Olfactory bulb neurons were dissociated from neonatal rats and plated at low density on a confluent layer of olfactory bulb astrocytes. Intracellular stimulation of presumptive mitral/tufted (M/T) cells evoked monosynaptic excitatory postsynaptic potentials (EPSPs) in adjacent neurons. Whole-cell recording techniques and a flow-pipe drug delivery system were used to compare EPSPs with voltage-clamp recordings of currents evoked by excitatory amino acids (EAA) including N-acetylaspartylglutamate (NAAG), a putative mitral cell transmitter. Cultured olfactory bulb neurons were morphologically and physiologically distinct. Large pyramidal-shaped neurons were present, which were NAAG immunoreactive; stimulation of these neurons invariably evoked EPSPs, suggesting that they were M/T cells. The majority of small bipolar neurons were glutamic acid decarboxylase (GAD) immunoreactive consistent with granule or periglomerular γ-aminobutyric acid (GABA)ergic interneurons. Monosynaptic EPSPs between M/T cells could be separated into fast and slow components by the use of EAA receptor antagonists. A fast component with a time-to-peak of 7.7 ± 1.0 (SE) ms and half-width of 31.8 ± 7.4 ms was blocked by the non-NMDA receptor antagonist 6-cyano-2,3-dihydroxy-7-nitro-quinoxaline (CNQX, 2.5 μM). The slow component (time-to-peak = 41.4 ± 7.2 ms; half-width = 218.9 ± 40.4 ms) was blocked by the N-methyl-D-aspartate (NMDA) receptor antagonist DL-2-amino-5-phosphonovaleric acid (AP5, 100 μM). Under voltage clamp, flow-pipe applications of NAAG (10 - 1,000 μM) evoked inward currents at a holding potential of -60 mV in Mg-free solutions. In the presence of AP5 (100 μM), the NAAG-evoked current was reduced to 15 ± 3% of control; in nominally glycine-free solutions the current was reduced to 10 ± 2% of control. This is consistent with selective activation of NMDA receptors. Concentration-response curves fitted with the logistic equation yielded an EC50 for NAAG of 666 μM, compared to 29 μM for NMDA. 2-amino-4-phosphonobutyrate (L-AP4, 50 μM) reduced the peak amplitude of monosynaptic EPSPs (35 ± 1% of control) but did not antagonize responses evoked by application of NAAG. The action of L-AP4 could be attributed to presynaptic inhibition because the ratio of the test EPSP to the conditioning EPSP increased in response to paired-pulse stimulation (138 ± 6%, AP4/control. Reductions in extracellular Ca from 2.0 to 0.75 mM had similar effects (144 ± 6%, low Ca/high Ca). The contribution of non-NMDA and NMDA receptors to the monosynaptic EPSP suggests that the transmitter released by cultured M/T cells is an EAA that acts as a mixed agonist. this is most consistent with L-glutamate rather than aspartate or NAAG as the M/T cell transmitter. We suggest that the slow time course of the NMDA-receptor-mediated EPSP has led to an underestimation of the role of these receptors in modulating excitability in the olfactory system.
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