Sound Rhythms Are Encoded by Postinhibitory Rebound Spiking in the Superior Paraolivary Nucleus

All SPON neurons display postinhibitory rebound spiking

The responses of 55 SPON neurons to injections of depolarizing and hyperpolarizing current pulses were investigated in vitro. Neurons were sampled from throughout the SPON, and they exhibited stable resting membrane potentials (mean resting membrane potential, −65 ± 0.7 mV) throughout the recordings and all had fast-rising, overshooting spike amplitudes (mean spike amplitude, 68 ± 1.4 mV). Current-step injections around the resting potential were used to estimate the input resistance (116 ± 8.6 MΩ), the membrane time constant (15 ± 1.4 ms), and the membrane capacitance (116 ± 10 pF). SPON cells were never spontaneously active in vitro, but depolarization from rest elicited highly variable spike patterns (data not shown). In contrast, in all recorded SPON neurons, hyperpolarizing currents triggered consistent responses, namely prominent rebound depolarization (RD), which reached threshold for fast action potentials (Fig. 1B) [for definition of RD, see Aizenman and Linden (1999)]. Therefore, this study focuses on the effects of hyperpolarization in these neurons. Most cells (47 of 55) had precisely timed RD that resulted in only one or two action potentials, independent of stimulus amplitude (Fig. 1B,C). Interestingly, in what seems to be a distinct subclass of SPON cells (n = 8), the RDs resulted in complex spike patterns with one overshooting action potential followed by several small spikelets (Fig. 1D). This less common spiking response was restricted to neurons located in the dorsolateral quadrant of the SPON, corresponding to the low-frequency region in vivo (Kulesza et al., 2003). Quantitative comparisons of these subtypes revealed no differences in resting membrane potential, input resistance, capacitance, latency to the first spike, or RD duration; data from both rebound types were therefore pooled.

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Figure 1.

SPON neurons display precisely timed postinhibitory rebound spiking. A, Schematic diagram of SPON circuitry. SPON neurons receive excitatory inputs from octopus cells and multipolar stellate cells in the contralateral ventral cochlear nucleus (VCN). Globular bushy cells in the same VCN drive the inhibitory, glycinergic MNTB projection to the SPON. The vertical dashed line indicates the midline. B, Responses to hyperpolarizing current injections applied in 100 pA increments to an SPON neuron demonstrate the low variability in the latency of the rebound spikes at three current levels. C, Schematic diagram of superior olive shows location of SPON neurons characteristically displaying one or two rebound spikes. D, A subset of SPON neurons displayed a complex rebound spiking pattern with an overshooting spike followed by a few small spikelets (arrowheads). A reconstruction of the distribution of these neurons reveals their location in the dorsolateral quadrant of the SPON. E, Responses to a series of hyperpolarizing currents varying in amplitude (between 0 and −220 pA in −10pA current increments) or duration (between 0 and 260 ms in 10 ms increments with an amplitude of −80 pA in an SPON neuron). F, The average normalized RD area (the integral of the RD response overshooting the resting membrane potential) increases proportionally with increasing current amplitudes. G, The average normalized latency of the peak RD response is plotted as a function of current level. Larger current injections result in earlier RD responses (n = 28). H, The average normalized RD area increases in a linear fashion up to 150 ms durations, after which the response saturates. I, The average normalized latency to the peak RD decreases exponentially with a time constant of 50 ms with increased hyperpolarization duration (n = 28).

The average number of overshooting spikes at the maximal RD response for the entire population of SPON neurons was 1.4 ± 0.1 (n = 55), indicating that a brief RD spiking response is a very robust property of these neurons. The RD spiking response peaked within 5–60 ms (23 ± 2.4 ms; n = 55) after the offset of the hyperpolarizing current step and lasted for 83 ± 6.8 ms (range, 7–152 ms; n = 55).

