Firing properties of ANF and NM units and block of SON activity
shows the averaged rate-intensity function for ANF and NM units when a CF tone stimulus was applied. The spontaneous firing rate of NM units was significantly higher than for ANF units (NM, 101.9 ± 2.1 spikes/s, n = 413; ANF, 48.7 ± 2.1 spikes/s, n
= 201, p
< 0.0001). The spontaneous firing rate, the threshold sound level (NM, 31.34 ± 0.7 dB; ANF, 30.3 ± 1.0 dB) and maximal firing rate (NM, 260.9 ± 4.2 spikes/s; ANF, 370.0 ± 7.9 spikes/s) were comparable to those of previous reports (Warchol and Dallos, 1990
; Salvi et al., 1992
; Fukui et al., 2006
). The firing rate of ANF units increased with the sound level and the rate-intensity relationship was monotonic in almost all units recorded. In contrast, the firing rate of NM units reached a maximum at an intermediate sound level and was slightly decreased or saturated at higher input levels (ANF). The depression index was defined as the firing rate at 90 dB SPL divided by the maximum firing rate of a unit. Thus low values indicate strong suppression. The depression index was lower than 0.95 in 72.7% of NM units, and only 6.7% of ANF units. Interestingly, although there was substantial variation in the depression index between units of similar CF, the depression index of NM units depended on the CF of units. Depression of spike rate was seen primarily in the middle to high CF NM units (), in contrast to units with CFs < 1 kHz. Consistent with these findings, as described below, the influence of inhibition (or SON input) was most clearly observed in neurons with CFs above 1kHz. Furthermore, there was a weak, but significant relationship between threshold and firing rate depression at high sound levels; units having low acoustic thresholds tended to have low depression indexes (larger depression; ).
To test the influence of the SON in NM, the activity of the SON was blocked by local iontophoretic injection of 0.5mM TTX leaving the contralateral SON intact (Suppl. Fig. 3
) and the responses of NM neurons were tested for 7 chickens. The depression of the NM units ipsilateral to the TTX inactivated SON was reduced compared to that seen in control NM neurons and, particularly, that observed in contralateral NM units (). The effect of inactivating the SON input was only apparent at high intensities (> approximately 60 dB) and was much more dramatic for neurons with high frequency CFs than low CF units. Moreover, the depression of the ipsilateral NM after the TTX injection to SON was significantly smaller than that of the control, while that of the contralateral NM after the TTX injection was larger than that of the control for units with CFs above 1 kHz (). ANOVA followed by individual comparisons revealed no significant differences for units with CFs < 1 kHz. For units with CFs ≥ 1 kHz, depression at high stimulus levels was significantly less on the ipsilateral side than controls (p
< 0.05) and reliably greater on the contralateral side than controls (p
< 0.001). Similarly, the difference between the average depression index ipsilateral and contralateral to the TTX blockade of SON activity was highly significant (p
< 0.001). This difference between ipsilateral and contralateral depression index was not due to differences in CF distribution for the two populations of neurons. The means and SDs did not differ significantly [< 1 kHz: ipsilateral CF = 767.2 ± 48.7 Hz (n
= 13); contralateral CF = 616.5 ± 81.7 Hz (n
= 7). ≥ 1 kHz: ipsilateral CF = 1745.0 ± 129.7 Hz (n
= 27); contralateral CF = 1619.3 ± 192.9 Hz (n
Blocking SON activity by TTX altered the degree of depression for NM neurons
There was no significant difference in the spontaneous rate in NM (ipsilateral 127 ± 4.7 spikes/s, n = 40; contralateral 131.7 ± 9.3 spikes/s, n = 15; p = 0.62) or the threshold sound level (ipsilateral 20.0 ± 1.2 dB, n = 40; contralateral 20.3 ± 2.7 dB, n = 15; p = 0.91). However, the NM spontaneous rates were higher for the TTX treated population than for the control data reported in (control 103.2 ± 2.3 spikes/s, n = 346; TTX 128.3 ± 9.3, n = 55, p < 0.01). The maximal firing rate was not significantly different between three groups for CFs < 1 kHz (control, ipsilateral, contralateral; ANOVA, p = 0.13). For CFs ≥ 1 kHz, the mean maximum rate of ipsilateral TTX treated NM neurons (347.8 ± 10.3 spikes/s, n = 27) was significantly higher than control (274.6 ± 3.7 spikes/s, n = 304, p < 0.001), but that of contralateral TTX treated was not (319.2 ± 22.6 spikes/s, n = 8). ANF activity was not significantly influenced by TTX injection into SON; spontaneous rate (control 43.5 ± 39.4 spikes/s, n = 31, +TTX 36.8 ± 18.0 spikes/s, n = 7, p = 0.66); rate at 90 dB SPL (control 356.0 ± 98.6 spikes/s, n = 31, +TTX 427.8 ± 84.5 spikes/s, n = 7, p = 0.08).
