Olfactory nerve-evoked, metabotropic glutamate receptor-mediated synaptic responses in rat olfactory bulb mitral cells

Abstract

The group I metabotropic glutamate receptor (mGluR) subtype, mGluR1, is highly expressed on the apical dendrites of olfactory bulb mitral cells and thus may be activated by glutamate released from olfactory nerve (ON) terminals. Previous studies have shown that mGluR1 agonists directly excite mitral cells. In the present study, we investigated the involvement of mGluR1 in ON-evoked responses in mitral cells in rat olfactory bulb slices using patch-clamp electrophysiology. In voltage-clamp recordings, the average EPSC evoked by single ON shocks or brief trains of ON stimulation (six pulses at 50 Hz) in normal physiological conditions were not significantly affected by the nonselective mGluR antagonist LY341495 (50-100 microM) or the mGluR1-specific antagonist LY367385 (100 microM); ON-evoked responses were attenuated, however, in a subset (36%) of cells. In the presence of blockers of ionotropic glutamate and GABA receptors, application of the glutamate uptake inhibitors THA (300 microM) and TBOA (100 microM) revealed large-amplitude, long-duration responses to ON stimulation, whereas responses elicited by antidromic activation of mitral/tufted cells were unaffected. Magnitudes of the ON-evoked responses elicited in the presence of THA-TBOA were dependent on stimulation intensity and frequency, and were maximal during high-frequency (50-Hz) bursts of ON spikes, which occur during odor stimulation. ON-evoked responses elicited in the presence of THA-TBOA were significantly reduced or completely blocked by LY341495 or LY367385 (100 microM). These results demonstrate that glutamate transporters tightly regulate access of synaptically evoked glutamate from ON terminals to postsynaptic mGluR1s on mitral cell apical dendrites. Taken together with other findings, the present results suggest that mGluR1s may not play a major role in phasic responses to ON input, but instead may play an important role in shaping slow oscillatory activity in mitral cells and/or activity-dependent regulation of plasticity at ON-mitral cell synapses.

Fig. 1

Metabotropic glutamate receptor (mGluR) antagonists have modest effects on olfactory nerve (ON)–evoked excitatory postsynaptic currents (EPSCs) in mitral cells in normal media. A, top: EPSCs evoked by 50 or 300 μA ON stimulation; top row shows responses to single-pulse stimulation, whereas the bottom row shows responses elicited by train of 6 ON pulses at 50 Hz. Each panel shows responses in: control artificial cerebrospinal fluid (ACSF, a), the presence of the nonselective mGluR antagonist LY341495 (100 μM, b), and the presence of LY341495 and 10 μM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), 100 μM (±)-2-amino-5-phosphopentanoic acid (APV), and 10 μM gabazine (c). Traces in all panels are averages of 5 responses to ON stimulation from the same mitral cell. Bottom bar graph shows the integral (see METHODS) of the ON-evoked EPSCs from 11 mitral cells (small horizontal lines plot the level of reduction by LY341495 for the 11 individual cells) for single-pulse stimulation and 5 mitral cells for 50-Hz stimulation. Note that LY341495 elicited a small but nonsignificant reduction (P > 0.05) in the mean size of the evoked EPSCs, whereas ionotropic glutamate and γ-aminobutyric acid (GABA) receptor antagonists eliminated the evoked EPSCs. *P < 0.001 vs. EPSC in control ACSF or in the presence of LY341495; Newman–Keul post hoc comparisons following a one-way ANOVA for drug treatment (P < 0.001). B: bar graphs of group data from similar experiments investigating the effect of the selective mGluR1 antagonist, LY367385 (100 μM), on ON-evoked EPSCs elicited by single-pulse or 50-Hz ON stimulation (n = 5 mitral cells). LY367385 did not affect the integral of the evoked EPSCs (P > 0.05). *P < 0.002 vs. EPSC in control ACSF or in the presence of LY367385; Newman–Keul post hoc comparisons following a one-way ANOVA for drug treatment (P < 0.001).

Fig. 2

Inhibition of glutamate uptake reveals significant mGluR-mediated ON-evoked excitatory postsynaptic potentials (EPSPs) in mitral cells. A: ON-evoked EPSPs (a) were not significantly affected by the mGluR antagonist LY341495 (b) but were blocked by the fast synaptic blockers: CNQX + APV + gabazine (c). Right bar graphs: group data from 5 mitral cells. *P < 0.001; Newman–Keul post hoc comparisons following a one-way ANOVA for drug treatment (P < 0.001). B: ON-evoked EPSPs were almost completely blocked by the fast synaptic blockers (a). Application of the glutamate uptake blockers D-threo-β-benzyl-oxyaspartate (TBOA) + D-threo-b-hydroxyaspartic acid (THA) (b) significantly enhanced the evoked EPSP, an effect that was reversed by the mGluR antagonist LY341495 (c). Right bar graph: group data from 5 mitral cells in each condition. *P < 0.001; Newman–Keul post hoc comparisons following a one-way ANOVA for drug treatment (P < 0.001).