To systematically investigate how the hyperpolarization affects the RD response, a series of hyperpolarizing current injections at 10 pA increments (Fig. 1E) were used. The area of the RD (above the resting membrane potential) increased in proportion to the hyperpolarization amplitude with a half-maximal current of 90 pA (Fig. 1F). The latency to the RD peak response decreased exponentially with hyperpolarizing amplitude with a similar half-maximal current of 87 pA (Fig. 1G). To assess the effects of the hyperpolarization duration on the RD, the time course of a hyperpolarizing step of fixed amplitude was varied from 0 to 260 ms in 10 ms increments (Fig. 1E). The RD area increased proportionally with the hyperpolarizing durations up to 150 ms, after which the response saturated (Fig. 1H). The RD latency decayed exponentially as a function of the hyperpolarization duration with a time constant of 50 ms (Fig. 1I). The variability in these data, as indicated by the small confidence intervals, was low, confirming that the RD is a consistent feature of SPON neurons.

I_h and low-voltage-activated Ca²⁺ currents are active at subthreshold voltages in SPON neurons

All SPON neurons displayed a prominent depolarizing voltage sag in response to the onset of the hyperpolarizing current pulses (Fig. 1E). This voltage sag is indicative of the hyperpolarization-activated inward cation current (I_h) that is implicated to play a role in RDs of other auditory neurons (Sivaramakrishnan and Oliver, 2001; Koch et al., 2004 or Koch and Grothe, 2003; Tan et al., 2007; Sun and Wu, 2008; Nagtegaal and Borst, 2010). In rats, the lateral superior olive (LSO) and the SPON predominantly express the HCN1 and HCN2 subunits of the hyperpolarization-activated cyclic nucleotide-gated channel complex (Koch et al., 2004; Notomi and Shigemoto, 2004). Immunohistochemistry was used to investigate the expression of both subunits in the mouse SPON. The antiserum directed against HCN1 produced robust staining in the somata (Fig. 2A, arrowheads) and dendrites (Fig. 2A, arrow) of neurons throughout the SPON. Likewise, the antiserum directed against HCN2 also generated strong immunolabeling in SPON neurons (Fig. 2B, arrowheads).

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Figure 2.

SPON neurons express HCN1 and HCN2 channels and have large I_h inwardly rectifying (IR) currents. A, HCN1 immunoreactivity in the superior olivary complex of an adult mouse. The low-magnification micrograph illustrates that the lateral superior olive expresses the strongest HCN1-like immunostaining, the SPON displays moderate levels of expression, and the MNTB shows the weakest immunoreactivity. At higher magnification, it is evident that both somata (arrowheads) and dendrites (arrow) of SPON neurons are immunopositive. Asterisks denote landmarks in slice. Scale bars: left, 200 μm; right, 50 μm. B) HCN2 is most strongly expressed in the MNTB, whereas expression in the LSO is weakest. At higher magnification, it is evident that SPON neurons are also immunopositive. Asterisks denote landmarks in slice. Scale bars: left, 200 μm; right, 50 μm. C, Current traces recorded from an SPON neuron induced by hyperpolarization from a holding current of −62 to −122 mV, in −10 mV voltage steps, under control conditions (top) and during pharmacological blockade of I_h with 20 μm ZD7288 (bottom). D, The size of the IR current was measured at steady state (i.e., ∼1.35 s after induction) at each hyperpolarizing voltage (n = 42). The IR currents were significantly diminished by ZD7288 (n = 6) in the SPON neurons. ***p < 0.001, Student's t test. E, Average activation time constants of the IR currents in SPON neurons were obtained by fitting a single-exponential function to the current traces.

The magnitude of inwardly rectifying currents was measured during hyperpolarizing voltage steps, when large sustained inward currents were observed in SPON neurons (Fig. 2C). At a steady state of −120 mV, this inward current amplitude measured, on average, 1.50 ± 0.09 nA (n = 42) (Fig. 2C), and it was abolished by the I_h antagonist ZD7288 (Fig. 2C,D; 20–50 μm; n = 6). Activation kinetics of the inward current were estimated by fitting single-exponential functions, which revealed an average time constant of 160 ± 12 ms at −120 mV (n = 42) (Fig. 2E). Such intermediate time constants are compatible with heterodimers of HCN1 and HCN2 isoforms (Chen et al., 2001; Ulens and Tytgat, 2001), which is supported by the immunolabeling results.

