GABAergic Inhibition Controls Neural Gain in Inferior Colliculus Neurons Sensitive to Interaural Time Differences

Influence of GABAergic inhibition on pure-tone ITD sensitivity in IC neurons

Figure 1A shows the response of an IC neuron to interaurally delayed tones over ITDs in the range of ±2467 μs, equivalent to ±1.5 cycles of the stimulus period (1645 μs for a carrier frequency of 608 Hz, the CF of the neuron). ITD functions are shown for responses before (○), during (•), and after recovery from the effects of (▵) iontophoretic addition of the GABA_A antagonist gabazine. The same data are shown in Figure 1B but collapsed onto a single cycle of IPD. Blocking GABAergic inhibition with gabazine produced, maximally, a fourfold increase in peak discharge rate compared with predrug levels (Fig. 1A, B). When functions were normalized to their maximum discharge rate (Fig. 1C), ITD tuning at 50% maximum response was broader (Fig. 1D)—a wider range of ITDs evoked discharge rates of at least 50% of the maximum. This analysis suggests a role for GABAergic inhibition in sharpening the tuning of IC neurons for spatial cues, consistent with conclusions reached by others (Fujita and Konishi, 1991; Sterbing et al., 2005).

Figure 2 shows the response of an IC neuron to interaurally delayed tones before (○), during (•), and after recovery from the effects of (▵) iontophoresis of GABA itself (Fig. 2A, B). Iontophoresis of GABA had the opposite effect to iontophoresis of gabazine, with discharge rates being substantially reduced compared with control discharge rates. After normalization to maximum discharge rate, ITD tuning appeared to be sharpened (Fig. 2C,D) compared with control responses.

The data described thus far suggest blocking GABAergic inhibition broadens neural tuning for ITD, whereas adding GABAergic inhibition sharpens such tuning. However, this conclusion depends on the normalization procedure applied. All normalization procedures have underlying mathematical assumptions as to the form of the processes responsible for generating and controlling spike output. In the normalization to maximum discharge rate described thus far and used previously in studies suggesting that ITD tuning is sharpened by GABAergic inhibition (Fujita and Konishi, 1991; Sterbing et al., 2005), these assumptions are unknown, and the processes are not quantified. However, by the assumption of two general and well established neural processes, additive and multiplicative gain control, it is possible to provide a satisfactory explanation of the responses of delay-sensitive IC neurons to changes in inhibitory input.

To apply the concept of neural gain to the responses of IC neurons, a simple procedure was developed to attempt an appropriate scaling of interaural delay functions, based on the relationship between the discharge rates evoked under control conditions and those evoked under drug and recovery conditions. For each neuron under conditions of GABA block or addition of exogenous GABA, discharge rates evoked at each ITD/IPD were plotted as a function of control discharge rates before application of drug. The resulting gain functions (Figs. 1E, 2E) were used to quantify the extent to which multiplicative and additive mechanisms could account for any changes in the response.

The effect of blocking GABAergic inhibition on neural gain is illustrated in Figure 1E. Iontophoresis of gabazine increased the slope of the function describing the discharge rate during GABA block as a function of the discharge rate during control (predrug) conditions above unity (Fig. 1E, •) (slope, 1.96). Conversely, iontophoresis of exogenous GABA reduced the slope of the gain function to 0.39 (Fig. 2E). Together, this suggests that GABAergic inhibition scales discharge rates multiplicatively (divisively), because the discharge rate was multiplied by a common factor when GABA was blocked—the slope of the gain function increased—and divided by a common factor when exogenous GABA was added—the slope of the gain function decreased.

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Figure 2.

TDFs recorded from an IC neuron in response to iontophoresis of GABA. Control, GABA, and recovery responses indicated by key. Format as in Figure 1. A, Raw TDFs recorded over range of ITDs equivalent to ±1.5 cycles of the stimulus period of the carrier frequency (390 Hz in this case). B, Smoothed and folded TDFs, plotted over ± 0.5 cycles IPD. C, TDFs plotted normalized to the peak of the functions in B. D, Fifty percent tuning width (cycles of IPD) of normalized IPD functions recorded under control, drug, and recovery conditions. E, Gain functions with linear regressions fitted. F, TDFs scaled to control levels, based on the regression parameters from E. G, TDFs plotted normalized to the peak of the scaled functions in F. H, Fifty percent tuning width (cycles of IPD) of scaled and normalized IPD functions recorded under control, GABA, and recovery conditions. sp, Spike; C, control; R, recovery.

