Rethinking tuning: in vivo whole-cell recordings of the inferior colliculus in awake bats

Abstract

Tuning curves were recorded with patch electrodes from the inferior colliculus (IC) of awake bats to evaluate the tuning of the inputs to IC neurons, reflected in their synaptic tuning, compared with the tuning of their outputs, expressed in their discharge tuning. A number of unexpected features were revealed with whole-cell recordings. Among these was that most neurons responded to tones with inhibition and/or subthreshold excitation over a surprisingly broad frequency range. The synaptic tuning in many cells was at least 1.5-2.0 octaves wide and, on average, was more than twice as wide as the frequency range that evoked discharges even after inhibition was blocked. In most cells, tones evoked complex synaptic response configurations that varied with frequency, suggesting that these cells were not innervated by congruent excitatory and inhibitory projections. Synaptic tuning was not only wide but was also diverse, in which some cells were dominated by excitation (n = 20), others were dominated by excitation with sideband inhibition (n = 21), but most were dominated by inhibition with little evidence of excitation (n = 31). Another unexpected finding was that some cells responded with inhibition to the onset and offset of tones over a wide frequency range, in which the patterns of synaptic responses changed markedly with frequency. These cells never fired to tones at 50 dB sound pressure level but fired to frequency-modulated sweeps at that intensity and were directionally selective. Thus, the features revealed by whole-cell recordings show that the processing in many IC cells results from inputs spectrally broader and more complex than previously believed.

Figure 1.

Neurons with V-shaped tuning curves (A, B) and upper-threshold tuning curves (C, D). The tuning curves in A and C were recorded with patch electrodes and show both the discharge and synaptic tuning of two IC neurons. The tuning curves in B and D are from two IC cells that were recorded with extracellular electrodes in a previous study by Xie et al. (2005). A, A neuron with a V-shaped discharge tuning curve with wide range of synaptic tuning and sideband inhibition. Notice the frequency increments are in steps of 4.0 kHz. Tone bursts were 50 ms, indicated by the time bars. Nonspiking responses are averages of 10 trials, and spikes are single traces. Resting potential was −48 mV. A more detailed and amplified view of the tuning at 60 dB SPL is shown in Figure 6B. B, V-shaped tuning curve recorded extracellularly that expanded markedly on both high- and low-frequency sides of the BF when inhibition was blocked. The scale of the histograms in the control and tuning curves when inhibition was blocked are not the same. The higher spike counts evoked when inhibition was blocked are indicated by the numbers in each tuning curve, which show the highest spike count of each curve. Tone duration was 10 ms as shown by time bars. C, Tuning curve of an upper-threshold cell recorded with a patch electrode showing the prominent inhibition evoked by most frequency–intensity combinations. Resting potential was −44 mV. Time bars, 50 ms. Nonspiking responses are averages of five trials, and spikes are single traces. D, An upper-threshold neuron recorded with an extracellular electrode before (control) and the release from inhibition that allowed an underlying suprathreshold excitation to be expressed when inhibition was blocked by bicuculline (Bic) and strychnine (Strych). Time bars, 15 ms.

Figure 2.

Tuning curves of two null-tuned cells recorded with patch electrodes (A, C1) and from another null-tuned cell recorded with an extracellular electrode (B). A, Null tuning curve from a cell whose responses were dominated by inhibition. Traces are averages of five trials. Time bars, 50 ms. Resting potential was −46 mV. B, Null tuning curves recorded with an extracellular electrode. When inhibition was blocked by bicuculline (Bic) and strychnine (Strych), the underlying suprathreshold excitation that was completely suppressed by inhibition was released and allowed the cell to express a V-shaped tuning curve. Time bars, 15 ms. C1, Another null-tuned neuron recorded with a patch electrode whose responses were dominated by subthreshold excitation. Time bars, 50 ms. C2, Responses of the same cell in C1 evoked by injections of depolarizing current steps. Current injections of +60 pA evoked a single action potential (gray trace), and higher current steps (+150 pA) evoked multiple action potentials. Lower currents were subthreshold. C3, Enlarged trace showing the threshold response evoked by +60 pA current injection (gray trace) and the subthreshold depolarization evoked by 18 kHz tone at 50 dB SPL (black trace), the largest tone-evoked response recorded. Resting potential was −55 mV.

