Post-episode depression of GABAergic transmission in spinal neurons of the chick embryo.

Whole cell recordings were obtained from ventral horn neurons in spontaneously active spinal cords isolated from the chick embryo [embryonic days 10 to 11 (E10-E11)] to examine the post-episode depression of GABAergic transmission. Spontaneous activity occurred as recurrent, rhythmic episodes approximately 60 s in duration with 10- to 15-min quiescent inter-episode intervals. Current-clamp recording revealed that episodes were followed by a transient hyperpolarization (7 +/- 1.2 mV, mean +/- SE), which dissipated as a slow (0.5-1 mV/min) depolarization until the next episode. Local application of bicuculline 8 min after an episode hyperpolarized spinal neurons by 6 +/- 0.8 mV and increased their input resistance by 13%, suggesting the involvement of GABAergic transmission. Gramicidin perforated-patch recordings showed that the GABAa reversal potential was above rest potential (E(GABAa) = -29 +/- 3 mV) and allowed estimation of the physiological intracellular [Cl(-)] = 50 mM. In whole cell configuration (with physiological electrode [Cl(-)]), two distinct types of endogenous GABAergic currents (I(GABAa)) were found during the inter-episode interval. The first comprised TTX-resistant, asynchronous miniature postsynaptic currents (mPSCs), an indicator of quantal GABA release (up to 42% of total mPSCs). The second (tonic I(GABAa)) was complimentary to the slow membrane depolarization and may arise from persistent activation of extrasynaptic GABAa receptors. We estimate that approximately 10 postsynaptic channels are activated by a single quantum of GABA release during an mPSC and that about 30 extrasynaptic GABAa channels are required for generation of the tonic I(GABAa) in ventral horn neurons. We investigated the post-episode depression of I(GABAa) by local application of GABA or isoguvacine (100 microM, for 10-30 s) applied before and after an episode at holding potentials (V(hold)) -60 mV. The amplitude of the evoked I(GABA) was compared after clamping the cell during the episode at one of three different V(hold): -60 mV, below E(GABAa) resulting in Cl(-) efflux; -30 mV, close to E(GABAa) with minimal Cl(-) flux; and 0 mV, above E(GABAa) resulting in Cl(-) influx during the episode. The amplitude of the evoked I(GABA) changed according to the direction of Cl(-) flux during the episode: at -60 mV a 41% decrease, at -30 mV a 4% reduction, and at 0 mV a 19% increase. These post-episode changes were accompanied by shifts of E(GABAa) of -10, -1.2, and +7 mV, respectively. We conclude that redistribution of intracellular [Cl(-)] during spontaneous episodes is likely to be an important postsynaptic mechanism involved in the post-episode depression of GABAergic transmission in chick embryo spinal neurons.

NMDA-induced dendritic oscillations during a soma voltage clamp of chick spinal neurons.

An investigation of dendritic membrane properties was performed by whole-cell patch measurements of the biophysical properties of intact chick spinal neurons that are involved in rhythmogenesis. A whole-cell voltage clamp of the somatic membrane was used to block NMDA-induced voltage oscillations from the cell body, thus partially isolating the intrinsic oscillatory properties of dendritic membranes from those of the soma. An experimental approach was developed that takes into account the complexity of the dendritic tree in an environment as normal as possible, without the need for cell isolation or slice preparations. A computational study of the experimentally determined model showed that excitatory amino acid receptors on dendrites can dynamically control the electrotonic length of the dendrites through the activation of negative slope conductances. These experiments demonstrate the presence of NMDA receptors on the dendrites and that they induce intrinsic oscillations when the synaptic input from other cells is significantly reduced.

Mechanisms of spontaneous activity in developing spinal networks.

