Activation of BC mGluRs
We made current-clamp recordings from BCs and bath-applied the group I mGluR agonist DHPG (). DHPG depolarized BCs from −61.1 ± 0.1 mV to −56.0 ± 0.2 mV (39 cells, P < 0.001, ). This depolarization was unaffected by TTX, suggesting a direct effect on BCs (6 cells, ). Similar depolarization occurred in perforated-patch experiments (7 cells, ), indicating our whole-cell recordings did not disrupt the intracellular signaling environment. Similar effects were also found in P42 animals, suggesting that mGluRs play a role in mature auditory function (3 cells, ).
We evaluated the contributions of different group I mGluR isoforms by applying specific blockers 3–5 min before DHPG. Vrest was corrected to −61 mV as needed during this period, but not thereafter. Pre-application of the mGluR5-specific blocker MPEP significantly reduced the depolarization (P < 0.001, unpaired t-test, 5 MPEP vs. 39 control cells, ). The mGluR1-specific blocker CPCCOEt also decreased the depolarization (P < 0.005, unpaired t-test, 5 CPCCOEt vs. 39 control cells, ). Co-application of MPEP and CPCCOEt completely blocked the depolarization by DHPG (P < 0.001, unpaired t-test, 4 MPEP+CPCCOEt vs. 39 control cells, ). Thus, DHPG depolarizes BCs primarily through mGluR5, with a smaller contribution through mGluR1.
We also applied MPEP and CPCCOEt in the absence of DHPG, and observed a small but significant hyperpolarization (7 cells, P < 0.002, ). Furthermore, application of the glutamate reuptake inhibitor TBOA (in the presence of CPP and NBQX, which are NMDA- and AMPA-receptor antagonists, respectively) significantly depolarized the BC (9 cells, P < 0.001, ). TBOA-induced depolarization was blocked in MPEP+CPCCOEt (3 cells, P > 0.1, ). These results indicate that mGluRs on BCs are sensitive to fluctuations in ambient glutamate concentration.
At the calyx of Held, mGluR activation by DHPG drives release of endocannabinoids, which reduce presynaptic neurotransmitter release (Kushmerick et al., 2004
). We tested this possibility at the endbulb by making voltage-clamp recordings from BCs and stimulating presynaptic AN fibers with pairs of pulses at different intervals. Application of DHPG had no significant effect on the amplitude or kinetics of the first EPSC (EPSC1
) () or the second EPSC in a pair (P
> 0.2, 6 cells, ). These results indicate that mGluR activation does not affect the probability of release at the endbulb.
No presynaptic effect of DHPG at the endbulb of Held
We also confirmed that other aspects of synaptic transmission were unaffected by mGluR activation by examining mEPSCs in the presence of TTX (). Neither the frequency (P > 0.2, ) nor the amplitude (P > 0.4, 12 cells, ) of mEPSCs changed significantly with DHPG application. This indicates that mGluR activation had no effect on postsynaptic AMPA receptors nor on the presynaptic release machinery.
Effect of mGluR activation on postsynaptic firing
We next examined how mGluRs influenced spike generation in BCs. In an example experiment, a 0.2 nA depolarizing current-pulse triggered a spike in the presence of DHPG but not in control conditions (, left). A greater current-pulse (0.6 nA) led to spiking in both cases, but DHPG application led to an additional spike (, right). On average, mGluR activation increased the number of spikes and decreased the latency of the first spike (33 cells, P < 0.002, ) without significantly affecting the AP peak or threshold voltage (P > 0.2, ). These effects could have resulted simply from depolarization bringing the BC closer to threshold. We used a small holding current to depolarize BCs to a Vrest of −56 mV in the absence of DHPG. Subsequent application of current pulses under these conditions also led to increased spiking, similar to that in DHPG (8 cells, ). This indicates that the principal effects of mGluR activation on spiking are mediated through depolarization.
