[PRESYNAPTIC SEROTONERGIC MODULATION OF SPONTANEOUS AND MINIATURE SYNAPTIC ACTIVITY IN THE FROG LUMBAR MOTONEURONS].

In this study we investigated the effect of 5-HT on the spontaneous and miniature synaptic activity in lumbar motoneurons from isolated frog spinal cord using intracellular recording. 5-HT increased the frequency of the spontaneous and miniature postsynaptic potentials (mPSPs). The effect of 5-HT on different subpopulations of mPSPs was multidirectional: it increased the frequency of glutamatergic excitatoty mPSP by 18 % and decreased the frequency of glycinergic inhibitory mPSP by 28 %, but had no effect on the frequency of GABAergic inhibitory mPSP. The amplitude and kinetic parameters of any subpopulation of mPSPs did not change. The data obtained show that 5-HT regulates the probability of glutamate and glycine release from presynaptic terminals ending at the frog spinal motoneurons. 5-HT shifts the ba- lance between synaptic excitation and inhibition in the spinal neural network toward excitation. Thus, 5-HT participates in control of motor output and provides its facilitation.

[THE INHIBITORY EFFECT OF GLYCINE AND GABA ON MONOSYNAPTIC EPSPs IN THE FROG SPTNAL MOTONFUCTYNMR].

In this research we have studied the inhibitory effect of glycine and GABA on the monosynaptic EPSPs induced by microstimulation of presynaptic fibers close to the frog lumbar motoneurones. Monosynaptic EPSPs had two components mediated by AMPA/KA (78%) and NMDA (22%) receptors. Both inhibitory mediators considerably decreased EPSP's decay time by 33.4 +/- 4.0% (n = 18) for glycine and by 40.2 +/- 3.6% (n = 18) for GABA. The decrease of EPSP's decay time was reduced after blockade ofNMDA receptors by AP5. The time characteristics were recovered in the normal saline. Glycine and GABA inhibited NMDA component of EPSPs to a greater extent than AMPA/KA component. It was confirmed by our previous data about the predominant inhibitory effect of glycine and GABA on responses induced by NMDA (as compared with AMPA and KA) application to motoneurone membrane.

[Inhibitory regulation of glutamate receptors in the frog motoneuron].

The interaction of exogenously applied excitatory (glutamate and their agonists NMDA, AMPA, kainate) and inhibitory (glycine and GABA) amino acid effects was studied intracellularly in the motoneurones of the isolated frog spinal cord. During simultaneous glycine or GABA bath applications GLU-, AMPA-, KA- and NMDA-evoked responses were, respectively, decreased up to 45.8 +/- 2.9% (n = 12) and 67.8 +/- 3.9% (n = 16), 13.9 +/- 4.3% (n = 9) and 32.1 +/- 8.3% (n = 12), 36.8 +/- 8.2% (n = 7) and 48.0 +/- 11.8% (n = 6), 7.7 +/- 3.5% (n = 9) and 18.1 +/- 3.8% (n = 14) from the control. Sequential applications of EAA after glycine or GABA as well as the applications of EAA-agonist and glycine (GABA) mixture demonstrated similar results. The decrease of EAA-responses by glycine and GABA was abolished by selective GlyR antagonist strychnine (1 microM) and the selective GABAR antagonist SR95531 (gabazine, 20 MM), respectively. The data revealed differences in inhibitory effect of glycine and GABA on the excitation responses mediated by different types of glutamate receptors in the frog motoneurones: the predominant inhibitory effect of glycine and GABA on NMDA-responses and weak inhibitory effect on KA- and GLU-responses. Inhibitory effect of glycine was twice as much as that of GABA at the same concentration.

[The contribution of glycine and GABA(A) receptors to generation of the inhibitory postsynaptic potentials in the frog spinal cord motoneurones].