In addition to a change in slope, there was also a change in the offset of the delay functions that applied at all IPDs. This was seen as an increase in the IPD-insensitive baseline discharge rate during GABA block (Fig. 1A, B) and a decrease during addition of exogenous GABA (Fig. 2A, B). It was manifest as a vertical shift in the gain functions describing drug-versus-control responses (Figs. 1E, 2E) and was quantified by the position of the intercept on the abscissa of each linear regression. This constitutes an additive (subtractive) change in discharge rate.

The slope and intercept from linear fits to the gain functions were used to transform IPD functions, scaling down to the control function when GABA was blocked (Fig. 1F), and scaling the control functions down to GABA levels, when exogenous GABA was added (Fig. 2F). Once scaled in this manner, IPD functions recorded under control, drug, and recovery conditions appear very similar; there were no significant deviations in average scaled discharge rate (z test; p > 0.05) when these linear factors were applied to the drug-affected delay functions. Without any additional normalization, it is clear that there is little difference in ITD tuning width measures once possible gain factors are accounted for. The scaled IPD functions were then normalized (Figs. 1G, 2G), only to facilitate the calculation of 50% tuning width (Figs. 1H, 2H). In contrast to the nonscaled IPD functions, once the drug-induced changes in discharge rate were properly accounted for by the linear scaling procedure, there was little or no change in the 50% tuning width of functions recorded under drug conditions compared with control and recovery states.

Recovery of control discharge rates after iontophoresis of gabazine or GABA is confirmed by plotting discharge rates as a function of control discharge rates after termination of the ejection current (Figs. 1E, 2E;▵). Gain functions plotting recovery versus predrug discharge rates, predictably, lie close to unity (Figs. 1E, 2E; dotted lines).

Another important feature of the ITD functions in Figure 1 is the asymmetry around the peak response; the function on the side facing negative ITDs has a steeper slope than the function on the side facing positive ITDs. Such asymmetries, previously reported in IC responses to ITDs in noise stimuli (Kuwada et al., 1997; McAlpine et al., 2001), have been considered to be the result of selective sharpening of ITD functions around midline (i.e., close to zero) ITDs by GABAergic inhibition from the DNLL. The DNLL receives ITD-sensitive input from the medial superior olive (MSO) on the same side of the brain, and projects to both ICs, although with a bias favoring the contralateral IC. Thus, ITD-sensitive inputs from the DNLL to the IC might be expected to reflect more the opposite MSO to that providing excitatory inputs to the IC (Kuwada et al., 1997). The potential influence of such an inhibitory input was examined by measuring the slope of the ITD function. A measure of the steepness of the ITD function was obtained by measuring the slope of the function over three data points around each ITD (i.e., each ITD and its two immediate neighbors). The steepest slope on the side of the function facing negative ITDs (i.e., left of the peak response in Fig. 1G) was compared with the steepest slope on the side facing positive ITDs (i.e., right of the peak response in Fig. 1G). Blocking GABAergic inhibition had no effect on the relative steepness of the ITD function on either side of the peak response. The relatively steeper slope facing negative ITDs (ratio of 1.38:1 compared with the slope facing positive ITDs) before gabazine was applied (○) was maintained during GABA block (•) with a ratio 1.45:1.

Influence of GABAergic inhibition on sensitivity to ITDs in noise in IC neurons

Responses of two IC neurons to interaurally delayed noise (NDFs) before (○), during (•), and after recovery from (▵) iontophoresis of gabazine (top row) or GABA (bottom row) are shown in Figure 3. In each case, control NDFs showed a main peak and damped side peaks (Fig. 3A, F), consistent with previous reports (Yin et al., 1987). There was also a strong asymmetry around the main peak of the NDF, with the slope on the side facing negative ITDs being steeper than that facing positive ITDs. For the neuron in the top row, the ratio of the steepest slope on either side of the peak was 1.42:1, favoring the side facing negative ITDs. For the neuron in the bottom row, the ratio was 1.85:1. Neither the damping nor the asymmetry were altered when GABAergic inhibition was blocked (asymmetry ratio, 1.38:1 for the neuron in the top row of Fig. 3) or when exogenous GABA was added (1.66:1 for the neuron in the bottom row of Fig. 3). Gain functions (Fig. 3B, F) indicated multiplicative (or divisive) changes in gain during iontophoresis (•), producing well scaled functions (C,G) with little or no change in ITD tuning width (D,H).

