Glycinergic "inhibition" mediates selective excitatory responses to combinations of sounds

Abstract

In the mustached bat's inferior colliculus (IC), combination-sensitive neurons display time-sensitive facilitatory interactions between inputs tuned to distinct spectral elements in sonar or social vocalizations. Here we compare roles of ionotropic receptors to glutamate (iGluRs), glycine (GlyRs), and GABA (GABA(A)Rs) in facilitatory combination-sensitive interactions. Facilitatory responses to 36 single IC neurons were recorded before, during, and after local application of antagonists to these receptors. The NMDA receptor antagonist CPP [(+/-)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid], alone (n = 14) or combined with AMPA receptor antagonist NBQX (n = 22), significantly reduced or eliminated responses to best frequency (BF) sounds across a broad range of sound levels, but did not eliminate combination-sensitive facilitation. In a subset of neurons, GABA(A)R blockers bicuculline or gabazine were applied in addition to iGluR blockers. GABA(A)R blockers did not "uncover" residual iGluR-mediated excitation, and only rarely eliminated facilitation. In nearly all neurons for which the GlyR antagonist strychnine was applied in addition to iGluR blockade (22 of 23 neurons, with or without GABA(A)R blockade), facilitatory interactions were eliminated. Thus, neither glutamate nor GABA neurotransmission are required for facilitatory combination-sensitive interactions in IC. Instead, facilitation may depend entirely on glycinergic inputs that are presumed to be inhibitory. We propose that glycinergic inputs tuned to two distinct spectral elements in vocal signals each activate postinhibitory rebound excitation. When rebound excitations from two spectral elements coincide, the neuron discharges. Excitation from glutamatergic inputs, tuned to the BF of the neuron, is superimposed onto this facilitatory interaction, presumably mediating responses to a broader range of acoustic signals.

Figure 1.

Blockade of NMDARs reduces excitation but does not eliminate facilitation in an IC neuron. Response to BF tone (A), low-frequency (LF) tone (B), and combinations of tones at different delays (C). PSTHs show spike timing (D). Test sequence: PRE (predrug), NMDAR Block (CPP, −40 nA, 18 min), +GlyR Block (+strychnine, +40 nA, 20 min), and REC (recovery, 21 min). Tone levels in C: 59.3 kHz at 15 dB SPL, 28.0 kHz at 69 dB SPL. Recovery not shown in (A–C) for clarity. In D, black rectangles above PSTHs indicate timing and duration of tones; in the bottom row of PSTHs, bottom rectangle represents lower-frequency tone. Numbers indicate spikes per 32 stimuli. In A and B, spike counts are based on a 200 ms window, whereas spike counts in C and D are based on a 75 ms window. Bin width of PSTHs, 1 ms.

Figure 2.

NMDARs contribute to excitation but not facilitation in combination-sensitive IC neurons. In A and B, facilitatory strength is based on the interaction index: numbers ≥0.09 indicate facilitation, whereas numbers less than or equal to −0.11 indicate inhibition (see Materials and Methods). A, NMDAR blockade rarely eliminated facilitation and had no effect on the average strength of facilitation (PRE mean, 0.39; NMDA Block mean, 0.39; t₍₁₃₎ = −0.48; p = 0.96, paired t test). B, Subsequent application of strychnine (+GlyR Block) to a subset of these neurons almost always eliminated facilitation and significantly reduced the average strength of facilitation (NMDA Block mean, 0.50; +GlyR Block mean, − 0.09, t₍₆₎ = 4.47; *p < 0.01, paired t test). C, Mean spike discharge for recorded population under different drug conditions for response to BF tone (BF spikes, open histograms) and response to combination of tones at best delay (BD Spikes; filled histograms). Sample sizes under different drug conditions apply to both BF and BD spikes. NMDAR Block significantly reduced BF spikes (PRE mean, 27.9; NMDAR Block mean, 9.1; t₍₁₃₎ = 2.95; p < 0.01, paired t test), whereas addition of strychnine significantly increased BF spikes (NMDA Block mean, 9.1; +GlyR Block mean, 43.0; t₍₆₎ = −2.87; p < 0.01, paired t test). BD spikes were significantly reduced by NMDAR Block (PRE mean, 64.2; NMDA Block mean, 43.9; t₍₁₃₎ = 2.11; p < 0.05) and also by addition of strychnine (NMDA Block mean, 43.9; +GlyR Block mean, 21.8; t₍₆₎ = 4.89; p < 0.01, paired t test).

