Deafening drives cell-type-specific changes to dendritic spines in a sensorimotor nucleus important to learned vocalizations

Abstract

Hearing loss prevents vocal learning and causes learned vocalizations to deteriorate, but how vocalization-related auditory feedback acts on neural circuits that control vocalization remains poorly understood. We deafened adult zebra finches, which rely on auditory feedback to maintain their learned songs, to test the hypothesis that deafening modifies synapses on neurons in a sensorimotor nucleus important to song production. Longitudinal in vivo imaging revealed that deafening selectively decreased the size and stability of dendritic spines on neurons that provide input to a striatothalamic pathway important to audition-dependent vocal plasticity, and changes in spine size preceded and predicted subsequent vocal degradation. Moreover, electrophysiological recordings from these neurons showed that structural changes were accompanied by functional weakening of both excitatory and inhibitory synapses, increased intrinsic excitability, and changes in spontaneous action potential output. These findings shed light on where and how auditory feedback acts within sensorimotor circuits to shape learned vocalizations.

Figure 1. Deafening causes a cell type-specific decrease in the size of dendritic spines in HVC_X neurons

A) Examples of a lentivirally-labeled HVC_RA neuron (top, arrowhead) and HVC_X neuron (bottom), double-labeled with red and blue retrograde tracers, respectively. Scale bars, 20 μm. B) Representative images showing spine size measurements (see Experimental Procedures) from an HVC_X neuron before and after deafening. Spines tend to increase in size over 24h intervals prior to deafening (size index > 1) and decrease in size following deafening (size index < 1). Scale bar, 5 μm. Note that image contrast has been enhanced for figure presentation; adjustments to brightness and contrast were not made to images used for data analysis. Although the size of all clearly visible spines was scored, for clarity, the scores for only a few representative spines are indicated in this and subsequent figures. C) Deafening causes a cell type-specific decrease in spine size index in HVC_X neurons (p = 0.03, Wilcoxon signed-ranks test) but not in HVC_RA neurons (p = 0.67). Numbers in parentheses indicate the total number of each PN type analyzed. See also Supplemental figure 1.

Figure 2. Analysis of song spectral features reveals that significant song degradation occurs within several days after deafening

A) Example of syllable-by-syllable analysis carried out for a bird whose song contains four syllables, A–D. Although syllable entropy and EV were both analyzed, only EV is shown for clarity. Song behavior is shown for two pre-deafening days (−2 and 0) and post-deafening days 1, 2, 4, and 9. Red arrowhead indicates the first syllable to undergo significant spectral degradation (syllable C, shown in red, defined as onset of degradation using one-way ANOVA). B) Representative spectrograms of the same song analyzed in A. Syllable C (red bars) underwent degradation starting at 2d post-deafening. EV values are included below each rendition of the syllable. Frequency range (y axis), 0.4–9 kHz, scale bar (white bar, bottom spectrogram), 150 ms. C) The onset of song degradation (day of post-deafening song) was not related to the age at which birds were deafened (p = 0.82, linear regression). D) Older birds sang a larger number of motifs before their songs underwent significant degradation (p < 0.01, linear regression). These data come from a subset of the birds analyzed in C (8/19). See also Supplemental figure 2.

Figure 3. Deafening-induced decreases in spine size in HVC_X neurons precede song degradation

A) Deafening causes a cell type-specific decrease in HVC_X spine size index that occurs prior to the onset of song degradation (time > 0 is post-degradation). Data are grouped into 4 time bins (2 bins before onset of song degradation and 2 bins after), the average time of deafening is just prior to night −2, and asterisks indicate a significant difference (p < 0.05, two-factor ANOVA) between the two PN types for a particular time bin. Numbers in parentheses indicate the total number of each PN type analyzed. B) Spines from HVC_X neurons and HVC_RA neurons from longitudinally-imaged, age-matched control birds do not exhibit decreases in size index. See also Supplemental figure 3.

Figure 4. Decreases in HVC_X spine size index predict the severity of song degradation on the subsequent day of singing

A) Normalized spine size index measurements from HVC_X neurons were compared to the amount of song degradation on the following day of singing (day +1). The amount of song degradation on each day was defined for each bird by calculating the percentage decrease from the baseline value of entropy or EV for the syllable that degraded first. This comparison reveals that larger decreases in spine size index predict a larger magnitude of song degradation on the subsequent day (p < 0.001, linear regression). B) Comparisons were made between spine size index measurements from HVC_X neurons and the amount of song degradation on days preceding, intervening, or following the 24h size index measurement, and correlation coefficients for those comparisons are plotted. Only comparison of spine size changes to the following day’s song degradation yielded a significant relationship.

Figure 5. Deafening causes a cell type-specific decrease in spine stability that follows the onset of song degradation

A) Representative images showing spine stability measurements from an HVC_X neuron before and after deafening. ‘S’ indicates spines that were stable over 2h, ‘+’ indicates spines that were gained, and ‘−’ indicates spines that were lost. Although the stability of all clearly visible spines was scored, for clarity, the scores for only a few representative spines are indicated in this figure. Scale bar, 5 μm. B) Deafening causes a cell type-specific decrease in spine stability in HVC_X neurons that occurs after the onset of song degradation (time > 0 is post-degradation). Data are grouped into 4 time bins, the average time of deafening is just prior to night −2, and asterisks indicate a significant difference (p < 0.05, two-factor ANOVA) between the two PN types for a particular time bin. Numbers in parentheses indicate the total number of each PN type analyzed. C) Spine gain and spine loss both tend to increase in HVC_X neurons following deafening. D) Spines from HVC_X and HVC_RA neurons in longitudinally-imaged, age-matched controls do not exhibit decreases in stability. See also Supplemental figure 4.

Figure 6. Deafening decreases the amplitude of spontaneous synaptic activity and alters spontaneous action potential activity in HVC_X neurons

A) Top: example traces of depolarizing postsynaptic potentials (dPSPs) from HVC_X neurons recorded in current-clamp configuration in anesthetized hearing control and deafened birds. Deafening drives a significant decrease in the amplitude of spontaneous dPSPs (bottom left, p < 0.0001, KS test) but has no significant effect on dPSP frequency (bottom right, p = 0.30, Mann Whitney U test). B) Top: example traces of spontaneous action potential activity from HVC_X neurons recorded in anesthetized hearing control and deafened birds. Deafening drives a significant decrease in ISI duration (bottom left, p < 0.0001, KS test) but has no significant effect on mean spontaneous action potential frequency (bottom left, p = 0.25, Mann Whitney U test). Synaptic activity was measured while injecting tonic hyperpolarizing current into the recorded cell; action potential activity was measured without current injection. See also Supplemental figure 5.

Figure 7. Deafening decreases the amplitude of both excitatory and inhibitory miniature synaptic currents in HVC_X neurons

A) Left: Confocal images of a retrogradely-labeled HVC_X neuron that was recorded in whole-cell voltage-clamp configuration in vitro and filled with Alexa 488 (green). Arrowheads in the lower image indicate retrogradely-labeled HVC_X neurons (red); retrograde labeling underwent bleaching over the course of the experiment. Scale bar, 20 μm. Right: Representative mIPSCs and mEPSCs are shown for HVC_X neurons recorded in brain slices from control and deafened birds. B) Deafening significantly decreases the amplitude of mEPSCs (left) and mIPSCs (right) in HVC_X neurons (KS test: p < 0.01 for mEPSCs, p < 0.0001 for mIPSCs). Numbers in parentheses indicate the total number of HVC_X neurons analyzed in each experimental group.