Effects of group I mGluR activation on spike generation in BCs. Filled symbols indicate values significantly different from control conditions (P < 0.05)
We next studied how DHPG affected BC spiking during AN activity (). We activated AN fibers using trains of 20 stimuli at physiological firing rates (100, 200 and 333 Hz). In control conditions, BCs fired reliably early in 100 Hz trains, but became less reliable at later pulses (, left), presumably because of synaptic depression. DHPG application increased the probability of spiking for those later pulses (, middle). We considered the effects of mGluR activation on spike probability and timing for the first pulse, as well as for pulses 11 to 20 where the EPSC amplitudes are near steady-state levels of depression. We quantified spike latency from each stimulus to the immediately following spike, and spike jitter as standard deviation in the latency. In DHPG, the spike probability increased for the steady-state part of the train, and spike latency and jitter both decreased (7 cells, P < 0.05, ). Repolarizing the BC to −61 mV using current injection, in the continued presence of DHPG, reversed the changes in firing probability, latency and jitter (9 cells, ). Furthermore, depolarizing the BC to −56 mV in the absence of DHPG had nearly identical effects (8 cells, P < 0.05, ), suggesting that the increase in firing could be accounted for by simple depolarization.
We also examined how the endogenous, tonic activation of mGluRs influenced BC firing. Application of MPEP+CPCCOEt decreased the spike probability for 200 and 333 Hz trains, while the latency of the first pulse and of 100 Hz trains increased significantly (7 cells, P < 0.05, ). There was no significant change in jitter. Thus, the tonic mGluR-dependent depolarization had a measurable impact on the firing properties of BCs.
We wanted to understand how mGluR activation could interact with the larger modulatory environment of the AVCN, particularly the inhibitory modulator GABA. Application of 50 µM GABA blocked EPSC1
by over 75% () and changed short-term plasticity from depressing to facilitating (), reflecting a drop in the presynaptic release probability (Chanda and Xu-Friedman, 2010a
). Further application of DHPG had no additional effect (), indicating that the two modulators have no synergistic presynaptic interaction. Application of CGP55845 restored the EPSC to control levels (), confirming that GABA acted through presynaptic GABAB
Interaction between GABABR- and mGluR-mediated modulation
We examined the consequences of these effects on the EPSC using current-clamp recordings. Single AN stimuli caused reliable spiking (, top traces), but after applying GABA, many EPSPs failed to elicit spikes (middle traces in , open red symbols in ). This did not result from postsynaptic effects of GABA as there were no significant changes in Vrest (, middle traces, −60.6 ± 0.2 mV in control vs. −60.9 ± 0.2 mV in GABA, P > 0.05, 6 cells), action potential threshold (−42.9 ± 0.9 mV in control vs. −43.9 ± 1.2 mV in GABA, P > 0.05, 4 cells) or input resistance (40.9 ± 2.9 MΩ in control vs. 42.3 ± 6.4 MΩ in GABA at −61 mV, P > 0.3, 4 cells). Furthermore, GABA had similar effects on BC firing even in the presence of GABAA-receptor antagonist bicuculline (data not shown). Thus, the drop in spiking was likely caused by the decrease in EPSP amplitude following GABABR activation.
When we next added DHPG, firing was restored to a considerable extent (, bottom traces). In six experiments, GABA application reduced the firing probability throughout the train (, P < 0.003), and mGluR activation significantly restored it (P < 0.005, , open blue symbols). We confirmed that GABA activated GABABRs using CGP55845: the firing probability in DHPG alone was the same as in DHPG+GABA+CGP (Pspike = 1 ± 0 in both conditions for pulse 1, and 0.91 ± 0.07 vs. 0.90 ± 0.08 for pulses 11–20, P > 0.5, 3 cells). Similarly, in five experiments, spiking was strongly blocked by the GABABR-specific agonist baclofen (P < 0.001), and subsequent DHPG application caused significant recovery (P < 0.02, , closed symbols).