Figure 3.

Histograms showing the percentage of V-shaped, upper-threshold, and null tuning curves recorded with patch electrodes in the present study compared with the percentages recorded in the previous study of the IC by Xie et al. (2005) with extracellular electrodes.

Figure 4.

Distributions of widths of discharge tuning from cells recorded in a previous extracellular study and synaptic tuning recorded in this study. Discharge tuning is widths of tuning curves at 50 dB SPL while inhibition was blocked. Synaptic tuning is widths of tuned regions at 50 dB SPL from 49 IC cells and from the tuned regions of 18 other IC cells at 40 dB SPL. The tuned regions at 50 dB SPL were not determined in the 18 cells because their tuning curves were generated in increments of 20 dB, at 0, 20, 40, and 60 dB SPL. The tuning width at 40 dB was used in those cells. Notice that the distribution of BFs of the two samples was similar. Widths of synaptic tuning of cells shown at the far left are for cells in which a BF could not be determined because there were no spikes in their tuned regions at 50 dB SPL. The tuning widths of those cells are included because their widths were evaluated with a similar range of frequencies as those presented to other neurons, from ∼10 to 30 kHz. Average discharge tuning width was 7.1 ± 5.4 kHz (n = 49). Average synaptic tuning was 17.6 ± 6.0 kHz (n = 67).

Figure 5.

Two neurons whose tuned regions were dominated by EPSPs. A, Only subthreshold and suprathreshold excitations were evoked by 10 ms tone bursts at 50 dB SPL. Resting potential was −50 mV. B, Another EPSP-dominated neuron in which 10 ms tones at 50 dB SPL evoked suprathreshold EPSPs from ∼15 to 25 kHz and mostly subthreshold EPSPs from ∼26 to 40 kHz. Small IPSPs that preceded the EPSPs were evoked on the low-frequency side of the tuned region, from ∼13 to 10 kHz. Resting potential was −50 mV. All traces are single trials in both neurons.

Figure 6.

Two cells in which a range of frequencies evoked predominantly EPSPs and sideband frequencies evoked either IPSPs or a subthreshold EPSP–IPSP configuration. A, Cell that responded to “low” frequencies, from 13 to 18 kHz, predominantly with EPSPs and discharges. Higher frequencies, from 19 to 25 kHz, evoked EPSPs that appeared to be suppressed to subthreshold levels by longer-lasting IPSPs. Synaptic responses evoked by frequencies from ∼26 to 29 kHz were largely small IPSPS. Tone bursts were 30 ms at 50 dB SPL. Resting potential was −50 mV. B, Cell with central excitatory region flanked on both high low sides by inhibition. Frequencies immediately below the excitatory region, at 14 kHz, evoked a synaptic configuration similar to the higher, suprathreshold frequencies, but the inhibition appeared to suppress the excitation. Lower frequencies, from 12 kHz and below, evoked strong inhibition. Similar features were seen on the high frequencies, from ∼30 to 34 kHz. Insets on right show magnified views of PSP responses. Tone bursts were 50 ms at 60 dB SPL. Resting potential was −48 mV. This is the same cell whose tuning curve is shown in Figure 1B. In both neurons, nonspiking traces are averages of 10 trials, and spiking traces are single trials.

Figure 7.

Integrated PSP values for cells dominated by excitation, inhibition, and excitation with inhibitory sidebands. In theses graphs, the normalized PSP responses are aligned to the largest (absolute value) PSP area (F_max) and graphed as responses evoked by frequencies above and below the frequency that evoked F_max. A, Normalized PSP values in the tuned regions of 14 EPSP-dominated cells. Integrated PSP values are positive (depolarized) for responses evoked by almost every frequency in all cells. B, Normalized PSP values in the tuned regions of 17 cells that had excitatory regions flanked by inhibitory surrounds. The responses to F_max were excitatory in all of these cell, but frequencies above and or below F_max evoked strong inhibitions. C, Normalized PSP values in the tuned regions of 31 IPSP-dominated cells. Three cells have a prominent excitatory peak, appearing to be similar to the excitatory-dominated cells with sideband inhibition in B. The reason for the excitatory peaks is that a larger and longer-lasting excitation than inhibition was evoked by one or two frequencies in their tuned regions, whereas the responses evoked by all other frequencies were inhibitory. D, Average PSP areas in the tuned regions of cells dominated by IPSPs and cells dominated by EPSPs. The average PSP area in every IPSP-dominated cell was negative (hyperpolarized), whereas the average PSP area in every EPSP-dominated cell was positive (depolarized). Differences in the absolute values among EPSP-dominated or among IPSP-dominated cells were attributable to differences in PSP amplitudes and durations. Some of the differences were a consequence of the durations of the tone bursts, which were not the same in all cells and ranged from 10 to 50 ms among cells.