To investigate the contribution of other currents to the activation of the RD, we measured currents induced at the stimulus offset in response to hyperpolarizing steps in voltage-clamp mode, which triggered large inward currents (Fig. 3A). These currents were reduced by <20% by adding the Na⁺ and I_h blockers TTX and ZD7288, respectively (Fig. 3A). The remaining currents were efficaciously antagonized by Ni²⁺ (100–300 μm), a known blocker of low-voltage-activated (LVA) Ca²⁺ currents (Perez-Reyes, 2003) (Fig. 3A,B; n = 5). These results demonstrate that SPON neurons express LVA Ca²⁺ currents at the offset of hyperpolarization. The average LVA Ca²⁺ current isolated with TTX and ZD7288 at the offset of a hyperpolarization to −120 mV was −627 ± 468 pA (n = 9). Although there was large variability in the size of this current (range, −222 to −1546 pA), this parameter was not correlated with the number of RD spikes (R² = 0.0203) or RD duration (R² = 0.0356). The voltage dependence of the steady-state inactivation of the LVA Ca²⁺ current was estimated by fitting a Boltzmann function to the normalized currents, which generated a half-inactivation voltage, V_1/2 = −92.1 mV, with a slope factor of 6.3 (Fig. 3C; n = 9). This indicates that most of the LVA Ca²⁺ channels in SPON neurons are inactivated at rest and require strong hyperpolarization to reactivate, consistent with the RD dependence on strong or long hyperpolarizations (Fig. 1E).

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Figure 3.

LVA Ca²⁺ currents of T type contribute to the RD response in SPON neurons. A, Voltage-clamped SPON neurons were stepped to −120 mV in 10 mV steps from a holding potential of −60 mV and depolarized back to −60 mV, which triggered large inward currents underlying a rebound response in control conditions (left; n = 9). These rebound currents were partially reduced by TTX (1 μm) and ZD7288 (20 μm; middle) but dramatically reduced by the LVA Ca²⁺ channel blocker Ni²⁺ (right). B, Average rebound current in SPON neurons at hyperpolarizing voltages when isolated with TTX and ZD7288 (black symbols; n = 12) and after application of Ni²⁺ (gray symbols; n = 5; **p < 0.01, Student's t test). C, Voltage dependence of the steady-state inactivation (n = 9) was estimated by fitting a Boltzmann function to the normalized currents, which generated a V_1/2 of −92 mV and a slope factor of 6.3. D, Ca²⁺ measurements from the soma of an SPON neuron during a RD response triggered by preceding hyperpolarizing current injections in −100 pA steps. The horizontal black bar marks the duration of the hyperpolarization. E, Normalized average peak ΔF/F as a function of the amplitude of the preceding current step triggering the RD response (n = 6). F, Somatic Ca²⁺ transients are almost abolished by Mibefradil, indicating that T-type LVA Ca²⁺ currents are active in SPON somata during the RD response.

To determine whether LVA Ca²⁺ currents contribute to the somatic Ca²⁺ response during the RD, a series of Ca²⁺-imaging experiments were also conducted. Analysis of the relative increase in fluorescence compared with background levels (ΔF/F) showed that the RD response generates robust Ca²⁺ transients (Fig. 3D) that increase systematically with the magnitude of the hyperpolarizing current (Fig. 3E). Moreover, a large part of these Ca²⁺ transients are blocked by Mibefradil (Fig. 3F), a specific antagonist of T-type Ca²⁺ channels (Bezprozvanny and Tsien, 1995). Only a small fraction of the ΔF/F, triggered by a −300 pA hyperpolarizing step, remains after blockade with 3 μm Mibefradil (control, 14.5 ± 3.9; Mibefradil, 1.1 ± 0.9; n = 6; p < 0.01). These results demonstrate that I_h currents and T-type LVA Ca²⁺ currents work in tandem to generate the temporally precise RD spiking in SPON neurons.

I_h and LVA Ca²⁺ currents act in tandem to generate the RD in SPON neurons

We next investigated the contribution of the I_h and LVA Ca²⁺ currents in the RD response by applying pharmacological antagonists in current clamp. Blocking I_h currents with bath application of ZD7288 (20–50 μm) abolished RD spiking in some neurons (Fig. 4A; n = 8), but not in others, and therefore the reduction in RD spiking was not statistically significant (Fig. 4B). The main effect of antagonizing I_h was a wider, less temporally precise RD (Fig. 4A), which manifests as a significantly longer RD latency (Fig. 4C). As a result of the wider response, the RD area increased somewhat with application of ZD7288 (Fig. 4D). These results indicate that I_h is an important contributor to generating the RD and that it determines the latency of the RD.