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Figure 3.

NDFs from IC neurons before, during, and after recovery from the effects of gabazine and GABA. Control, drug, and recovery functions are indicated by the key. The top row indicates results from application of GABA antagonist. A, Raw NDFs recorded over ±1.5 cycles IPD (with regard to characteristic frequency of the neuron). B, Gain functions under drug and recovery conditions (excluding small points, from IPDs less than-0.25 cycles and more than 0.75 cycles). C, NDFs (plotted over ±0.5 cycles IPD) scaled down to control levels. D, Fifty percent tuning width (cycles of IPD) of scaled NDFs. The bottom row indicates results from iontophoresis of GABA. E-H follow the same scheme as those described above for A-D. sp, Spike; C, control; R, recovery.

GABAergic inhibition and dynamic binaural cues: responses to binaural beats

A common stimulus used to quantify delay sensitivity in binaural neurons is the binaural beat, created using a small (usually 1 Hz) difference in the frequency of tones presented to the two ears. The dynamic change in IPD that results generates the percept of sound motion. Most studies using binaural beats report similar delay sensitivity to the dynamic IPDs of binaural beats as to static ITDs (Yin and Kuwada, 1983). Figure 4, A and F, shows responses of two IC neurons to a 3 s binaural beat stimulus. Iontophoresis of gabazine increased discharge rates above control levels (Fig. 4A, •). Responses to two complete cycles (500-2500 ms) of the beat response in Figure 4A were averaged and plotted as a function of IPD in Figure 4B. This neuron differs from those previously in that it showed a peak response at negative, rather than positive, delays.

Blocking GABAergic inhibition scaled the discharge rate multiplicatively, with little additive shift (Fig. 4C, •). Using the slope of the gain function to scale the response (Fig. 4D) revealed little change in ITD tuning (E). Note also that the relative symmetry of the function around the peak response, which was opposite to that observed for neurons with peak responses at positive delays, was maintained during GABA block.

Ionotophoretic application of exogenous GABA reduced discharge rate in response to the binaural beat stimulus (Fig. 4F,G; •), in this case, apparently by divisive scaling (H, •). Once the IPD functions were scaled (Fig. 4I), there was no discernible change in 50% tuning width (J). Again, the asymmetry of the function was maintained after the addition of GABA.

In both cases, excellent recovery of predrug neural responses to the binaural beat was obtained after termination of the iontophoretic current (Fig. 4,▵).

Summary of GABAergic effects of ITD tuning in IC neurons

Iontophoretic blockade of GABA receptors and addition of exogenous GABA had predictable effects on the discharge rates evoked during stimulation with interaurally delayed tones and noise and with binaural beats. Application of GABA receptor antagonists increased average discharge rates of TDFs (n = 14), NDFs (n = 4), and binaural beats (n = 9) (Fig. 5A, D,G, respectively), which returned to control levels on recovery from drug application. Similarly, application of GABA produced a reduction in average discharge rates of TDFs (n = 11), NDFs (n = 1), and binaural beats (n = 4) (Fig. 6A, D,G, respectively).

The mean best IPD for the population of neurons was unaffected by iontophoretic blockade of GABAergic inhibition or iontophoretic application of exogenous GABA, either to stimulation by interaurally delayed tones, interaurally delayed noise, or binaural beats (Figs. 5B, E,H; 6B, E,H; •). For TDFs, the absolute change in mean IPD compared with predrug responses was 0.029 cycles (±0.037), and 0.019 cycles (±0.008) compared with recovery functions during GABA block. Additionally, there was little influence on best IPDs to binaural beats, with an absolute change in mean best IPD of 0.014 cycles (±0.01) compared with predrug responses and 0.006 (±0.006) compared with recovery functions. These changes were similar to those observed during iontophoretic application of GABA itself [e.g., 0.027 (±0.022) for TDFs during GABA iontophoresis compared with predrug responses]. Because application of GABA itself should not have ITD-dependent effects, this suggests that these small changes in best IPD are mostly within the variability of repeated measurements of ITD functions. In addition, there was no effect of application of either class of drug, on the position (in terms of cycles of IPD) at which the neural response reached 50% of maximum, on either side of mean best phase (50% border position) (Figs. 5B, E,H; 6B, E,H; ○). The 50% tuning width of the TDF, NDF, and binaural beat functions were similarly unaffected by application of GABA antagonist or GABA (Figs. 5C,F,I; 6C,F,I; •). Together, these observations form a compelling argument that GABAergic inhibition in the guinea pig IC does not play a role in sculpting neural responses to interaurally delayed sounds in terms of their tuning sharpness or in terms of the positioning of their receptive fields in ITD/IPD space. Additionally, large increases in discharge rate on GABA block were not accompanied by changes in the symmetry of ITD functions, which has previously been proposed to reflect ITD-sensitive GABAergic inputs from the DNLL sculpting ITD functions in the IC by means of lateral (in ITD terms) inhibition.

