N-Methyl-D-Aspartate Receptors at Parallel Fiber Synapses in the Dorsal
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.
SUMMARY AND CONCLUSIONS
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