The contribution of glycine and GABA(A) receptors to generation of the inhibitory postsynaptic potentials (IPSPs) evoked by microstimulation of the inhibitory fibers was studied intracellularly in the motoneurones of the isolated frog spinal cord. IPSPs were isolated by bloking EPSPs with kynurenate or CNQX and AP-5. The reversion under the small depolarising current (1-10) nA was used for the identification of IPSPs. The selective GlyR antagonist strychnine (1-5 microM) reduced the amplitude of IPSPs by a factor of 4.7 on the average in all studied motoneurones, while the selective GABA(A)R antagonist bicuculline (50-70 microM)--only by a factor of 1.6 and had no effect in 44% of motoneurones. Sequential applications of strychnine and bicuculline completely blocked the IPSPs. The results suggest that postsynaptic inhibition in the frog motoneurones is mediated by glycine (predominantly) and GABA(A) (to a smaller extent) receptors. It is possible the GABA(A) receptors are partly extrasynaptic.

Differences in the activation of inhibitory motoneuron receptors in the frog Rana ridibunda by GABA and glycine and their interaction.

Intracellular recording of potentials was used in isolated spinal cord segments from the frog Rana ridibunda to compare the inhibitory effects of GABA and glycine on the motoneuron membrane. At equal concentrations, the response (a change in membrane potential) to application of glycine was 1.5-2 times greater than the response to GABA in terms of amplitude, and EC(50) values were 0.75 and 1.57 mM, respectively. The response to simultaneous application of GABA and glycine averaged 79.1 +/- 2.4% (n = 19) of the sum of the individual responses and 130.1 +/- 1.5% (n = 19) of the glycine response (partial occlusion). Preliminary application of glycine decreased the GABA response by 85.3 +/- 0.2% (n = 10), while preapplication of GABA decreased the glycine response by only 52.9 +/- 0.3% (n = 11). The glycine and GABA responses were specifically suppressed by strychnine and bicuculline. These results provide evidence that as in mammals, amphibian motoneurons have both glycine (predominantly) and GABA(A) receptors; they also show that asymmetrical cross inhibition can occur.

[Differences in activation of the motoneurone inhibitory receptors in a frog Rana ridibunda by GABA and glycine and their interaction].

The membrane potential responses evoked by GABA and glycine bath applications were studied intracellularly in the motoneurons of the isolated frog spinal cord. The amplitude of glycine-evoked responses was 1.5-2.0 larger than that of GABA-evoked response at the same concentration. EC50 were 0.75 mM and 1.57 mM for glycine and GABA, respectively. The amplitude of responses induced by the simultaneous applications of both agonists were 79.1 +/- 2.4% (n = 19) of the sum of individual responses and 130.1 +/- 1.5% (n = 19) of individual glycine-induced responses (partial occlusion). GABA-evoked responses were decreased by 85.3 +/- 0.2% (n = 10) as a result of glycine preliminary application while preapplication of GABA reduced glycine-evoked responses only by 52.9 +/- 0.3% (n = 11). Glycine- and GABA-evoked responses were selectively suppressed by strychnine and bicuculline, respectively. These results suggest that amphibian spinal motoneurones express (less specifically than those of mammals) both glycine (predominantly) and GABAA receptors, with asymmetric cross-inhibition possibly taking place in them.

Three types of inhibitory miniature potentials in frog spinal cord motoneurons: possible GABA and glycine cotransmission.

Miniature inhibitory postsynaptic potentials (mIPSP) of motoneurons in isolated frog spinal cord were recorded in conditions of blockade of the conduction of nerve spikes and ionotropic glutamate receptors (TTX, 1 microM, CNQX, 25 microM, D-AP5, 50 microM). Three types of mIPSP were identified: those with fast and slow time characteristics and mIPSP with two-component decays. Two-component mIPSP accounted for 8.7% of all selected responses, fast mIPSP for 64.5%, and slow mIPSP for 26.8%. Blockade of GABA(A) receptors with bicuculline (20 microM) led to decreases in the numbers of slow and two-component mIPSP and an increase in the number of mIPSP with fast kinetics. Strychnine (1 microM), a blocker of glycine receptors, led to a reduction in the number of fast receptors and an increase in the number of slow potentials. These data suggest that frog spinal cord motoneurons have three types of inhibitory mIPSP, mediated by GABA, glycine, and simultaneous release of these two transmitters from the same presynaptic terminals.

[Three types of miniature inhibitory potentiaes in the frog spinal cord motoneurons: the possibility of GABA and glycine co-release].