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Figure 4.

Binaural beat responses from IC neurons exposed to GABA and gabazine. Control, drug, and recovery functions are indicated by the key. The top row indicates results from iontophoresis of gabazine. A, Raw poststimulus time (PST) histograms to three cycles of the binaural beat stimulus. B, Smoothed beat period histograms, plotted relative to the IPD (±0.5 cycles) of the binaural beat stimulus. C, Gain functions under drug and recovery conditions. D, Beat period histograms scaled down to control levels. E, Fifty percent tuning width (cycles of IPD) of scaled IPD functions. The bottom row indicates results from application of GABA. F-J follow the same scheme as those described for A-E. sp, Spike; C, control; R, recovery.

Serial recordings in the same neuron, and effects of discharge rate adaptation/saturation

In many neurons, a series of sequential recordings were made demonstrating the effect of increasing drug concentration (i.e., increasing ejection current or increasing time after the onset of the ejection current), and in some cases, recordings were made during the exposure to both drug classes, with an intervening period of recovery. Figure 7 illustrates two such examples.

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Figure 5.

Summary of the effects of GABA blockade on IPD tuning. Top, middle, and bottom rows indicate data from TDFs (A-C), NDFs (D-F), and binaural beats (G-I), respectively. Changes in average discharge rate are indicated in the left column (A, D, G). Gray lines indicate data from individual neurons. Black symbols and the line indicate mean of group data (±SD). Effects of drug application on IPD tuning are indicated in the middle and right columns. B, E, H, Group-averaged mean best phase (±SD) is indicated by black circles. Open circles represent group average (±SD) for IPD positions at which the response crosses 50% of the maximum point for borders at IPDs above and below the mean best phase. C, F, I, Fifty percent tuning widths of IPD functions are plotted. Gray lines indicate data from individual neurons. Black circles and lines indicate the group average tuning width (±SD). “ns” indicates no significant difference between drug and control/recovery conditions. Significant effects (ANOVA) are indicated as follows: ^*p < 0.05 and ^**p < 0.01. BP, Best phase; sp, spike; C, control; R, recovery.

The neuron in the top row of Figure 7, A-C, was exposed, first to GABA, and then to bicuculline. From control conditions (Fig. 7A, •), GABA was ejected using currents of 20, 10, and 5 nA (Fig. 7A; see key). For the larger GABA ejection currents, there was a larger reduction in discharge rate compared with control conditions (down to <6% of the control rate) (Fig. 7A-C), which recovered to control levels after termination of the ejection current (Fig. 7A-C). Only very minor changes in 50% tuning width were noted under these conditions (<0.05 cycles IPD) (Fig. 7C). The reduction in discharge rate was brought about primarily by divisive changes in the gain functions (Fig. 7B). The slope fell from 1.0 in control conditions (by definition) to 0.07 with 20 nA GABA. The slope increased to 0.19 with 10 nA GABA, to 0.34 with 5 nA GABA, and recovered to 1.01 after the termination of the GABA ejection current. This implies that primarily multiplicative gain changes were observed in this neuron with application of GABA.

