphological and age-dependent differentiation of cells. 7. Of the investigated cells 78% were sensitive to both glycine and L-glutamate application. A negative correlation between glycine- and L-glutamate-activated current densities (correlation coefficient, -0.71) was revealed by means of statistical analysis. 8. We conclude that these changes in chemosensitivity of the spinal cord neurones may underlie the increase in locomotor activity of the chick embryo observed on the 9th day of incubation.

Effect of noradrenaline precursors (L-alanine, L-phenylalanine, L-tyrosine, L-DOPA and dopamine) on glycine and NMDA receptors of freshly isolated spinal neurones of chick embryo were investigated by means of the whole cell patch-clamp and concentration clamp techniques. L-alanine and L-DOPA were found to be glycine agonists which can potentiate NMDA responses. L-tyrosine does not activate glycine receptor but potentiate NMDA one. L-phenylalanine and dopamine do not interact with both glycine and NMDA receptors.

Transient and steady-state components of L-glutamate-activated membrane currents were investigated using intracellular perfusion, voltage clamp, and concentration clamp techniques in spinal cord neurons of 6–11 day chick embryos. Hill's coefficient was found to equal 1 for transient and 2 for steady-state components. It was shown that the L-glutamate-activated receptors are present, which appear in the membrane of spinal neurons at the early stages of development.

The pattern of change in the density of L-glutamate- and glycine-activated ionic currents through the spinal cord neuronal membrane of 6–11 day chick embryos was traced using an enzymatic technique for isolating cells during voltage-clamp experiments. A general trend towards increased current density was found during cell differentiation, followed by a substantial reduction in the density of glycine-activated current on the 9th day of incubation. A reciprocal relationship between densities of L-glutamate- and glycine-activated currents were characteristic of individual neurons. The functional significance of the patterns identified is discussed in connection with the evolution of spinal cord motor function during embryogenesis.

Isolated spinal cord neurons of 6-11 days chick embryo were investigated under conditions of intracellular perfusion and voltage clamp. It was found that densities of sodium inward currents through the neuronal membrane increase during development of chick embryo with morphological and time-dependent differentiation of cells.

Isolated spinal cord neurons of 6-11 day old chick embryos were investigated under conditions of intracellular perfusion and voltage clamp. Glycine-activated chloride conductivity characteristics of neuronal membranes did not change at the investigated stages. Concentrations evoking half-effect (7.5 X 10(-5) mol/l) and Hill's coefficients (1.65-1.95) were also constant at these stages.

Isolated cells from the spinal cord of chick embryo were investigated under conditions of intracellular perfusion and voltage clamp using the method of rapid application of extracellular solutions. Two components were found in the L-glutamate-activated current: a fast-inactivating component and a steady one. The dose-response relationship demonstrated one-to-one interaction between L-glutamate and membrane receptor with Kd values about 0.75 X 10(-5) mol/l for the steady component and 3.2 X 10(-5) mol/l for the fast one.

Ionic currents induced by application of L-3, 4-dihydroxyphenylalanine (L-DOPA) and acidic amino acids: L-glutamate, kainate, N-methyl-D-aspartate (NMDA) were investigated in experiments on isolated spinal cord neurons of the lamprey by means of whole-cell recording and concentration clamp methods. L-DOPA was found to activate glycine, but not excitatory amino acid receptors.

Transient and steady-state L-glutamate-activated membrane currents were investigated by means of intracellular perfusion, voltage clamp techniques and concentration clamp in spinal cord neurons of 6-11 day chick embryos. The Hill coefficient was found to equal 1 for transient and 2 for steady-state components. Concentration of L-glutamate evoking half effect decreased during development more than 10 times for the steady-state component whereas it remained constant for the transient component. Existence of two types of glutamate-activated receptors in the membrane of spinal cord neurons at early stages of embryonic development is supposed.

Effects of inhibitory mediators on the membranes of isolated lamprey spinal cord neurones were investigated by means of whole-cell recording and concentration clamp techniques. Glycine and γ-aminobutyric acid (GABA) applications evoked desensitizing chloride currents. The concentrations at half-maximum effects were 16 μM for glycine- and 1.5 mM for GABA-activated currents. Ionic responses to applications of both amino acids demonstrated full cross-desensitization. Strychnine and bicuculline suppressed both glycine- and GABA-activated conductances to the same degree. We suggest that in the membranes of lamprey spinal cord neurones glycine and GABA act on the same receptor-channel complexes.

The changes in densities of L-glutamate- and glycine-activated currents through the membrane of spinal neurons were investigated in 6-11 day chick embryos using enzymatic isolation of cells and voltage clamp technique. General tendency of the current density increase during cell differentiation was found. A considerable decrease of the glycine-activated current density against a background of such an increase was observed on the 9th day of incubation. Reciprocal relation between L-glutamate- and glycine-activated current densities was found for a single neuron. Functional significance of the revealed density changes is discussed in connection with the development of the locomotor function of the spinal cord.