Deafening alters neuron turnover within the telencephalic motor pathway for song control in adult zebra finches

Abstract

In the telencephalon of adult songbirds, projection neurons are lost and replaced within the efferent pathway controlling learned vocal behavior. We examined the potential role of auditory experience in regulating the addition and long-term survival of vocal control neurons in adult male zebra finches. Deafened and control birds were injected with the cell birth marker [(3)H]thymidine and then killed 1 or 4 months later. At the 1 month survival time, the number of [(3)H]-labeled neurons present in the high vocal center (HVC) was 70% lower in deafened birds compared with controls. This was true for all [(3)H]-labeled HVC neurons, as well as the subset that projected to the robust nucleus of the archistriatum. Over the next 3 months, two-thirds of the [(3)H]-labeled HVC neurons in control birds were lost, presumably through cell death. Surprisingly, deafened birds showed no loss over this interval. The total number of HVC neurons did not differ between control and deafened birds at either survival time. Nuclear diameters of [(3)H]-labeled HVC neurons decreased with cell age in both control and deafened birds, a process that may relate to the eventual death and replacement of these cells. These results suggest that experience influences the addition and also the longer-term fate of neurons formed in adulthood. We propose that auditory deprivation decreases the incorporation of new neurons and prolongs their life span. Alterations in the neuronal replacement cycle may relate to the gradual deterioration in song that occurs after deafening in adult zebra finches.

Fig. 1.

A, Low-magnification fluorescence (UV) photomicrograph of a 6 μm parasagittal HVC section. Over half of all HVC neurons project to RA, and when these cells are retrogradely labeled by Fluoro-Gold (white), HVC clearly stands out from surrounding areas. Dorsal is up, and caudal is to the left. The hippocampus, which normally overlies HVC, was lost during tissue processing. B, C, Higher power combined fluorescence–bright-field photomicrographs of the same field viewed with different fluorescence filters. InB, exposed silver grains (black) overlying the nucleus of two cells indicate that these cells were produced at or shortly after injections of [³H]thymidine. One of these cells (right) also contains Fluoro-Gold in the cytoplasm (whitish stipple) when visualized with UV fluorescence, indicating that it was an adult-formed RA-projecting HVC neuron. InC, the same field is viewed with rhodamine fluorescence to show all cells counterstained with fluorescent cresyl violet. The [³H]-labeled cell on the left is not easily discernable in C because it is not in the same focal plane as the silver grains overlying its nucleus. Scale bars: A, 200 μm; B, C, 10 μm.

Fig. 2.

Mean ± SEM estimates of HVC volume (A), total neuron number (B), and total number of RA-projecting HVC neurons (C) as defined by Fluoro-Gold (Fg) backfills from RA. Two to 3 weeks after deafening in adulthood, experimental birds and controls were injected with [³H]thymidine and then killed either 1 or 4 months after the last injection. No significant differences were found on any of these measures between deafened and control birds.

Fig. 3.

Mean ± SEM estimates of the total number of new HVC neurons ([³H]) and new RA-projecting HVC neurons ([³H]+Fg) at two survival times after [³H]thymidine injections. The 1 month survival time was designed to measure the effects of deafening on HVC neuronal incorporation. Comparisons between the 1 and 4 month survival times permitted an analysis of the subsequent survival of these cells. There was a dramatic difference between deafened and control birds in the total number of new HVC neurons (p = 0.004) and the fraction that projected to RA, as defined by Fluoro-Gold (Fg) backfills from RA, at the 1 month survival time (p < 0.016). In contrast, there were no significant group differences at the 4 month survival time. This was because of a significant decrease in total new HVC neurons and new RA-projecting cells in control (p < 0.04) but not deafened birds (p > 0.3) between the two survival times.

Fig. 4.

Sonograms from one control bird (birdBWB24; top two panels) and one deafened bird (bird PINK35; bottom two panels). Song recordings were made before deafening (PRE-RECORDING) and 4 months after deafening (POST-). Control birds were recorded at the same times. Song begins with one to several repetitions of the same short introductory note (i), followed by a regularly repeating series of distinct notes that differ by their duration, frequency envelope, and/or extent of frequency modulation (labeled1–4 for both birds). In hearing birds, note sequences and morphology are stable from one rendition to the next as shown by the two recordings, spaced 4 months apart. In contrast, note morphology was substantially degraded in deafened birds between the two recording times. Notes 1 and 4 in the preoperative song may correspond to postoperative notes A andD, respectively. However, notes B andC bear little similarity to notes 2 and3.