Bicuculline facilitated the firing activity of NM units
SON is known to make a GABAergic projection to NM. We further evaluated the effects of GABAergic inhibition by locally blocking GABAA
receptors in NM with the iontophoretic application of bicuculline using a multibarrel piggy-back electrode while recording from NM neurons. Firing rates before, during, and after the application of bicuculline were measured. It should be stressed that this manipulation did not evaluate possible contributions of GABAB
receptors to the firing properties of NM neurons, and it is known that GABAB
receptors are found both pre-and postsynaptically in NM (Burger et al., 2005a
). The results of blocking GABAA
receptors for a typical neuron are shown in . For this neuron, following 1–4 min of bicuculline iontophoresis (−60 nA), the firing rate in response to a 90 dB CF tone gradually increased, and saturated within 6 min of bicuculline application (). Again, the increase in the firing rate was observed only for stimuli above about 60 dB SPL, and was not effective at either moderate (50 dB SPL) or subthreshold (10 dB) levels (). After 2–4 min from the suspension of bicuculline application, the firing rate at 90 dB SPL decreased to the control rate, and no decrease was observed at 10 or 50 dB SPL (). The complete rate-intensity function was similar to the control after 6–10 min of recovery from bicuculline application (c in ). In 24 NM neurons tested, a significant increase of the firing rate was observed at sound levels higher than 60 dB SPL and the mean firing rate at 90 dB SPL was increased 1.38 ± 0.04 times (), while the spontaneous firing rate was not significantly changed (p
= 0.73, ). Iontophoretic application of the pH adjusted isotonic vehicle solution (154 mM NaCl, pH 3.0) had no significant effect on the firing activity (n
= 2, data not shown). The largest increases in the firing rate following bicuculline application were found in units with the lowest depression index values in the control (p
< 0.001; ). The firing rate of ANF units was not affected by the application of bicuculline (n
= 2, data not shown). Despite the large changes in firing rate at high sound levels, the overall firing pattern was only marginally changed.
Bicuculline-induced increase in NM neuron firing rate was level-dependent
shows one example of the PSTH in the control (Aa) and during the application of bicuculline (Ab), and the averaged difference of firing rate (B). After application of bicuculline, the firing rate increased during the entire 80 ms CF tone stimulus with the exception of the onset response (, vertical dotted line). In this example, the increase became significant starting at around 8 ms following stimulus onset and continued to be statistically significant until offset of the response to sound (). The averaged increase in firing rate between 10–80 ms was 54.9 ± 8.4 spikes/s to this 90 dB SPL CF stimulus. That corresponds to a 44.2% above the firing rate of prior to the bicuculline treatment (124.3 ± 9.9 spikes/s, n
= 24). show average firing rates for the early portion of the PSTH and sustained response portion, respectively, as a function of stimulus level for the control condition and with bicuculline treatment. It is clear that the bicuculline treatment dramatically enhances responses to high intensity stimuli during the sustained response, but has little or no effect during the onset response. While PSTH analyses show little change in the overall pattern of response, analyses of the temporal precision in responses to tone stimuli was dependent on GABAergic input. Vector strength (VS) is a measure of the degree of phase selectivity of a response (Goldberg and Brown, 1969
). shows one example of period histograms of spikes in under control conditions and during bicuculline treatment. Phase-locking was observed in the control with a VS of 0.46. During bicuculline application the period histogram broadened and the VS fell to 0.33, while the firing rate increased to 1.55 times the control at 90 dB SPL. Ratios of VS measured at 90 dB SPL in each condition are plotted in . The averaged ratio of VS at 90 dB SPL was 0.92 ± 0.02 for all units studied in both conditions (n
= 24, p
< 0.001). Again, the decrease in VS was more striking for higher CF units (r = −0.47, p
< 0.05). VS decreases were also level-dependent, a priori
comparisons were made between control and bicuculline condition at each intensity. At 70, 80 and 90 dB stimulus levels the difference was significant (p
< 0.05). At lower intensities, the small differences in VS ratios were not reliable ().
