Sex and age differences in brain-derived neurotrophic factor and vimentin in the zebra finch song system: Relationships to newly generated cells

Abstract

The neural song circuit is enhanced in male compared with female zebra finches due to differential rates of incorporation and survival of cells between the sexes. Two double-label immunohistochemical experiments were conducted to increase the understanding of relationships between newly generated cells (marked with bromodeoxyuridine [BrdU]) and those expressing brain-derived neurotrophic factor (BDNF) and vimentin, a marker for radial glia. The song systems of males and females were investigated at posthatching day 25 during a heightened period of sexual differentiation (following BrdU injections on days 6-10) and in adulthood (following a parallel injection paradigm). In both HVC (proper name) and the robust nucleus of the arcopallium (RA), about half of the BrdU-positive cells expressed BDNF across sexes and ages. Less than 10% of the BDNF-positive cells expressed BrdU, but this percentage was greater in juveniles than adults. Across both brain regions, more BDNF-positive cells were detected in males compared with females. In RA, the number of these cells was also greater in juveniles than adults. In HVC, the average cross-sectional area covered by the vimentin labeling was greater in males than females and in juveniles compared with adults. In RA, more vimentin was detected in juveniles than adults, and within adults it was greater in females. In juveniles only, BrdU-positive cells appeared in contact with vimentin-labeled fibers in HVC, RA, and Area X. Collectively, the results are consistent with roles of BDNF- and vimentin-labeled cells influencing sexually differentiated plasticity of the song circuit.

Keywords: AB_514483; AB_528504; AB_630940; neurogenesis; radial glia; sexual differentiation; songbird.

Figure 1

Estimates of the total number of BDNF+ cells in the HVC (top) and RA (bottom) of 25-day-old and adult zebra finches. Means + SEM are indicated, and sample sizes are in parentheses. In HVC, a main effect of sex was detected, as was an interaction between age and sex. Males had more BDNF expressing cells than females at both ages, but the difference is larger in adults. In RA, a main effect of age and a significant effect of sex were detected. The two variables also interacted. For each brain region, common symbols over individual bars denote significant differences between them in pairwise comparisons.

Figure 2

Photographs of HVC in males and females at 25 days of age (d25) and in adulthood (Ad). The nissl stained sections (left) and those exposed to immunohistochemistry for BDNF and BrdU (right) are adjacent. Arrows indicate the borders of the brain region. Inserts (right column) show examples from the same animals of double-labeled cells in the two males, neighboring BrdU+ (blue/gray) and BDNF+ (brown) single labeled cells in the juvenile female, and a single labeled BDNF+ cell in the adult female.

Figure 3

Photographs of RA in males and females at 25 days of age (d25) and in adulthood (Ad). Pairs of nissl stained sections (left) and those exposed to immunohistochemistry for BDNF and BrdU (right) are adjacent. Arrows indicate the borders of the brain region. Inserts (right column) indicate cells labeled for both BrdU (blue/gray, nuclear) and BDNF (brown, cytoplasmic).

Figure 4

Average cross-sectional area covered by vimentin labeling (left) and of the full brain region defined by nissl staining (right) in the HVC (top), RA (middle) and Area X (bottom) of juvenile and adult zebra finches of both sexes. Means + SEM are indicated, and sample sizes are in parentheses. In HVC, main effects of both sex and age were detected for the area covered by vimentin labeling. A main effect of sex also existed for the overall cross-sectional area defined by nissl staining, and an interaction between sex and age was present. In RA, an increase in the vimentin covered area and a decrease in the nissl defined area were found in females compared to males. Significant effects of age for vimentin, and interactions of age and sex in both markers, were also detected. In Area X, juveniles had a larger area covered by vimentin and a smaller nissl defined area than adults. The brain region could not be defined in females. Asterisks spanning bars indicate effects of age within a sex; # denotes a sex difference within adults; ^ indicates a sex difference within juveniles.

Figure 5

Photographs of HVC in males and females at 25 days of age (d25) and in adulthood (Ad). The nissl stained sections (left column) and those exposed to immunohistochemistry for vimentin and BrdU (middle column) are adjacent. Arrows in these sections indicate the borders of the brain region. The column on the right contains photos of higher magnification from near the center of HVC in the immunohistochemical sections immediately to their left. Arrows indicate BrdU+ nuclei and vimentin+ fibers that appear to be in contact, and the arrowhead in the top right image points to an isolated BrdU+ cell. Birds in the top two images (25-day-old) received scores of “2” in the qualitative assessment of contacts between BrdU− and vimentin-positive cells. The bottom two (adults) both received scores of “0”.

Figure 6

Photographs of RA in males and females at 25 days of age (d25) and in adulthood (Ad). The nissl stained sections (left column) and those exposed to immunohistochemistry for vimentin and BrdU (middle column) are adjacent. Arrows in these sections indicate the borders of the brain region. The column on the right contains photos of higher magnification from near the center of RA in the immunohistochemical sections immediately to their left. Arrows indicate BrdU+ nuclei and vimentin+ fibers that appear to be in contact. Birds in the top two images (25-day-old) received scores of “3” in the qualitative assessment of contacts between BrdU− and vimentin-positive cells. The bottom two (adults) both received scores of “0”.

Figure 7

Photographs of Area X in a 25-day-old (d25) and an adult (Ad) male. The nissl stained sections (left column) and those exposed to immunohistochemistry for vimentin and BrdU (middle column) are adjacent. Arrows in these sections indicate the borders of the brain region. The column on the right contains photos of higher magnification from near the center of Area X in the immunohistochemical sections immediately to their left. Arrows indicate BrdU+ nuclei and vimentin+ fibers that appear to be in contact, and the arrowheads in the top right image points to isolated BrdU+ cells. The bird in the top image (25-day-old) received a score of “1” in the qualitative assessmemt of contacts between BrdU− and vimentin-positive cells. The bottom individual (adult) received a “0”.