Specific and rapid effects of acoustic stimulation on the tonotopic distribution of Kv3.1b potassium channels in the adult rat

Abstract

Recent studies have demonstrated that total cellular levels of voltage-gated potassium channel subunits can change on a time scale of minutes in acute slices and cultured neurons, raising the possibility that rapid changes in the abundance of channel proteins contribute to experience-dependent plasticity in vivo. In order to investigate this possibility, we took advantage of the medial nucleus of the trapezoid body (MNTB) sound localization circuit, which contains neurons that precisely phase-lock their action potentials to rapid temporal fluctuations in the acoustic waveform. Previous work has demonstrated that the ability of these neurons to follow high-frequency stimuli depends critically upon whether they express adequate amounts of the potassium channel subunit Kv3.1. To test the hypothesis that net amounts of Kv3.1 protein would be rapidly upregulated when animals are exposed to sounds that require high frequency firing for accurate encoding, we briefly exposed adult rats to acoustic environments that varied according to carrier frequency and amplitude modulation (AM) rate. Using an antibody directed at the cytoplasmic C-terminus of Kv3.1b (the adult splice isoform of Kv3.1), we found that total cellular levels of Kv3.1b protein-as well as the tonotopic distribution of Kv3.1b-labeled cells-was significantly altered following 30 min of exposure to rapidly modulated (400 Hz) sounds relative to slowly modulated (0-40 Hz, 60 Hz) sounds. These results provide direct evidence that net amounts of Kv3.1b protein can change on a time scale of minutes in response to stimulus-driven synaptic activity, permitting auditory neurons to actively adapt their complement of ion channels to changes in the acoustic environment.

Figure 1. Illustrations of auditory stimuli

(a) Spectrogram of the complex control stimulus (dynamic moving ripple; “DMR”) composed of 0.5 octave-wide sounds centered on either low (3.36–4.76 kHz; “4 kHz”) or high (26.9–38.06 kHz; “32 kHz”) frequencies. The DMR is smoothly and randomly modulated both in time (0–40 Hz) and frequency (spectral contrast between 0–0.5 cycles/octave). Scale bars represent 1 second and 0.25 octaves along the horizontal and vertical arms, respectively. (b) AM sound stimuli were carried by either 4 kHz or 32 kHz pure tones and modulated at either low rates (60 Hz ± 5 %) or high rates (400 Hz ± 5 %).

Figure 2. Effects of sound amplitude modulation and carrier frequency on Kv3.1b levels in the MNTB

(a) Pseudocolor images showing the pattern and intensity of Kv3.1b labeling in representative MNTB sections from rats belonging to each of the six stimulus groups. Scale bar: 100 μm. (b) Kv3.1b labeling intensity following 30 minutes of acoustic stimulation varies according to both the carrier frequency and modulation rate of the stimulus, with the highest levels of Kv3.1b immunoreactivity observed when the high-frequency tone is modulated at the fastest rate. Data are presented as means ± S.E. The number of cells (N) in each stimulus condition were as follows: N (4 kHz: DMR) = 1,333; N (4 kHz: 60 Hz AM) = 1,763; N (4 kHz: 400 Hz AM) = 1,481; N (32 kHz: DMR) = 1,970; N (32 kHz: 60 Hz AM) = 2,430; N (32 kHz: 400 Hz AM) = 1,535. Differences between groups were statistically evaluated using both a one-way and two-way ANOVA followed by Bonferroni post-hoc analyses. * indicates p < 0.01 compared to other 4 kHz stimuli; ** indicates p <0.001 compared to other 32 kHz stimuli; ## indicates two-way interaction of p <0.001 between carrier frequency and modulation rate. (c) High-magnification confocal images of Kv3.1b immunofluorescence in representative cells from each sound stimulation condition. The subcellular pattern of Kv3.1b immunofluorescence was consistent with the channel’s known membrane and cytoplasmic localization (Li et al., 2001; Elezgarai et al., 2003) and did not vary across stimulation conditions. Scale bar: 0.25 μm.

Figure 3. Effects of carrier frequency on distribution of Kv3.1b immunoreactivity along the tonotopic axis of the MNTB

(a) The medial-to-lateral ratio of Kv3.1b-labelled cells varies according to the carrier frequency of the stimulus. Data are presented as means ± S.E. * indicates p < 0.01. The dotted line represents the average medial-to-lateral ratio across all groups (1.30 ± 0.08). The number of sections (n) in each stimulus condition were as follows: n (4 kHz: DMR) = 15; n (4 kHz: 60 Hz AM) = 14; n (4 kHz: 400 Hz AM) = 15; n (32 kHz: DMR) = 19; n (32 kHz: 60 Hz AM) = 23; n (32 kHz: 400 Hz AM) = 13. Differences between groups were statistically evaluated using a two-way ANOVA (b) Probability histograms of pooled cells in each condition demonstrate that the 400 Hz AM stimuli had a stronger effect (D = 0.15) on the distributions of Kv3.1 in the MNTB than either the 60 Hz AM (D = 0.049) or the DMR (D = 0.076) stimuli. * indicates p < 0.02, ***** indicates p < 4.0 × 10⁻¹². Lines above the probability histograms span the median 50% of cells in each distribution (solid lines represent 32 kHz stimuli, dotted lines represent 4 kHz stimuli). The number of cells (N) in each stimulus condition were as follows: N (4 kHz: DMR) = 1,384; N (4 kHz: 60 Hz AM) = 1,637; N (4 kHz: 400 Hz AM) = 1,475; N (32 kHz: DMR) = 1,828; N (32 kHz: 60 Hz AM) = 2,264; N (32 kHz: 400 Hz AM) = 1,118.