When 25 nA of bicuculline ejection current was applied to the same neuron, discharge rate increased to >250% of control levels after 17 min and 40 s of current application (Fig. 7A-C; see key). There was a modest increase in the slope of the gain function (Fig. 7B, C) from 1.01 to 1.38 over the same time period, indicating some multiplicative gain, and a substantial increase in the baseline, ITD-insensitive, discharge rate, indicating some additive gain. Despite these dramatic discharge rate effects, there was negligible effect on the 50% tuning width of the functions (<0.02 cycles increase) (Fig. 7C). When the bicuculline ejection current was terminated, these parameters returned to levels close to the initial control recordings.

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Figure 6.

Summary of the effects of GABA on IPD tuning. Top, middle, and bottom rows indicate data from TDFs (A-C), NDFs (D-F), and binaural beats (G-I), respectively. Figure follows conventions detailed for Figure 5.

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Figure 7.

Serial recordings from individual neurons in response to GABA and GABA block. Folded ITD functions are shown for two neurons in A and D. Filled circles indicate control and recovery responses; triangles indicate responses with iontophoresis of GABA (labeled G in key) or GABA antagonist (labeled BIC or GZ in key). In the key, iontophoresis of GABA (G) is labeled additionally with the ejection current used; iontophoresis of GABA antagonist is labeled additionally with the time after the onset of the drug ejection current. Gain functions are shown in B and E for the same neurons. C and F show composite plots illustrating changes in 50% tuning width, average discharge rate, and gain function slope (indicated in key) with changing drug/recovery condition, from control, through exposure to GABA, recovery from GABA effects (recovery 1), GABA blockade, and finally to recovery from all iontophoresed drugs (recovery 2). Dotted lines indicate control value for each parameter for comparison at each drug condition. sp, Spike; DR, discharge rate.

A second example of serial recordings is shown in the bottom row of Figure 7, D-F. From control levels, 5 nA GABA ejection current was applied and there was a resulting fall in discharge rate (Fig. 7D-F). Concomitantly, the slope of the gain function was reduced from 1.0 to 0.50, with little offset shift, indicating a primarily divisive gain change. However, there was, again, negligible effect on 50% tuning width of the scaled ITD functions (from 0.32 to 0.30 cycles). On termination of the GABA current, discharge rate recovered, the slope of the gain function returned close to unity (0.95), and the 50% width returned to 0.32 cycles. When a gabazine ejection current was applied, to block inhibition, there was a clear increase in discharge rate, with the largest rates recorded 17 min after the onset of the current. For this series of recordings, sustained blockade of GABAergic inhibition pushed the neurons discharge rate into saturation. After 11 min of gabazine ejection current, the neuron maintained a response that was well modulated with ITD (Fig. 7D, ▵). However, as the average discharge rate increased further with time, the response became less well modulated with ITD (Fig. 7D, ▴). There was also a tendency for peak responses to fall slightly below those of the ITD function recorded at 11 min into the ejection current (with a lower average discharge rate). We interpret these observations as the effects of saturation on the input-versus-output function of the neuron [firing rate (f)-input current (I) curve] (see Discussion) and possibly spike frequency adaptation. The randomized presentation of different ITDs coupled with the high spike rate at all ITDs means that the neuron is likely forced into an adapted state, even at less favorable ITDs. Because recovery from adaptation in ITD-sensitive IC neurons is a slower process than the adaptation itself (Ingham and McAlpine, 2004), the relatively fast repetition rate of ITD presentation (250 ms duty cycle) means that the neuron was not able to recover from the adaptation induced by the high spike rates, resulting in spike rates at favorable ITDs that could be lower than those recorded for the same ITDs in functions in which the average discharge rate was lower. Strictly, we cannot differentiate between saturation and adaptation effects, but the higher baseline rate and lower peak rate had a significant effect on the gain function describing this particular condition for the neuron. As average discharge rate increased, the slope of the gain function was seen to increase from 0.95 to 1.56 (after 11 min) with accompanying increases in function offset (Fig. 7E), indicating a combined additive and multiplicative gain control. With an additional increase in average discharge rate (at 17 min), the gain function flattened, showing a slope of only 0.55 and a large offset. This effect is consistent with the influence of saturation and/or spike frequency adaptation described above. It should be noted that the 50% tuning width is principally unaffected by the addition of gabazine, increasing from 0.32 cycles (recovery 1) to 0.36 cycles (after 11 min of gabazine ejection). Only when the ITD modulation of discharge rate breaks down, and thus the gain function scaling of the function performs less well, is there any degree of change in 50% tuning width (increasing to 0.46 cycles after 17 min). However, this tuning width measure returns to 0.34 cycles when the neuron was fully recovered from the effects of gabazine. Similarly, after the termination of the gabazine current, the gain function slope returned to control values as did discharge rate (Fig. 7F).