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Figure 4.

I_h and LVA Ca²⁺ currents have complementary roles in the RD response of SPON neurons. A, Top, Voltage responses of an SPON neuron to hyperpolarizing current injections in −100 pA steps in aCSF. Bottom, Voltage response of the same neuron during pharmacological blockade of I_h with ZD7288 (20 μm). The horizontal bar with arrowheads indicates the increase in latency to peak response compared with the control condition. B, C, Average number of RD spikes (B) or latency to peak RD response (C) before (open columns) and after (filled columns) application of ZD7288 at three different current levels. D, Average RD area (above resting membrane potential) before (black symbols) and after (gray symbols) application of ZD7288 at three different current levels. E, Top, Voltage response of an SPON neuron to hyperpolarizing current injections in −100 pA steps in aCSF. Bottom, Voltage response of the same neuron during pharmacological blockade of LVA Ca²⁺ channels with Ni²⁺ (100 μm). Blockade of LVA Ca²⁺ currents abolished RD spiking as indicated by the arrow. F, G, Average number of RD spikes (F) or latency to peak RD response (G) before (open columns) and after (filled columns) application of Ni²⁺ at three different current levels. H, Average RD area (above resting membrane potential) before (black symbols) and after (gray symbols) application of Ni²⁺ at three different current levels. I, Top, Voltage response of an SPON neuron to hyperpolarizing current injections in −100 pA steps in aCSF. Bottom, Voltage response of the same neuron during pharmacological blockade of T-type LVA Ca²⁺ channels with Mibefradil (3 μm). Blockade of T-type LVA Ca²⁺ currents abolished RD spiking as indicated by the arrow. J, K, Average number of RD spikes (J) or latency to peak RD response (K) before (open columns) and after (filled columns) application of Mibefradil at three different current levels. L, Average RD area (above resting membrane potential) before (black symbols) and after (gray symbols) application of Mibefradil at three different current levels. n = 8, 8, and 6 for the respective experiments. *p < 0.05; **p < 0.01, Student's t test.

The residual RD in the presence of ZD7288 (Fig. 4A) indicated that at least one additional current contributes to the response. Blocking LVA Ca²⁺ currents with Ni²⁺ (50–300 μm) abolished RD spiking (Fig. 4E; n = 8). Whereas RD spiking (Fig. 4F) and the RD area (Fig. 4H) decreased significantly with Ni²⁺ application, the latency of the RD was unaffected (Fig. 4G). Mibefradil (3 μm) also abolished rebound spiking (Fig. 4I; n = 6) and had qualitatively similar effects on the RD response as Ni²⁺ application (Fig. 4J–L). Together, I_h currents and T-type LVA Ca²⁺ currents are complementary when generating the temporally precise RD spiking in SPON neurons.

I_h and LVA Ca²⁺ currents contribute to offset responses of SPON neurons in vivo

We recorded responses in vivo from seven neurons in the SPON of young mice (P21–P24; n = 7) to determine the degree to which I_h and LVA Ca²⁺ currents contributed to sound-evoked spiking. After obtaining responses to a CF pure tone under baseline conditions, either the I_h current blocker ZD7288 or the LVA Ca²⁺ current blocker Mibefradil was administered alone, followed by the addition of the second drug. The order of drug application was alternated to assess the actions of each drug in isolation. After responses were obtained with both drugs delivered simultaneously, the neuron was allowed to recover. The complete recording procedure, from baseline to recovery, was completed for four neurons, with ZD7288 applied first in two cases and Mibefradil first in the other two cases. A partial protocol was run for an additional three units, in which data from baseline conditions and either the ZD7288 (n = 1) or the Mibefradil (n = 2) condition were collected. However, in these cases, single-unit isolation was lost before application of the second drug.