Miniature inhibitory postsynaptic potentials (mlPSPs) were recorded from motoneurons of the frog isolated spinal cord after blocking action potentials and ionotropic glutamate receptors (TTX 1 mcm: CNQX 25 mcm, D-AP5 50 mcm). Three types of mlPSPs were selected by their time characteristics) fast, slow and mlPSPs with two decay time constants. We classified 8.7% of mlPSPs as dual-component, 64.5% as fast mlPSPs, and 26.8% as slow mlPSPs. The GABA(A)R blocker bicuculline (20 mcm) diminished the number of the slow and dual-component events while the number of mlPSP with a fast kinetics was increased. The GlyR antagonist strychnine (1 mcm) reduced the frequency of fast mlPSPs and increased this parameter of slow mlPSPs. These data suggest existence of three different mlPSP groups distinguished by their kinetics and sensitivity to receptor antagonists: fast events mediated by glycine, slow events mediated by GABA and dual-component mlPSPs corresponding to glycine and GABA co-release.

[The effect of excitatory amino acid antagonists on postsynaptic potentials in the motoneurons of the frog Rana ridibunda]. / Vliianie antagonistov vozbudaiushchikh aminokislot na postsinapticheskie potentsialy v motoneironakh liagushki Rana ridibunda.

The effects of excitatory amino acids on postsynaptic potentials (PSP) generated by activation of three synaptic inputs in isolated frog Rana ridibunda spinal cord motoneurons were investigated. PSP evoked by dorsal root (DR) and reticular formation (RF) stimulation were blocked by kynurenate (0.5-1 mM) and CNQX (1 mkM). However, the resistivity of RF PSP and DR PSP to kynurenate and CNQX was recorded in 7 and 4 cases respectively. APV blocked the late phase of DR and RF PSP, but did not decrease the amplitude of the early phase. Lateral column (LC) PSP generated in the same cell by LC stimulation was not decreased by kynurenate or CNQX at all. LC PSP was blocked by APV in 1 of 6 cells and it was blocked by the mixture of strychnine and APV in 2 of 6 cells. DR and RF PSP resistant to kynurenate were also blocked by the mixture of strychnine, picrotoxin and APV in 3 of 17 cells. Strychnine itself did not block LC PSP or resistant DR and RF PSP. Moreover, LC PSP was depressed, whereas RF and DR PSP were potentiated while glutamate was added into superfusing solution.

[The potentiation of postsynaptic potentials under the influence of glutamate and agonists in the motoneurons of the frog Rana ridibunda]. / Potentsiatsiia postsinapticheskikh potentsialov pod vliianiem glutamata i agonistov v motoneironakh liagushki Rana ridibunda.

Effects of glutamate and agonists (aspartate, NMDA, quisqualate, AMPA, kainate) on dorsal root and reticulomotoneuronal excitatory postsynaptic potentials (EPSPs), as well as on spontaneous postsynaptic potentials (PSP), were studied in the motoneurons of isolated frog spinal cord. Depolarizing responses were evoked by glutamate or agonists bath application. Amplitude of the response decreased in conditions of TTX-block or replacement of Ca2+ by Mn2+, Mg2+ or Co2+ in perfusing solution. Excitatory amino acid antagonists (kynurenate, CNQX, APV, argiopine) also reduced depolarizing response amplitude. DR and RF EPSPs significantly increased in amplitude (and spontaneous PSP in amplitude and frequency) during depolarization, evoked by glutamate or agonists. The potentiation reached up to 300 %. Potentiation diminished with depolarization decay. Sometimes several minutes depression of EPSPs was observed after the depolarizing response. There was no potentiation of the spontaneous PSPs in conditions of TTX-block or replacement of Ca2+ by Mn2+ in perfusing solution. The data obtained suggest rather presynaptic, than postsynaptic mechanism of the potentiation. We found no depressant effect of glutamate or agonists on postsynaptic glutamate receptors, at least for 10 minutes contact. Effects of more prolonged applications and some changes of EPSP amplitude after depolarizing response appear to be associated with other types of glutamate receptors.

[The heterogeneity of the excitatory synaptic inputs in the spinal motor neurons of the frog Rana ridibunda]. / Geterogennost' vozbuzhdaiushchikh sinapticheskikh vkhodov v spinal'nykh motoneironakh liagushki Rana ridibunda.