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Figure 8.

ITD discrimination by single neurons, the responses of which were modified by blocking, or adding exogenous, GABA inhibition. A-C, Folded ITD functions for three neurons, indicating average discharge rate (±SD). For the neuron in the left column (A, D, G), data are plotted for two levels of GABA blockade (gray lines) as well as a control (black line). For the neuron in the middle column (B, E, H), data are plotted for responses during iontophoresis of gabazine (light gray lines) and during iontophoresis of GABA (dark gray lines). For the neuron in the right column (C, F, I), data are plotted for two different GABA ejection currents (gray lines) and control (black line). D-F, The standard separation, D (see Materials and Methods), for each ITD away from zero cycles is plotted for the three neurons. G-I, Cumulative D (for functions in D-F) is plotted as ITD increases from zero cycles. Dotted lines indicate a value of 1.0, above which ITD becomes discriminable from zero. J, Averaged threshold D—the jnd—calculated from TDFs plotted as a function of discharge rate ratio (values <0.85 indicating discharge rates during iontophoresis of GABA; values >1.15 indicating discharge rates during GABA blockade). Error bars represent SEM.

These serial recordings of the effects noted provide additional evidence that GABAergic inhibition in the IC serves to regulate the output of the IC neurons with no apparent ITD-specific effects on the sharpness of ITD tuning in these neurons. It should be noted that any recordings obtained indicating strong evidence of saturation/adaptation effects were not included in the summary data shown in Figure 5.

GABAergic inhibition and ITD discrimination

Although GABAergic inhibition does not sharpen ITD functions by lateral inhibition—it has no effect on preferred interaural delays or 50% tuning widths—it does, nevertheless, modulate ITD discrimination thresholds, as would be expected from a mechanism that adjusted neural discharge rates per se. Figure 8, A-C, shows responses of three IC neurons to interaurally delayed tones. Blocking or adding GABAergic inhibition modulated discharge rates and produced multiplicative and/or additive changes in neural gain (as described above) (gain functions not shown). These gain changes were accompanied by altered ITD discrimination performance. For each neuron, a measure of discrimination—the standard separation or D (see Materials and Methods)—was calculated over ITDs equivalent to ±0.5 cycles of IPD around zero delay, corresponding to the auditory midline. Discrimination at each delay was calculated between neighboring values in either direction away from zero delay. Values of D < 1.0 (Fig. 8D-F, horizontal lines) are considered below discrimination threshold. Consistent with recent reports in the guinea pig (Shackleton et al., 2003), sounds leading at the ipsilateral (to the IC) ear are most discriminable at the neural level. Figure 8, D-F, indicates that, under control conditions (black lines), a shift of 0.05 cycles from zero toward negative (ipsilateral leading) delays were more detectable (i.e., higher D value) than a shift of 0.05 cycles toward contralateral delays.

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Figure 9.

Gain control models. A-C, Main panel indicates input-output or f-I curves, under control conditions (1, solid line), conditions of reduced GABA (2, dashed line), and conditions of additional GABA (3, dotted line). The gray area indicates the range of input current produced by an ITD-modulated stimulus and the corresponding range of neural output. Small panels on the right indicate ITD functions that might be produced under the same conditions.

For the neuron shown in the left column of Figure 8, blocking GABAergic inhibition (gray lines) reduced discrimination across the range of delays as discharge rate increased, particularly when, with sustained gabazine current, discharge rates started to saturate at favorable delays (light gray line). This is reflected in the lower D values for these conditions, plotted in Figure 8D. The same effect was noted for the neuron plotted in the middle column of Figure 8. Blocking inhibition (light gray line) increased discharge rate (Fig. 8B) and reduced D values (i.e., discrimination) across all ITDs compared with the control data (Fig. 8E, black line).

Discrimination was usually reduced with iontophoresis of exogenous GABA (Fig. 8, middle column, dark gray lines), presumably as spike count decreased and the peak-to-trough modulation depth of the delay function was reduced. Figure 8E (dark gray lines) indicates that D values were reduced at all ITDs compared with control values (black lines).