SPON neurons showed distinct changes in spiking behavior after ZD7288 and Mibefradil application (Fig. 5). In one example, a robust offset response was observed with little spiking during the stimulus under baseline conditions (Fig. 5A), but after ZD7288 application, the magnitude of offset spiking decreased and spiking that occurred during the stimulus presentation increased substantially, particularly near the onset of the tone (Fig. 5C). When Mibefradil was added along with ZD7288, virtually all spiking was abolished, apart from a well timed onset response (Fig. 5E). After the drugs were allowed to wash out, a large portion of the offset component recovered, but the response duration remained truncated compared with baseline (Fig. 5A,G). For a second representative SPON unit, Mibefradil was applied first, followed by the addition of ZD7288. In this case, Mibefradil acted to reduce the magnitude and duration of offset spiking and decreased the onset response seen in the baseline condition (Fig. 5B,D). When ZD7288 was added, the offset response was abolished, and the onset response that was reduced by Mibefradil increased (Fig. 5F). After both drugs were removed, the onset and offset responses for this unit recovered to near-baseline levels (Fig. 5H).

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Figure 5.

ZD7288 and Mibefradil alter the spiking of SPON neurons in vivo. A–H, PSTHs were constructed for two SPON units under the following conditions: baseline before the application of drugs (A, B); iontophoretic application of either the I_h current blocker ZD7288 (C) or the LVA Ca²⁺ blocker Mibefradil (D) in isolation; combined application of both ZD7288 and Mibefradil simultaneously (E, F); and recovery after washout of the drugs (G, H). Horizontal bars show the location of the stimulus in the recording window. I, J, Spike counts (I) and first-spike latencies (J) were measured for offset responses before and during drug application. Error bars represent the SEMs. ZD, ZD7288; Mb, Mibefradil.

On average, action potentials occurring in the offset time window (5–35 ms poststimulus) decreased by 53% in the presence of ZD7288 compared with baseline conditions (Fig. 5I), and the application of Mibefradil alone resulted in an even larger reduction (78%) in offset spiking. Moreover, although both ZD7288 and Mibefradil applied in isolation acted to substantially limit the offset response, the combination of both drugs nearly eliminated all poststimulus spiking, with <2% of baseline spikes remaining after the combined drug application. In addition to the offset response, drugs also affected spiking during the stimulus presentation, particularly near the tone onset. The application of Mibefradil alone or ZD7288 plus Mibefradil decreased onset spiking by 91 and 77%, respectively, compared with baseline, similar to what was observed for offset responses (data not shown). However, when ZD7288 was applied alone, spiking during the stimulus presentation increased by 152% (Fig. 5A,C).

In addition to spike counts, mean first-spike latencies were also measured to determine the impact of each drug on the temporal precision of SPON offset responses (Fig. 5J). The application of ZD7288 alone elevated offset response first-spike latencies by 21% compared with baseline conditions, whereas Mibefradil alone resulted in an increase of only 6%. When ZD7288 and Mibefradil were combined, latencies increased to 18% above baseline. For onset responses, latencies were highly variable, ranging from 6.1 to 17.0 ms, compared with offset latencies, which ranged from 7.9 to 10.3 ms. In all drug conditions, onset response latencies were elevated compared with baseline conditions, even for ZD7288, which caused a substantial increase in onset spiking.

SPON neurons are intrinsically tuned to low-frequency-modulated current stimuli in vitro

The tuning of SPON neurons to acoustic patterns, such as envelope fluctuations demonstrated in vivo, may reflect specialized intrinsic membrane properties. For instance, SPON neurons express very large I_h currents, which are known to underlie slow (typically within θ range) neuronal oscillations in other parts of the brain, such as the cortex, hippocampus, and thalamus (Pape and McCormick, 1989; Strohmann et al., 1994; Dickson et al., 2000; Fisahn et al., 2002, Nolan et al., 2004).

