N. P. Veselkin

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 EC50 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 GABAA receptors; they also show that asymmetrical cross inhibition can occur.

Characteristics and Interaction of GABAergic and Glycinergic Processes in Frog Spinal Cord Neurons

Whole-cell patch clamp recordings from isolated spinal cord neurons from the frog Rana temporaria were made to study the interaction of processes induced by application of GABA and glycine. The amplitudes of currents evoked by application of glycine did not change with time, while the amplitudes of GABAmediated currents decreased two-fold during the first 15 min of the experiment and stabilized at the new level. Neuron responses to simultaneous application of GABA and glycine were always smaller than the sum of the responses to separate application of these neurotransmitters. On application of GABA and glycine at the same concentration (5 mM), the amplitude of the response to simultaneous application decreased with time, reaching the level of the glycine-mediated response. A mixture of glycine and GABA at 8 μM and 5 mM, respectively, gave settled responses which were larger than the largest individual response by more than obtained with other mixtures. These data provide evidence that frog motoneurons may express receptors activated by both GABA and glycine.

Effects of Glycine and GABA on Monosynaptic EPSP in Frog Motoneurons

We report here our studies of the effects of the inhibitory transmitters glycine and GABA on monosynaptic EPSP recorded in response to microstimulation of presynaptic fibers (PF PSP) close to the bodies of motoneurons in the isolated frog spinal cord. Monosynaptic PF PSP had two components, mediated by AMPA/KA and NMDA receptors. Blockade of NMDA receptors was used to analyze the characteristics of the AMPA/KA and NMDA components of PF PSP. The amplitude of the NMDA component was 20% of PF PSP amplitude. The areas under the curves of the AMPA/KA and NMDA components were 78% and 22% respectively of the area of the PF PSP. Glycine and GABA had little effect on PF PSP, but significantly decreased the decay time constant (τdecay), by an average of 33.4 ± 4.0% (n = 18) on application of glycine and by 40.2 ± 3.6% (n = 18) on application of GABA. Blockade of NMDA receptors with DL-2-amino-5-phosophonovaleric acid (AP5) decreased the effects of glycine and GABA on the time constant. Effects were reversible and the amplitude and time characteristics of responses recovered in normal solution. Assessment of the inhibitory influences of glycine and GABA on each of the components of PF PSP showed that suppression of the NMDA component produced a greater proportionate effect than suppression of the AMPA/KA component. These results show that the inhibitory effects of glycine and GABA on the monosynaptic EPSP of motoneurons are mediated mainly by the NMDA component, with a small influence from the AMPA/KA component.

Characteristics of the Desensitization of Glycine-Mediated Ion Currents in Frog Spinal Cord Neurons

Whole-cell patch clamp studies of isolated spinal cord neurons from the frog Rana temporaria in voltage-clamped conditions addressed the kinetics of ion channel desensitization evoked by glycine application. The ion current desensitization phase (decay from 90% to 10%), induced by periodic applications of glycine (1 mM), approximated an exponential function. Approximation results identified three types of glycine-mediated response. The decay constant of type 1 responses in the first minute of the experiment was τ1 = 1693 ± 135 msec (n = 8) and decreased from application to application during further recording. Type 2 responses had a decay constant τ2 = 364 ± 42 msec (n = 9) at the beginning of recording and showed virtually no change with time. Type 3 responses had a biexponential decay consisting of a rapid (τ2) component and a slow (τ1) component, acceleration of the desensitization phase of this response from application to application occurring as a result of a decrease in the larger decay constant τ1. These data suggest that the frog spinal cord membrane has at least two varieties of glycine receptor with different rates of desensitization and different dynamics of changes in properties during patch-clamp recording. Different neurons can evidently have these receptor varieties simultaneously or separately.