It was not always the case that control levels of GABA provided for best discrimination. The neuron in the right column in Figure 8, for example, showed a large ITD-insensitive component to its control response (black line), which was substantially reduced during iontophoresis of GABA (gray line). This increased the proportion of the response that was modulated with interaural delay, and improved the discrimination performance of the neuron (Figure 8F, gray line). However, with an additional increase in the GABA current (light gray line), discharge rate continued to fall, and discrimination was reduced as response modulation, in terms of absolute discharge rate, decreased.

Recent data suggest that slopes of interaural delay functions, rather than their broad peaks, are a critical factor in sound localization over the frequency range at which sensitivity to interaural delays is observed in mammals (Shackleton et al., 2003). These slopes are often positioned across the physiological range of ITDs (±180 μs in guinea pigs) around zero delay, with the result that modulating the interaural delay of a sound source, or head movement with respect to a fixed sound source, modulates the discharge rate up and down the slope of delay function. For each neuron, the “just-noticeable difference” (jnd)—the minimum detectable change in interaural delay from zero—was obtained by calculating the cumulative D either side of zero delay. The D value previously calculated between neighboring ITDs was summed for consecutive ITDs in either direction away from zero delay. The interaural delay at which D crossed the threshold value of 1.0 was considered the threshold interaural delay—the smallest change in ITD from zero that could be detected. This analysis is shown in the bottom row for each of the neurons in Figure 8. For the neuron in the left column of Figure 8, the minimum detectable change in ITD from zero (Fig. 8G), the threshold jnd from zero, corresponded to 0.039 cycles, equivalent to a minimum detectable interaural time difference of 78 μs at 500 Hz, the tone frequency used to obtain the delay function. As discharge rate increased with increasing GABA block, the minimum detectable change in IPD increased to 0.22 cycles (435 μs; dark gray line) and, finally, to 0.25 cycles (505 μs; light gray line) when the interaural delay function was close to saturation. For the neuron in the middle column of Figure 8, the jnd increased from 0.024 cycles (in control conditions) to 0.064 cycles when additional GABA was applied, or to 0.178 cycles when GABA was blocked (Fig. 8H). For the neuron in the right column of Figure 8, the minimum detectable change in ITD was obtained with the intermediate discharge rate evoked during GABA block (Fig. 8I).

The tendency for thresholds to be lowest for control levels of GABAergic inhibition is confirmed in Figure 8J, which shows the effect of blocking or adding exogenous GABA on minimum discriminable interaural delay in response to interaurally delayed pure tones for 15 neurons. Mean threshold ITD was elevated as discharge rate increased (>15% increase cf. control) with iontophoresis of gabazine (0.076 ± 0.044 cycles of IPD) or as discharge rate decreased (>15% decrease cf. control) with iontophoresis of GABA (0.061 ± 0.029 cycles of IPD) compared with the mean threshold for control discharge rates (0.048 ± 0.020 cycles of IPD). ANOVA revealed these differences to be significant (F = 4.55; p < 0.02).

GABAergic inhibition and sensitivity to interaural time differences

The second finding of this study is that GABAergic inhibition does not influence the sensitivity of IC neurons to ITDs, at least in terms in which such sensitivity is normally considered. Once changes in neural gain are accounted for, neural tuning for preferred ITD and tuning sharpness were unaffected by blocking GABAergic inhibition or by adding exogenous GABA. This is contrary to previous reports. Using a simple normalization procedure (to the peak discharge rate) of their ITD functions, Fujita and Konishi (1991) (in the barn owl) and Sterbing et al. (2005) (in the rabbit) concluded that GABAergic inhibition serves to sharpen delay tuning in IC neurons. However, such a normalization process is usually reported without mathematical justification and fails to take into account the influence of neural gain. With the assumption that GABA controls multiplicative and additive gain, changes in neural responses to static ITDs during GABA block or addition of exogenous GABA can be accounted for by scaling and/or offsetting neural responses by the appropriate factors. Reanalysis of previously published ITD functions (Fujita and Konishi, 1991) using our scaling procedure demonstrates that delay functions in barn owl IC scale in the same way as those seen in the current study, and that no changes in static ITD tuning are observed. Our data do not preclude the possibility that ITD functions may be sharper at successive stations in the ascending auditory pathway (suggested by Fitzpatrick et al., 1997), but it does not support a role for GABA in the sharpening process at the level of the IC.