We injected currents that increased linearly in frequency between 1 and 15 Hz (chirp stimuli). As expected, the SPON neurons fired action potentials at the peaks of the current chirps, driven by both the peak depolarization and the rebound from the preceding hyperpolarizing trough. To search for a preferred frequency, chirps were first tested at spike threshold followed by a systematic increase in current amplitude. Each SPON neuron displayed its own tuning to these frequency-modulated current injections, evidenced by firing one or a few action potentials at a preferred frequency at current threshold (Fig. 6A). We also noted that the neuron's preferred frequency was independent of the duration of the stimulus (Fig. 6A). Likewise, a reversal of the chirp stimulation, i.e., applying a stimulus with a linearly decreasing frequency, did not affect the tuning of these cells (Fig. 6B). After increasing the current levels, the cell's response became progressively less selective, until it fired at all frequencies. This is illustrated using raster plots of the action potentials in response to increasing stimulus strengths to generate a tuning curve for the neuron (Fig. 6C). To estimate the sharpness of the tuning, the bandwidth at the half-maximal response (also called the Q3dB value) was calculated, yielding an average Q3dB of 6.3 ± 2.9 (n = 6). Temperature-related effects on the intrinsic tuning to the chirps were excluded by recording from SPON neurons at 36°C, yielding no difference in tuning from neurons recorded at room temperature (room temperature: 8.2 ± 0.8 Hz, n = 31; 36°C: 9.0 ± 1.3 Hz, n = 12; p = 0.6). A distribution histogram demonstrates that the entire range of frequencies (1–15Hz) was represented in our sample (Fig. 6D), although there was a tendency for more SPON cells to be tuned to frequencies ≤5 Hz (35%). On average, the intrinsic tuning of SPON neurons was equivalent before (younger than P12: 8.1 ± 0.7 Hz, n = 40) and after (older than P12: 9.9 ± 1.5 Hz, n = 10; p = 0.22) the onset of hearing. We did, however, uncover a significant negative correlation between tuning and input resistance (p < 0.001; Fig. 6E), indicating that voltage-dependent ion conductances are involved in controlling the intrinsic frequency tuning of the SPON neurons.

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Figure 6.

SPON neurons are tuned to frequency-modulated current injections. A, Left, Voltage response of an SPON neuron to a current that increases linearly in frequency (chirp) between 1 and 15 Hz over 2 s with a current amplitude of 400 pA. Note that the neuron only fires an action potential at the end of the stimulation. Right, Extending the duration of the stimulus to 30 s using the same holding current does not affect the frequency tuning of the cell. B, Left, Another SPON neuron is tuned to fire at medium frequencies (freq) to a 1–15 Hz chirp stimulation injection of 30 s at a holding current of 10 pA. Right, Reversal of the chirp, i.e., injection of a stimulus that decreases linearly in frequency between 15 and 1 Hz does not affect the tuning of the neuron. C, Examples of tuning curves that were created by measuring the voltage response to the 30 s, 1–15 Hz chirp at the threshold current and increasing the current stepwise. Raster plots illustrate the progressive loss of frequency selectivity at higher current levels. The best frequency (BF), estimated by converting time to frequency for the first spike or the range of spikes at firing threshold, was 2.5 Hz (left) and 12–15 Hz (right), respectively. D, A histogram reveals that the intrinsic tuning of SPON cells to chirp stimuli were distributed over the entire bandwith used, although most cells (35%) were tuned to frequencies <5 Hz. E, The input resistance (R_in) of the SPON neurons was negatively correlated with their intrinsic tuning to the chirp stimuli.

SPON neurons are tuned to low-frequency-modulated acoustic stimuli in vivo

To assess the ability of SPON neurons to encode low-frequency periodic stimuli in vivo, responses were recorded from neurons in young (P20–P22, n = 13) and in adult (P58–P80, n = 19) mice, with CFs ranging from 11 to 40 kHz. Each unit was presented with SAM tones, with the carrier frequency set to the unit's CF and the MF systematically varied from 1 to 20 Hz.

Two types of SAM responses were frequently observed for neurons in both young and adult animals. One type was marked by significant phase-locked spiking to SAM tones with low MFs (Rayleigh test, p < 0.05) and a pronounced decrease in entrainment to cycles of MFs above ∼15 Hz; this type of modulation transfer function was observed in 38% of units in young animals (Fig. 7A) and in 42% of units in adult animals (Fig. 7B). A second type of SAM response, seen in 62 and 58% of units in young (Fig. 7C) and adult (Fig. 7D) animals, respectively, displayed significant phase-locking to all MFs presented. In addition, all neurons that responded to low MFs did so with clustered spiking restricted to approximately one-third of each modulation cycle, as shown by period histograms (Fig. 7, PSTH insets).

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Figure 7.