Developing networks of the chick spinal cord become spontaneously active early in development and remain so until hatching. Experiments using an isolated preparation of the spinal cord have begun to reveal the mechanisms responsible for this activity. Whole-cell and optical recordings have shown that spinal neurons receive a rhythmic, depolarizing synaptic drive and experience rhythmic elevations of intracellular calcium during spontaneous episodes. Activity is expressed throughout the neuraxis and can be produced by different parts of the cord and by the isolated brain stem, suggesting that it does not depend upon the details of network architecture. Two factors appear to be particularly important for the production of endogenous activity. The first is the predominantly excitatory nature of developing synaptic connections, and the second is the presence of prolonged activity-dependent depression of network excitability. The interaction between high excitability and depression results in an equilibrium in which episodes are expressed periodically by the network. The mechanism of the rhythmic bursting within an episode is not understood, but it may be due to a "fast" form of network depression. Spontaneous embryonic activity has been shown to play a role in neuron and muscle development, but is probably not involved in the initial formation of connections between spinal neurons. It may be important in refining the initial connections, but this possibility remains to be explored.

Blockade and recovery of spontaneous rhythmic activity after application of neurotransmitter antagonists to spinal networks of the chick embryo.

We studied the regulation of spontaneous activity in the embryonic (day 10-11) chick spinal cord. After bath application of either an excitatory amino acid (AP-5 or CNQX) and a nicotinic cholinergic (DHbetaE or mecamylamine) antagonist, or glycine and GABA receptor (bicuculline, 2-hydroxysaclofen, and strychnine) antagonists, spontaneous activity was blocked for a period (30-90 min) but then reappeared in the presence of the drugs. The efficacy of the antagonists was assessed by their continued ability to block spinal reflex pathways during the reappearance of spontaneous activity. Spontaneous activity ceased over the 4-5 hour monitoring period when both sets of antagonists were applied together. After application of glycine and GABA receptor antagonists, the frequency of occurrence of spontaneous episodes slowed and became highly variable. By contrast, during glutamatergic and nicotinic cholinergic blockade, the frequency of occurrence of spontaneous episodes initially slowed and then recovered to stabilize near the predrug level of activity. Whole-cell recordings made from ventral spinal neurons revealed that this recovery was accompanied by an increase in the amplitude of spontaneously occurring synaptic events. We also measured changes in the apparent equilibrium potential of the rhythmic, synaptic drive of ventral spinal neurons using voltage or discontinuous current clamp. After excitatory blockade, the apparent equilibrium potential of the rhythmic synaptic drive shifted approximately 10 mV more negative to approximately -30 mV. In the presence of bicuculline, the apparent equilibrium potential of the synaptic drive shifted toward the glutamate equilibrium potential. Considered with other evidence, these findings suggest that spontaneous rhythmic output is a general property of developing spinal networks, and that GABA and glycinergic networks alter their function to compensate for the blockade of excitatory transmission.

Mechanisms of spontaneous activity in the developing spinal cord and their relevance to locomotion.

The isolated lumbosacral cord of the chick embryo generates spontaneous episodes of rhythmic activity. Muscle nerve recordings show that the discharge of sartorius (flexor) and femorotibialis (extensor) motoneurons alternates even though the motoneurons are depolarized simultaneously during each cycle. The alternation occurs because sartorius motoneuron firing is shunted or voltage-clamped by its synaptic drive at the time of peak femorotibialis discharge. Ablation experiments have identified a region dorsomedial to the lateral motor column that may be required for the alternation of sartorius and femorotibialis motoneurons. This region overlaps the location of interneurons activated by ventral root stimulation. Wholecell recordings from interneurons receiving short latency ventral root input indicate that they fire at an appropriate time to contribute to the cyclical pause in firing of sartorius motoneurons. Spontaneous activity was modeled by the interaction of three variables: network activity and two activity-dependent forms of network depression. A "slow" depression which regulates the occurrence of episodes and a "fast" depression that controls cycling during an episode. The model successfully predicts several aspects of spinal network behavior including spontaneous rhythmic activity and the recovery of network activity following blockade of excitatory synaptic transmission.

Population behavior and self-organization in the genesis of spontaneous rhythmic activity by developing spinal networks.