Time course of the bicuculline sensitive response
Inhibitory tuning curve in NM
The acoustically evoked inhibition in NM, demonstrated by suppressing the excitatory synaptic input from ANFs, was frequency dependent (). We applied 20 μM DNQX onto the surface of the brainstem to partially block AMPA receptors in NM from activation by ANFs. The spontaneous firing rate of NM units was slightly lower in the presence of DNQX than in control conditions (in DNQX, 97.5 ± 4.2 spikes/s, n
= 58; in controls, 116.5 ± 6.2 spikes/s, n
= 49, p
< 0.05), while the spontaneous firing rates of ANF units was not significantly different (in DNQX, 52.6 ± 3.6 spikes/s, n
= 41; in control, 49.7 ± 4.7 spikes/s, n
= 35). After the application of DNQX, the auditory evoked discharges in NM units nearly ceased, only occurring at the onset of the sound stimulus for most neurons (). Surprisingly, the ongoing firing rate for remainder of the duration of the tone decreased below the spontaneous level. The onset responses enabled us to identify NM units and characterize their frequency selectivity. The zero crossing time and latency in spike triggered averaging (STA, Suppl. Fig. 1B
) of the click response corresponded to that of untreated NM units (Suppl. Fig. 1A
The excitatory and inhibitory response tuning was assessed for each NM unit in two time windows with respect to the stimulus. The excitatory onset responses were assessed in a window including the initial 15 ms while the inhibitory response area was measured from the area of response suppression in the remaining 15–80 ms period (see Methods). The excitatory response exhibited a relatively narrow tuning range at 90 dB SPL (, the frequency range above the threshold level was 1.32–2.07 kHz) when compared to the inhibitory tuning range (, the frequency range below the inhibitory threshold level was 0.45–3.75 kHz). shows one example of the tuning curve for the excitation measured from the onset response and the tuning curve of the inhibition measured from the ongoing response. The tuning curve of the excitatory onset response was sharp. shows another example of the inhibitory tuning curve in the unit for which the onset excitatory response was not detectable. The tuning curve of the inhibitory response was again very broad () and overlapped with that of the excitatory response at loud sound levels (). The W-shape of inhibitory tuning curve in is likely due to the remaining excitatory response that canceled the inhibition during the sound stimulus.
The frequency ranges of the excitation and the inhibition are compared in for DNQX treated NM neurons (n = 30). For most units (24 out of 30 units) the frequency range of the inhibition extended well beyond the frequency range of the excitation. In 3 out of 30 neurons the frequency range of the excitation could not be detected with the DNQX treatment. While a small onset response was detectable, it did not achieve our criterion for threshold across a range of frequencies. In contrast, evoked inhibition suppressed spontaneous activity in these neurons (top 3 units in ) across a broad range of frequencies. In two units (indicated by closed arrowheads in ), the inhibition extended only to the high frequency side, and in one unit (indicated by grey arrowhead in ) the inhibition extended only to the low frequency side.
Inhibitory tuning curves are broader than excitatory tuning curves in NM neurons
Tuning width was assessed for units using the sensitive frequency ratio (H/L), defined as the ratio of the high frequency limit of the response range divided by the low frequency limit. Population mean H/L ratios for ANF, NM, as well as the excitatory and inhibitory response areas for NM units treated with DNQX are shown in . ANF, NM, showed sharp frequency tuning. DNQX treated NM units (NM-exc in ) also showed sharp excitatory tuning, but the inhibitory area (NM-inh in ) was strikingly broader. H/L ratios for all four conditions were significantly different from each other (ANF 2.9 ± 0.2; NM 2.4 ± 0.1; NM-Exc. 1.8 ± 0.1; NM-Inh 9.7 ± 1.3). The broad tuning of the inhibitory response area was similar in scale to the excitatory tuning curves observed in recordings of individual SON units (data not shown).
The H/L of NM units was also CF dependent and had a tendency to be smaller than that of ANF units for CFs >1 kHz (). The range of sensitive frequencies, defined by the maximum and minimum frequencies evoking superthreshold responses at any intensity, was compared between the control and bicuculline conditions for a group of NM units. shows one typical example. The range of sensitive frequency (bounded by two arrows) became broader by bicuculline. For almost all 11 NM units studied in this way, the H/L ratio became larger after the application of bicuculline (). The average of H/L was 1.8 ± 0.3 in control and 2.5 ± 0.5 in the bicuculline (n = 11). Bicuculline broadened the H/L by 1.4 ± 0.2 (p < 0.05). The average of the lower and higher bound was 1154 ± 185 Hz and 1653 ± 127 Hz for the control, 1031 ± 172 Hz and 1958 ± 112 Hz for the bicuculline conditions. Both side of the bounds was significantly modified by the application of bicuculline (p < 0.01 for lower bound, p < 0.05 for higher bound). Thus, taken together with data from the tuning of excitation in NM while GABAergic input is blocked was much narrower than the tuning of inhibitory input while excitation was blocked.
Bicuculline sensitive current sharpened the frequency response