Summation of GABA- and Glutamate-Mediated Ion Currents in Isolated Rat Cerebral Cortex Neurons

Patch clamp experiments in the whole cell configuration were performed on isolated rat prefrontal cortex neurons to study the summation of ion currents evoked by application of glutamate and GABA. Ion currents were recorded using two different pipette solutions, based on cesium chloride and fluoride. In recordings made using the cesium chloride-based solution, the peak amplitude of the current evoked by simultaneous application of GABA and glutamate (each at 200 μM) coincided with the peak amplitude of the current evoked by application of GABA alone, and was significantly smaller than the arithmetic sum of the responses to application of the two neurotransmitters individually. When the pipette solution based on cesium fluoride was used, the response to simultaneous application of glutamate and GABA was essentially the same as the arithmetic sum of the individual responses. On exposure to these neurotransmitters at saturating concentrations (5 mM), the response recorded to simultaneous application was significantly smaller than the response to application of GABA alone. These results suggest that there is a mechanism of interaction between GABAA and ionotropic glutamate receptors (AMPA and kainate). On simultaneous application of glutamate and GABA, activation of GABAA receptors evidently has a greater influence on glutamate receptors than activation of glutamate receptors of GABAA receptors.

Mechanisms of Interaction between GABA- and Glycine-Medicated Responses of Frog Spinal Cord Neurons

Whole-cell patch-clamp recordings were made from isolated frog (R. temporaria) spinal cord neurons to study the interaction between processes evoked by application of glycine and GABA. When GABA and glycine were applied simultaneously, neuron responses were always smaller than the sum of the responses to separate application of these neurotransmitters. The mechanisms underlying this occlusion phenomenon were identified. Firstly, GABA could activate glycine receptors (EC50 = 1.2 mM) in frog spinal cord neurons relatively effectively. Secondly, studies of the effects of application of glycine on the course of “rundown” of GABA-mediated responses showed that activation of glycine receptors facilitated inhibition of GABAA receptors. Both of these mechanisms of interaction of neuron responses are presumptively mediated via simultaneous release of the neurotransmitters glycine and GABA in synapses.

Membrane Potential Oscillations Produced by Excitatory Amino Acids in Isolated Spinal Neurons of the Lamprey Lampetra fluviatilis

Membrane potential (MP) oscillations produced by excitatory amino acids (EAA) have been studied in branching neurons isolated by an enzymatic-mechanical method from the lamprey spinal cord. It was shown that (1) all studied EAA (glutamate, kainate, NMDA, aspartate, and quisqualate) evoke an ion current and a short-term reversible depolarization in studied cells; (2) EAA added to perfusion solution may produce MP oscillations, with kinetic parameters and duration of the oscillation depending on the amino acid used (the most effective are kainate and NMDA, the least effective, quisqualate); (3) oscillations can be irregular (of the type of a synaptic noise or of a long-term plateau of depolarization with action potentials—AP) or regular, with frequency of 0.5–1.5 Hz. Amplitude of both oscillation types depends on MP level, frequency is more steady for each cell and less depends on MP. In 68 out of 128 studied cells, oscillations could be evoked, which indicates that a significant part of lamprey spinal neurons have intrinsic capability for MP oscillations and probably pacemaker properties. The functional role of oscillations can be different. They can take cells out from the profound inhibition state, synchronize activity of rhythm generation neurons and/or be the base for trigger signals (AP firing) sent by locomotor neuronal circuits to trunk muscles.

Potentiation of postsynaptic potentials in motoneurons of the frog,Rana ridibunda, by gallamine

The potentiating effect of gallamine on postsynaptic potentials was studied intracellularly on the preparation of the isolated spinal cord of the frog,Rana ridibunda. The amplitude both of afferentPSP (stimulation of DR) and of descendingPSP (stimulation of LC and VC) increased to 200–600% of the initial value. The responses reached 20 mV, and a convulsive effect developed. Both early (mono- and disynaptic) and late components were considerably potentiated. New components, absent prior to the gallamine application, appeared. Responses to a shortterm application of glutamate were potentiated much less than PSP in the same cell. NMDAresponses were not potentiated. Brain treatment with EAA blockers showed that potentiation and convulsive effect might appear under conditions of blockage of NMDA receptors but not under conditions of blockage of non-NMDA receptors. Gallamine eliminated inhibition evoked by the agents that block EAA. After gallamine application the duration of antidromic and orthodromic AP did not change substantially, while after-depolarization increased considerably, which resulted in generation of 2-3 AP in response to a single stimulus. A competitive blocker of cholinergic transmission, d-tubocurarine, did not potentiate PSP but increased synaptic noise. Mechanisms of gallamine action are discussed.