Potential sources of GABAergic inhibition to the IC

Two potential circuits that could provide for ITD-sensitive inhibition are via the DNLL and via local interneurons in the IC itself. The DNLL receives input from the MSO on the same side of the brain and projects its primarily GABAergic inhibitory input bilaterally to the IC, although favoring the IC on the opposite side of the brain. Several studies have demonstrated DNLL neurons to be ITD sensitive (Aitkin et al., 1970; Brugge et al., 1970), and it is often assumed that DNLL neurons influence IC neurons in a delay-sensitive manner. Our data potentially support this contention but indicate that any delay-sensitive GABAergic input to IC neurons operates in a different manner to that previously imagined, in that it requires precise matching of the delay-sensitive inhibitory and excitatory inputs to produce the multiplicative gain effects we observe. This projection has been proposed as the candidate nucleus for sculpting responses of ITD-sensitive neurons, possibly providing them with their “saw-tooth” appearance (Kuwada et al., 1997), as well as for rendering IC neurons sensitive to the context in which IPD cues are presented (Spitzer and Semple, 1998). However, it was notable that no change in the shape of ITD functions occurred during GABA block; the relative symmetry around the peak responses of delay functions and binaural beats was maintained. Such asymmetries have been suggested previously to arise from the influence of a mismatch between the ITD sensitivity of the excitatory and inhibitory ITD inputs to IC neurons. However, our data demonstrate convincingly that GABAergic inhibition does not have the expected properties and appears remarkably well matched in input strength to the excitatory input across the range of excitatory input values.

An alternate and plausible explanation, given the requirement of precisely matched excitatory and inhibitory inputs, is that GABAergic inhibitory circuits, acting in feedforward or local (to the IC) feedback loops. This would provide a mechanism of gain control local to the IC that matches excitatory input levels to the range of discharge rates at which discrimination performance is best. Similarly, acoustic parameters other than binaural cues are likely to be subject to the same forms of gain control in the IC; ITD-sensitive neurons in the IC are also sensitive to changes in sound frequency and intensity. Local circuitry in the IC could modulate the efficacy of GABAergic inhibition for specific sound sources, enhancing detection and recognition of auditory objects in a complex acoustic environment.

Mechanisms of gain control in the IC

A potential candidate mechanism to control neural gain is shunting inhibition, mediated by a GABA-controlled Cl^- conductance. Shunting inhibition has long been considered a potential mechanism for gain control, although initial in vitro studies appeared to demonstrate that shunting inhibition produced only subtractive gain effects, and could not account for divisive gain (Brickley et al., 1996; Holt and Koch, 1997; Chance et al., 2002). Recent in vitro studies indicate that, when input trains mimic the noisy synaptic inputs known to exist in vivo, shunting inhibition produces divisive gain (Mitchell and Silver, 2003). In the current study, the divisive gain change observed on iontophoresis of GABA is particularly relevant, because it represents a gain change that is dependent on the discharge rate, and by extension the underlying voltage-current relationship of the neuron. Given the ITD-independent nature of exogenous GABA, this is consistent with a cellular mechanism of gain control, rather than the involvement of feedback/feedforward circuits. Such loops cannot be excluded, however, especially given the nature of the experimental procedure, which involved blocking all potential GABAergic inputs. It is possible that interneurons themselves are influenced by exogenous GABA, and their proximity, or otherwise, to the recorded neuron is unknown, as indeed is their existence. Exogenous GABA might also reduce the excitatory influence of the recorded neuron on any inhibitory interneurons acting in a feedback loop circuit, reducing their efficacy, and resulting in a compensatory increase in neural gain. However, in the absence of detailed knowledge concerning the influence of local inhibitory circuits in the IC, and the apparent lack of inputs mismatched in their ITD sensitivity relative to that of excitatory inputs, the most parsimonious explanation is one of shunting inhibition, which does not require extensive neural circuitry, relying instead on processes restricted to the neuron itself. Recent studies suggest that IC neurons in the barn owl multiply inputs sensitive to interaural time and interaural intensity differences (Pena and Konishi, 2001), and that inhibition is likely involved in modifying these responses (Pena and Konishi, 2002). The current data are not inconsistent with this hypothesis but also suggest a broader role for neural multiplication in controlling neural gain.