During development spinal networks generate recurring episodes of rhythmic bursting that can be recorded from motoneurons and interneurons. Optical imaging has identified a set of propriospinal interneurons that may be important in the production of this activity. These neurons are rhythmically active, are recurrently interconnected and have powerful projections to motoneurons. The excitability of this propriospinal network is depressed by activity and recovers in the interval between episodes. These and other observations have been formulated into a qualitative model in which population behavior and self-organization are responsible for the spontaneous activity generated by developing spinal networks.

Pharmacological characterization of the rhythmic synaptic drive onto lumbosacral motoneurons in the chick embryo spinal cord.

The isolated spinal cord of the chick embryo generates episodes of rhythmic bursting in which sartorius (hip flexor) and femorotibialis (knee extensor) motoneurons exhibit characteristic patterns of activity. At the beginning of each cycle both sets of motoneurons discharge synchronously. Following this brief synchronous activation sartorius motoneurons stop firing at the time of peak femorotibialis activity, producing a period of alternation between the two sets of motoneurons. Intracellular recording from motoneurons has suggested that the pause is mediated by a synaptically induced shunt conductance. However, the pharmacological basis for this shunt and the nature of the excitatory drive to motoneurons is unknown. To address these questions we have investigated the pharmacology of the rhythmic, synaptic drive to lumbosacral motoneurons using local and bath application of several excitatory and inhibitory antagonists, and documenting their effects on motor output in E10-E12 chick embryos. Local application of bicuculline or picrotoxin over sartorius motoneurons abolished the pause in firing recorded from the sartorius muscle nerve. As a consequence, the pattern of sartorius and femorotibialis activity was similar and the motoneurons were coactive. The pause in sartorius firing was shortened following local application of the glycine antagonist strychnine the nicotinic, cholinergic antagonists mecamylamine, and dihydro-beta-erythroidine and several excitatory amino acid antagonists. Application of the GABA uptake inhibitor nipecotic acid depressed the slow potentials and discharge recorded from the sartorius muscle nerve. These findings suggest that the pause is determined primarily by synaptic inputs acting at motoneuron GABAA receptors with contributions from glycinergic, cholinergic, and glutamatergic inputs. The actions of locally applied GABA onto spinal neurons are consistent with these findings because the neurotransmitter depolarizes spinal neurons and reduces their input resistance. Local application of bicuculline, but not strychnine, onto segments containing femorotibialis motoneurons altered the amplitude and duration of femorotibialis discharge and changed the profile of the slow potentials recorded from the muscle nerve. This finding implicates GABAergic inputs in the regulation of femorotibialis discharge. The pause in sartorius firing was still present and a pause in firing appeared in each cycle of femorotibialis discharge following bath application of bicuculline or strychnine. The pause in both sets of motoneurons could be abolished by local application of the NMDA receptor antagonist AP-5 onto the motoneurons, but not by local application of bicuculline. This action of AP-5 was in contrast to its activity in normal Tyrode's solution where it shortened the pause slightly.(ABSTRACT TRUNCATED AT 400 WORDS)

[The action of L-DOPA on the spontaneous activity generated by the isolated spinal cord of 16- to 20-day-old chick embryos]. / Deistvie L-DOFA na spontannuiu aktivnost', generiruemuiu izolirovannym spinnym mozgom 16--20-dnevnykh kurinykh émbrionov.

Effect of bath application of L-DOPA, dopamine (DA), noradrenaline (NA) on the spontaneous activity--cyclical oscillations of electrotonic potentials in dorsal and ventral roots (DR and VR, respectively) generated by isolated spinal cord of 16-20-day-old chick embryo was studied. L-DOPA in low concentration (30-150 mumol/l) activated spinal generator: suprathreshold rhythms appeared (i.e., spike activity in DR and VR). High concentration (200-1000 mumol/l) of L-DOPA depressed generator activity, but in the course of washing of the spinal cord spontaneous activity was enhanced and suprathreshold rhythms appeared. DA and NA (50 mumol/l) did not influence the activity but in concentration 100 mumol/l they depressed spontaneous activity in DR and VR. Bath application of 2-amino-5-phosphonovaleric acid (20 mumol/l) blocked activity induced by small concentration of L-DOPA. Activity generated by the neuronal network of the isolated dorsal horn was intensified after bath-application of L-DOPA in small concentration. Rhythmic activity in the isolated ventral horn did not appear after bath-application of L-DOPA. It is concluded that excitatory action of L-DOPA on the neuronal network of dorsal horn chick embryo was direct (i.e., did not depend on catecholamines).