SPON neurons exhibited two types of modulation transfer functions in response to SAM stimuli in vivo. A, B, One response type observed in SPON neurons of both young (A) (P20–P22; n = 5) and adult (B) (P58–P80; n = 8) animals was marked by temporally precise spiking to each modulation cycle of SAM tones ranging from 1 to 15 Hz, with a sharp decrease phase-locking to modulation frequencies above 15 Hz. C, D, A second type of response to SAM stimuli seen for neurons in young (C) (n = 8) and adult (D) (n = 11) animals consisted of phase-locked spiking to all modulation frequencies presented. Horizontal rippled bars in A–D represent the location of the SAM tone in the recording window. Period histograms (insets) were constructed from spikes that occurred during the stimulus presentation. E, Percentage of neurons in young and adult animals that demonstrated phase-locked responses to SAM stimuli as a function of modulation frequency, as indicated by a significant result of the Rayleigh test. F, G, Mean vector strengths for young (n = 13) and adult (n = 19) neurons in response to low (F) and higher (G) modulation frequencies. Error bars represent the SEMs. mod, Modulation; VS, vector strength.

In both young and adult animals, the highest percentage of phase-locked responses for SPON neurons occurred from 5 to 15 Hz (Fig. 7E). Overall, phase-locking did not significantly differ between units in young and adult animals at any of the MFs tested (1–600 Hz; Mann–Whitney U test, p = 0.07–0.95). However, at very low MFs (1–5 Hz), vector strengths in young animals were noticeably higher than in adults (Mann–Whitney U test, p = 0.07–0.13; Fig. 7F). In all but two cases, neurons that phase-locked to MFs up to 20 Hz (Fig. 7C,D) continued to exhibit significant entrainment to SAM modulations up to 200–400 Hz (Fig. 7G), similar to the phase-locking reported in neurons of the rat SPON (Kulesza et al., 2003; Kadner and Berrebi, 2008).

To assess the effects of I_h and LVA Ca²⁺ currents on the behavior of SPON neurons to periodic stimuli in vivo, responses to SAM stimuli were recorded before, during, and after the application of ZD7288 and/or Mibefradil (Fig. 8). In an example in which ZD7288 was applied first, we observed decreased spiking and phase-locking (Fig. 8A,C). In addition, ZD7288 acted to reduce the range of MFs over which the neuron phase-locked, limiting entrainment to MFs of 1–4 Hz, down from 1–15 Hz in baseline recordings. This finding indicates that I_h is only partially involved in determining the MF tuning of SPON neurons. When Mibefradil was added along with ZD7288, spiking was dramatically reduced, and phase-locking to all MFs was abolished (Fig. 8E). After allowing the drugs to wash out for 1 h, both vector strengths and spiking partially recovered (Fig. 8G). For a second unit, Mibefradil was applied first, resulting in decreased spiking and entrainment to SAM tones (Fig. 8B,D). Although vector strengths of responses to MFs of 6–12 Hz were lower in the presence of Mibefradil, they were still significantly phase-locked to the SAM stimuli (Rayleigh test; p = 0.01–0.001). However, when ZD7288 was combined with Mibedfradil, the spiking rate of the neuron was further reduced, and phase-locking was abolished at all MFs presented (Fig. 8F). After a period of recovery from the drugs, vector strengths returned toward baseline levels, but spiking remained depressed (Fig. 8H). These data demonstrate that the same ion currents that underlie the RD response in vitro are crucial for generating the offset response to tones in vivo.

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Figure 8.

I_h and LVA Ca²⁺ currents contribute to SAM tuning in vivo. Spike rate and vector strength modulation transfer functions (right), along with representative PSTHs (left; p values obtained from the Rayleigh test), are shown for responses to SAM tones recorded from two units under the following conditions: baseline before the application of drugs (A, B);either the I_h current blocker ZD7288 (C) or the LVA Ca²⁺ blocker Mibefradil (D) in isolation; combined application of both ZD7288 and Mibefradil simultaneously (E, F); and recovery after washout of the drugs (G, H). Horizontal rippled bars in each PSTH plot represent the location of the SAM tone in the recording window. Asterisks indicate significant phase-locking as determined by the Rayleigh test (p < 0.05). mod, Modulation; VS, vector strength.