Blockade and Recovery of Spontaneous Rhythmic Activity after Application of Neurotransmitter Antagonists to Spinal Networks of the Chick Embryo

Abstract

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.

Emergence of the Pre-Bötzinger Respiratory Rhythm Generator in the Mouse Embryo

“…Generation of this LF activity in the embryonic mouse brainstem does not seem to rely on single anatomical structures or specific circuits, because it was observed in both facial and medullary slices. Spontaneous activity occurring at low rate and invading large territories has been described in various species and a wide variety of developing structures in the CNS such as the spinal cord (O'Donovan and Landmesser, 1987;Chub and O'Donovan, 1998;O'Donovan, 1999;Branchereau et al, 2000;Hanson and Landmesser, 2003;Ren and Greer, 2003;Yvert et al, 2004), the cortex (Garaschuk et al, 2000;Peinado, 2000), the hippocampus (Ben-Ari, 2001), the retina (Galli and Maffei, 1988;Katz and Shatz, 1996), and the brainstem (Fortin et al, 1994;Abadie et al, 2000;Gust et al, 2003). The LF activity observed in the present study probably corresponds to an immature activity.…”

GABA A transmission is a critical step in the process of triggering homeostatic increases in quantal amplitude

“…Embryonic movements are likely to be a reliable indicator of spinal SNA because electromyographic recordings in ovo during embryonic movements are remarkably similar to motoneuron recordings during in vitro SNA (30)(31)(32)(33). It is also likely that SNA that recovers after antagonist injections in the egg is similar to SNA before the antagonists were added because similar findings have been observed in the in vitro spinal cord preparation (22). Further, we did not observe differences in several features of the in ovo embryonic movements in treated and control embryos, including total movement duration, bout duration, bout frequency, or recovery of the movements.…”

“…Doubling the concentration of any drug or the addition of a second bolus at E9 produced no additional effect on limb movements (data not shown). This finding suggested that the drugs were still effective but that the spinal networks controlling limb movements had compensated for the loss of transmission (22). These experiments demonstrate that GABA A or AMPA/NMDA receptor activation can be significantly reduced in ovo from E8 to E10, whereas levels of SNA remained largely intact, apart from a transient reduction after drug administration.…”

“…It is likely that several mechanisms are recruited to recover and maintain activity levels and that the different mechanisms may have different time courses (13). After glutamatergic blockade, activity recovers in part because of a fast loading of intracellular chloride, which strengthens the unblocked GABAergic currents (22,44). After GABAergic block, there may be fast changes in the probability of release (45) and changes in cellular excitability (46,47).…”

“…isolated spinal cord in vitro (22). Regardless, we wanted to test the possibility that changes in quantal amplitude in the embryonic spinal cord might occur faster than in other systems and contribute to the recovery of embryonic movements.…”