The synaptic responses induced in motoneurones by the stimulations of the dorsal root (DR), single afferent fibres and reticular formation (RF) were intracellularly recorded in the isolated frog spinal cord. It was shown that argiopine (the selective blocker of glutamate receptors of non-NMDA type) in concentrations ranging from 3.10(-7) to 1.10(-5) M effectively suppressed the di- and polysynaptic, but not the monosynaptic components of EPSP's induced by DR stimulation. The initial reaction to argiopine consisted of the increase of this monosynaptic component of EPSP. In the same concentrations range, argiopine reduced both mono- and polysynaptic EPSP, evoked by RF stimulation. 2-amino-phosphonovaleric acid (1.10(-4) M) did not affect, whereas the kinurenate (1--2.10(-3) M) completely blocked the amplitude of all kinds of synaptic responses. The various effects of argiopine on the responses induced by microstimulation of presynaptic nerve terminals were observed. The data obtained speak in favour of heterogeneity of monosynaptic excitatory inputs in the motoneurones of frog spinal cord. Being the glutamatergic by nature, the inputs differ in the properties of postsynaptic receptors. All of these receptors concerning to non NMDA-type can be divided to argiopine-sensitive and argiopine-resistant. The first seem to be involved in the monosynaptic connections of RF and the second--in those of primary afferents with motoneurones.

[Properties of the response of frog motoneurons to glutamate application]. / Svoistva otveta motoneironov liagushki na applikatsiiu glutamata.

The reversal potential (E) of the glutamate response produced by iontophoretically applied glutamate and that of the EPSP's recorded from three synaptic inputs produced by reticular formation (RF), dorsal roots (DS), and microstimulation of presynaptic terminals (PT) at the point of glutamate application were compared at the same motoneuron of the isolated frog spinal cord using current-chop technique. Following Cs injection the glutamate response and EPSP's were reversed. Different points of glutamate application were tested. Mean values of E were: Eglu = -16.9 +/- 1.7 mV (n = 13), EPT = -15.1 +/- 1.4 mV (n = 13), ERF = -6.8 +/- 1.7 mV (n = 13), EOR = -9.8 +/- 1.8 mV (n = 6). The summation of the glutamate responses and EPSP was close to linear. Increase of the input conductance did not exceed 10%. The results obtained suggest that glutamate induces depolarization via the receptors of the postsynaptic membrane and may serve as a neurotransmitter in all of the studied inputs.

[Reversal potential of the EPSP of frog motoneurons]. / Potentsial ravnovesiia monosinapticheskikh VPSP v motoneironakh liagushki.

The current-chop technique has shown that I-V characteristic of the membrane are nonlinear in lumbar motoneurons of isolated perfused frog spinal cord. Input resistance of the membrane decreased with depolarization when constant current was applied during 0.1-1.0 s. However, injection of current 40-60 nA during 1-2 min led to an increase of the membrane resistance to the initial value. As a result the membrane potential could be shifted to the positive level up to +50 mV and more. Monosynaptic excitatory postsynaptic potentials evoked by stimulation of the brainstem or by microstimulation of ventrolateral tract fibres were found to reverse completely at a positive level of the membrane potential. In most cases the reversal potentials ranged between 0 mV and -10 mV.

[Amplitude of miniature potentials of motor neurons in the frog Rana ridibunda]. / Almplituda miniatornykh potentsialov v motoneironakh liagushki Rana ridibunda.

Miniature synaptic potentials have been recorded from motoneurones of the isolated spinal cord of the frog Rana ridibunda. In normal Ringer's solution, their frequency varied from 5 to 50/sec, whereas their amplitude reached 2-5 mV. Only 50-300 microV (rarely 0.5-1.5 mV) potentials persisted when TTX was added to Ringer's solution and/or Ca was replaced by Mn. However, in Ca-free solution, TTX in combination with Mn did not decrease the amplitude of miniature potentials, provided the initial values varied within 50-300 microV. Noise fluctuations did not exceed 40-50 microV, and the ratio of the number of miniature potentials of 50 microV to the number of 50 microV noise potentials was about 10:1. The observed miniature potentials with an amplitude of 50-100 microV coincide with the quantal units calculated by other authors from statistical analysis of the unitary EPSPs evoked by primary afferents or by ventrolateral tract fibers.