Fig. 3

ON-evoked EPSCs elicited in the presence of glutamate uptake inhibitors are dependent on stimulation intensity and frequency. A: 4 top panels show plots of the peak amplitude of ON-evoked EPSCs as a function of ON stimulation intensity (10, 30, 40, 50, 60, 100, 300, 600, and 1,000 μA); each panel shows data for a different ON stimulation frequency. Insets (in each graph): traces of ON-evoked EPSCs in different pharmacological conditions; traces are averages of 5 ON-evoked EPSCs. Colored lines correspond to the pharmacological conditions indicated at the top; note that control data are not plotted on the line graphs. TBOA–THA significantly increases the ON-evoked EPSCs at a threshold intensity of 60–100 μA for all frequencies. Residual responses remaining in the presence of LY341495 and CNQX, APV, and gabazine were abolished by low-Ca²⁺ ACSF, as shown for the single-pulse and 200-Hz stimulation frequencies. *P < 0.05, #P < 0.001 vs. CNQX, APV, and gabazine. B: bar graph plots of the integral of ON-evoked EPSCs for 2 representative stimulation intensities. Note that the enhancement of the ON-evoked EPSCs is reversed by LY341495. *P < 0.001, #P < 0.05; Newman–Keul post hoc comparisons following a one-way ANOVA for drug treatment (P < 0.001).

Fig. 4

Enhancement of ON-evoked EPSCs by glutamate uptake inhibitors is reversed by the mGluR1 selective antagonist LY367385. Bar graph plots of the integral of ON-evoked EPSCs elicited by 50 or 300 μA intensity, single-pulse or 50-Hz stimulation (n = 5 mitral cells in each condition). Application of TBOA (100 μM) and THA (300 μM) enhanced ON-evoked EPSCs in the presence of CNQX (10 μM), APV (100 μM), and gabazine (10 μM); this effect reached statistical significance for the 300-μA-intensity stimulation. LY367385 reversed the TBOA–THA enhancement of evoked EPSCs. *P < 0.05; Newman–Keul post hoc comparisons following a one-way ANOVA for drug treatment (P < 0.001).

Fig. 5

Blockade of presynaptic inhibition does not alter ON-evoked EPSCs elicited in the presence of ionotropic glutamate and GABA receptor antagonists. A: bar graph shows the integral of ON-evoked EPSCs in mitral cells in the 4 pharmacological conditions indicated at the top of the figure. In the presence of CNQX, APV, and gabazine, additional application of the D₂ dopamine receptor antagonist sulpiride (100 μM) and the GABA_B receptor antagonist CGP55485 (10 μM) had no substantial effect on EPSCs elicited by 50 or 300 μA intensity, single-pulse or 200-Hz ON stimulation (n = 3 mitral cells in each condition). Subsequent addition of TBOA (100 μM) and THA (300 μM) enhanced the ON-evoked EPSCs, an effect reversed by application of LY341495. *P < 0.05; Newman–Keul post hoc comparisons following a one-way ANOVA for drug treatment (P < 0.001). B: bar graphs showing that the ON-evoked EPSC in the presence of TBOA–THA + CGP-sulpiride + CNQX–APV–gabazine (data from A above) are larger than those in TBOA–THA + CNQX–APV–gabazine (data from Fig. 3). *P < 0.01, unpaired t-test.

Fig. 6

Inhibition of glutamate uptake does not enhance EPSCs evoked by antidromic activation of mitral/tufted cells in the presence of ionotropic glutamate and GABA receptor antagonists. Line graph showing the peak amplitude of EPSCs in mitral cells evoked by stimulation of the lateral olfactory tract (LOT) at different intensities (10, 30, 40, 50, 60, 100, 300, 600, and 1,000 μA) with single pulses (left) or with 6 pulses at 50 Hz (right); n = 9 mitral cells in all conditions. Individual lines correspond to the pharmacological conditions indicated at the top left of the figure. Typical LOT-evoked EPSCs (stimulation intensity: 300 μA) are shown in insets in both panels and correspond to conditions indicated at top left in each panel by letters a–c. LOT-evoked EPSCs were largely attenuated by application of CNQX (10 μM), APV (100 μM), and gabazine. Subsequent application of TBOA (100 μM) and THA (300 μM) had no significant effect on the EPSCs, nor did further application of LY341495. LOT-evoked responses partially recovered to baseline values within 10–20 min after washout with normal ACSF (n = 5).