N-Methyl-D-Aspartate Receptors at Parallel Fiber Synapses in the Dorsal Cochlear Nucleus. Manis, Paul B. and Scott C. Molitor. Departments of Otolaryngology-Head and Neck Surgery, Biomedical Engineering, and Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, MD 21205.
APStracts 3:0086N, 1996.
1. N-methyl-D-aspartate (NMDA) binding and NMDA-receptor immunolocalization experiments have revealed an enhanced expression of these receptors in the outer two layers of the dorsal cochlear nucleus (DCN). The distribution of the receptors is congruent with the distribution of synapses produced by the granule cell-parallel fiber system. In order to determine the functional distribution and contribution of NMDA receptors at parallel fiber synapses, synaptic responses to parallel fiber stimulation were studied in in vitro brain slice preparations of the guinea pig and rat dorsal cochlear nucleus. 2. The field potential response to parallel fiber stimulation in guinea pigs includes three postsynaptic components. The short latency components (the P3 2 and N2 2 ) are blocked by general excitatory receptor antagonists, including the non-NMDA receptor blockers 6,7-dinitroquinoxaline-2,3-dione (DNQX) and 6- cyano-7-nitroquinoxaline-2,3-dione (CNQX), but are insensitive to NMDA receptor antagonists. 3. A slower component (P4 2 ) is revealed when the slices are washed with a low magnesium solution to eliminate the magnesium block of currents through NMDA receptors. This slow component is reduced by D- or DL-2-amino-5-phosphonovaleric acid (D-APV, DL-APV) and 3-[(+/-)-2- carboxypiperazine-4-yl] propyl-1-phosphonate (CPP), but is not blocked by DNQX or CNQX. Eliminating the voltage dependence of the NMDA receptors also results in a complex oscillatory response in some slices. This response exhibits the same pharmacological sensitivity as the slow potential. The pharmacologic sensitivity to NMDA receptor antagonists suggest that the slow component (P4 2 ) and the associated oscillatory response are mediated through activation of NMDA receptors. 4. Current-source density analysis of the parallel fiber- evoked field potentials was carried out in order to determine the relative spatial distributions of the fast and slow synaptic currents. Both synaptic components were associated with a superficial current sink and a deeper current source, localized within the superficial 250 [mu]m of the nucleus. The slow (APV-sensitive) current was slightly shifted in depth relative to the fast (DNQX-sensitive) current in 3 of 5 slices, with the maximum current sink and source occurring approximately 16 [mu]m further from the surface of the DCN. These data suggest that either the NMDA receptors are either not present at all of the synapses that generate the fast non-NMDA currents, or that postsynaptic cells with different dendritic distributions have different densities of NMDA receptors. 5. The types of cells in layers 1 and 2 exhibiting NMDA receptor-mediated synaptic potentials were investigated. Intracellular recordings with sharp electrodes in guinea pig slices showed that eliminating the voltage dependence of the NMDA receptors in low magnesium revealed a slow EPSP in both simple and complex spiking cells. The late phase of the EPSP could be reduced by APV in both cell types. These results could be explained by NMDA receptors on the postsynaptic cells or by NMDA receptors on excitatory interneurons. Attempts to demonstrate an appropriate voltage dependence of the parallel fiber synaptic response in normal magnesium medium under current-clamp were confounded by the intrinsic voltage-dependent conductances of the cells. 6. In order to determine whether NMDA receptors were present on postsynaptic cells, the direct sensitivity of DCN cells to NMDA application was examined during intracellular recording. Both simple spiking and complex spiking cells responded to NMDA with depolarization. The response to NMDA persisted when non-NMDA receptors were blocked with CNQX or DNQX. However in all cells tested, the response to NMDA was blocked by APV. These experiments further support the postsynaptic localization of NMDA receptors on both simple and complex spiking cells. 7. The voltage dependence of the EPSC produced by parallel fiber stimulation was measured in thin slices from 12-16 day old rat pups. The EPSC produced by parallel fiber stimulation showed an APV-sensitive slow component that activated near -50 mV and reversed at 0 mV with Cs-containing electrodes. The non-NMDA receptor-dependent component had a linear current-voltage relationship and also reversed near 0 mV. These effects were seen for EPSCs in both morphologically identified pyramidal and cartwheel cells. The voltage dependence of the response to glutamate was also measured under voltage clamp in acutely isolated cells from adult guinea pigs. A slow voltage-dependent and APV-sensitive component was identified in these experiments; the voltage dependence was similar to that seen in cells in rat DCN. 8. The results from these experiments suggest that ionotropic effects of the parallel fibers in the dorsal cochlear nucleus are mediated through both non-NMDA and NMDA receptors, and that these receptors are present on both simple spiking (pyramidal) and complex spiking (cartwheel) cells in layers 1 and 2 of the nucleus. Although cartwheel cells share many biochemical, anatomical, and physiological features with cerebellar Purkinje cells, the presence of NMDA receptors on cartwheel cells further distinguishes them from Purkinje cells.

Received 4 October 1995; accepted in final form 4 April 1996.
APS Manuscript Number J664-5.
Article publication pending J. Neurophysiol.
ISSN 1080-4757 Copyright 1996 The American Physiological Society.
Published in APStracts on 19 May 96