Role of excitatory amino acids in mediating burst discharge of red nucleus neurons in the in vitro turtle brain stem-cerebellum

Bursts of discharge have been recorded in the red nucleus in several species and are thought to represent the expression of motor commands. A cerebellorubral circuit comprised of recurrent connections among the cerebellum, red nucleus, and reticular formation was postulated to function as a positive feedback loop that generates these motor commands and transmits them to the spinal cord via the rubrospinal pathway. We have used an in vitro preparation from the turtle that leaves the circuitry connecting the cerebellum, brain stem, and spinal cord intact to study the role of excitatory amino acid neurotransmitters and recurrent excitation in mediating the generation of burst discharges in the red nucleus. Burst discharges were recorded extracellularly from single cells in the red nucleus in response to single pulse or brief train stimulation of the contralateral spinal cord or brief train stimuli applied to the ipsilateral cerebellar cortex. The firing characteristics and pharmacologic sensitivities of the bursts were independent of the type of stimulus used. The bursts had long durations ranging from 2 to 17 s and showed spike frequency adaptation. Transection of the cerebellar peduncle, which eliminates inhibition impinging onto the cerebellorubral circuit, greatly enhanced the spontaneous activity and burst discharges recorded in the contralateral red nucleus. Furthermore, bath application of a solution containing elevated levels of calcium and magnesium blocked the expression of burst discharges even though synaptic activation of the neurons was not blocked. The possibility that excitatory amino acid receptors mediate burst responses in the red nucleus was investigated in light of the antagonistic effects of elevated magnesium ions on bursting. Bath application of 100 μm DL-2-amino-5-phosphonovaleric acid (APV), a specific N-methyl-D-aspartate (NMDA) receptor antagonist; [10 μM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX)], a specific non-NMDA receptor antagonist; or 100 μM, DL-2-amino-4-phosphonobutyric acid (AP4), an agonist of a fourth class of excitatory amino acid receptor, blocked burst activity in the red nucleus. With a multibarreled pipette for simultaneous ejection of drug and recording, iontophoresis of APV or CNQX into the red nucleus blocked bursting whereas AP4 failed to show a significant effect. These data suggest that red nucleus neurons have both NMDA and non-NMDA receptors. The site of action of the AP4-sensitive receptor appears to be elsewhere in the cerebellorubral circuit. Iontophoretic application of excitatory amino acid receptor agonists NMDA and quisqualate (Q) induced excitation of red nucleus neurons. Excitation following iontophoresis of NMDA was blocked by bath application of APV but not CNQX. Similarly, excitation after ejection of Q was blocked by CNQX, but not APV, showing the effects of these drugs in the turtle red nucleus are selective. Iontophoretic or bath application of 100 μM NMDA did not induce bursting pacemaker activity in red nucleus neurons. In conclusion, excitatory amino acids appear to mediate red nucleus burst discharge. We postulate that non-NMDA receptors may be particularly important in the initiation of bursting. An initial depolarization mediated in this manner might then lead to the expression of known NMDA actions that serve to sustain the burst. Activation of negative slope conductance and slow excitatory postsynaptic potentials might produce sustained activity in individual neurons and in the recurrent loops comprising the cerebellorubral circuit. It is further postulated that Purkinje cell inhibition regulates activity in the cerebellorubral circuit, thus shaping motor programs destined for spinal motor centers.