[Effect of N-methyl-D-aspartate on the spontaneous activity generated by isolated spinal cord of 16 to 20-day-old chick embryos]. / Vliianie N-metil-D-aspartata na spontannuiu aktivnost' generiruemuiu izolirovannym spinnym mozgom 16-20-dnevnykh kurinykh émbrionov.

Bath-application of N-methyl-D-aspartate (NMDA) and antagonist of NMDA-receptors 2-amino-5-phosphonovaleric acid (2-APV) has been studied from the standpoint of its effect on the spontaneous activity in dorsal and ventral roots (DR and VR, respectively) generated by isolated spinal cord of 16-20-day-old chick embryo. Spontaneous activity consisted of synchronous oscillations of electrotonic potentials in DR and VR. Bath-application of NMDA (2-25 mumol/l) increased amplitude of the electrotonic potentials, induced spice discharges in DR and VR, a tonic component of electrotonic potentials. Bath-application of 2-APV (20 mumol/l) depressed spontaneous and NMDA-induced activity. The neuronal network of isolated dorsal horn (after splitting of the spinal cord) retained ability to generate spontaneous activity in DR which was intensified after bath-application of NMDA. No rhythmic activity appeared in the ventral part of the cord. The location of NMDA-sensitive neuronal network generating rhythmic (motor) activity in the spinal cord is discussed.

[The modification of the cyclic activity of the isolated spinal cord in l6- to 20-day-old chick embryos through changes in the Ca2+ and Mg2+ concentrations]. / Modifikatsiia tsiklicheskoi aktivnosti izolirovannogo spinnogo mozga 16--20-dnevnykh kurinykh émbrionov pri izmeneniiakh kontsentratsii Ca2+ i Mg2+.

Spontaneous rhythmic activity which appeared as synchronous oscillations of electrotonic potentials in dorsal and ventral roots (DR and VR, respectively) of isolated spinal cord sections of 16-20-day-old chick embryos was completely blocked with a rise of Mg2+ concentration from the control level (1.3 mmol/l) to 5 mmol/l. The similar effect was observed when Ca2+ concentration fell from 2.6 to 1 mmol/l, but in that case spike discharges could appear in VR. Moreover, during disappearance of the above spontaneous activity (after changes in Mg2+ or Ca2+ concentration) rhythmic activity of other type was observed in 6 from 8 experiments. That rhythmic activity consisted of oscillations of electrotonic VR potentials (amplitude to 200 microV and duration to 400 ms) which arose irrespective of oscillations of DR potentials. Maximum frequency of that activity was 0.4/s. Possible functional role of the found types of rhythmic activity is discussed.

[Role of various regions of the spinal cord in generating spontaneous motor activity in the chick embryo]. / Rol' razlichnykh oblastei spinnogo mozga v generatsii spontannoi dvigatel'noi aktivnosti kurinogo émbriona.

Experiments on the isolated spinal cord of 16-20 day old chick embryos using brain sections have shown that rhythm of the spontaneous motility was generated by the dorsal horn network.

[Rhythmic electrical activity of the isolated spinal cord of the chick embryo]. / Ritmicheskaia élektricheskaia aktivnost' izolirovannogo spinnogo mozga kurinogo émbriona.

Experiments on the isolated spinal cord have shown that even in early chicken embryogenesis, when first signs of the locomotor activity appeared, modulation of the presynaptic inhibition by the locomotor generator was present. Only few spinal cord segments are able to generate the locomotor rhythm.