Figure 8.

Tuned regions of two cells that were dominated by IPSPs. The tuned regions of this IPSP-dominated subtype were characterized by uniform PSP patterns evoked across frequency. The widths of the tuned regions differed among cells in this subgroup, illustrated by the narrow tuning in A and the much broader tuning in B. Tone burst durations were 10 ms in A and 20 ms in B. All tones were 50 dB SPL. Records in A are averages of 10 trials, and records in B were averages of three trials. Resting potential of the cell in A was −44 mV, and the cell in B was −46 mV.

Figure 9.

Responses evoked in an on–off neuron with two different tone burst durations, showing that the neuron responded with IPSPs to the onset and to the offset of the tone burst. Tone burst was 25 kHz at 50 dB SPL. Resting potential was −48 mV. Both records are averaged responses of 10 trials. The tuned region of this cell is shown in Figure 10A.

Figure 10.

Tuned regions of four IPSP-dominated cells that responded to tones with on–off IPSPs. All tones were 50 dB SPL with durations shown by the time bars below each tuned region. Resting potential was −48 mV (A), −50 mV (B), −48 mV (C), and −49 mV (D). All traces are averaged responses of 10 trials. For additional explanation, see Results.

Figure 11.

Intensity functions showing responses evoked by tones of increasing intensity at several frequencies for two on–off cells. The discharge features of both cells were upper-threshold and very narrowly tuned in that they fired only to one frequency and only at very low intensities. The amplitudes of the spiking responses evoked by 24 kHz at 10 dB in A and by 23 kHz at 10–20 dB in B are reduced for clarity. Inset on far right shows PSPs of the neuron in B evoked by increasing tone intensity but without spikes. Notice progressive increase in both the amplitude and duration of the initial hyperpolarization that appeared to quench the afterdepolarization. Tone burst durations were 50 ms in A and 20 ms in B and are shown by time bars below each intensity function. The tuned region of neuron A is shown in Figure 10C, and the tuned region of neuron B is shown in Figure 10D. In both neurons, nonspiking traces are averages of 10 trials, and spiking traces are single trials. Traces evoked by 23 kHz tone at 10–20 dB SPL in inset are averages of nonspiking trials.

Figure 12.

Directionally selective responses evoked by FM sweeps in an on–off neuron. The tuned region for tone bursts at 50 dB SPL is shown in A. The neuron responded with discharges to FM signals that swept downward from 45 to 15 kHz. Discharges were evoked by downward FM sweeps at all signal durations. The cell failed to discharge to the same FM signals that swept upward from 15 to 45 kHz. Insets show magnified view of synaptic responses to the downward and upward FM sweeps. FM duration is shown by time bars under each record. All FM sweeps were 50 dB SPL, the same intensity as the tone bursts in A that failed to evoked any discharge. All traces of tone burst responses in A were averages of five trials, whereas all FM responses were from single trials. Resting potential was −48 mV.

Figure 13.

Another on–off cell with directionally selective responses to FM sweeps. The tuned region for tone bursts at 50 dB SPL is shown in A. The cell discharged to downward FM signals at 50 dB SPL that swept from 45 to 15 kHz with durations ranging from 5.0 to 40 ms. The neuron responded to longer (50 ms) and shorter (2 ms) downward FM sweeps with subthreshold EPSPs. It did not fire to upward FM sweeps and responded to those sweeps with an initial IPSP followed by an EPSP. Presumably the initial hyperpolarization suppressed the subsequent excitation to subthreshold levels. Nonspiking traces are averages of 10 trials, and spiking traces are single trials. Resting potential was −52 mV.