Figure 3.

Combined blockade of iGluRs eliminated excitation but not facilitation in an IC neuron. Test sequence: PRE (filled circles), iGluR Block (CPP+NBQX, − 30 nA each, 17 min; open triangles), +GlyR Block (+30 nA, 7 min; light gray squares), and REC (31 min). Tone levels in C: 83.5 kHz at 54 dB SPL, 25.2 kHz at 59 dB SPL. Recovery not shown in A–C for clarity. For protocols, see Figure 1.

Figure 4.

Blockade of GABA_ARs had little effect on single tone and combination responses after iGluR blockade in an IC neuron. Test sequence: PRE (filled circles), iGluR Block (−30 nA each, 9 min; open triangles), +GABA_AR Block (+gabazine, +15 nA, 9 min; gray triangles), +GlyR Block (+30 nA, 9 min; light gray squares), and REC (10 min). Tone levels in C: 59.0 kHz at 34 dB SPL, 24.2 kHz at 64 dB SPL. Recovery not shown in A–C for clarity. In D, application of gabazine alone (GABA_AR Block alone) substantially increased response to 59.0 kHz tone compared with recovery. This indicates that gabazine was successfully applied to the neuron, even if it had little effect on facilitation. For protocols, see Figure 1.

Figure 5.

iGluRs mediated all spikes in response to single tones but did not contribute to facilitation in combination-sensitive neurons. A, Combined iGluR Block never eliminated facilitation. The significant increase in measured facilitatory strength was attributable to the elimination of responses to single tones resulting from iGluR blockade. B, Addition of GABA_AR blocker gabazine (solid lines; n = 10) never eliminated facilitation, whereas addition of bicuculline eliminated facilitation in two of four neurons (dotted lines). C, Addition of GlyR blocker (with GABA_AR Block, dashed lines; or without, solid lines) always eliminated facilitation. In B and C, the number of visible lines are fewer than the sample size because several neurons had overlapping values, resulting from facilitation values of +1.0 after iGluR blockade. *p < 0.01. D, E, Mean spike discharge for recorded population under different drug conditions for response to BF tone (BF spikes; open histograms) and response to combination of tones at best delay (BD Spikes; filled histograms). Sample sizes under different drug conditions apply to both BF and BD spikes. Addition of GABA_AR and GlyR blockade to iGluR blockade never revealed a weak iGluR-mediated excitation to BF tones that might survive iGluR Block.

Figure 6.

GlyR-mediated facilitatory spikes (Glutamate Independent) have latencies that match iGluR-mediated latencies in response to single tones (Glutamate Dependent). Glutamate-independent first-spike latencies are obtained in response to combination tones at best delay during application of iGluR blockers NBQX and CPP. In both measures, first-spike latency is measured from the onset of the BF tone.

Figure 7.

Multiple glycinergic inputs create facilitatory responses in IC. A, Glycinergic facilitation may result either from subthreshold depolarization activated by BF and low-frequency (LF) inputs (“glycinergic depolarization”) or by “postinhibitory rebound” activated by each of BF and LF glycinergic inputs. This schematic example is for a facilitated neuron with best delay of 6 ms. Membrane potentials displayed are only those activated by facilitating glycinergic inputs. In the postinhibitory rebound example, similar duration signals may activate inhibition of different durations, leading to differences in timing of rebound. B, The present results strongly suggest that excitatory glutamatergic inputs and inhibitory GABAergic and glycinergic inputs are functionally and anatomically segregated from facilitatory glycinergic inputs.