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Comparative Study
. 2012 Aug 1;520(11):2531-44.
doi: 10.1002/cne.23061.

Astrocytes in the rat medial amygdala are responsive to adult androgens

Ryan T Johnson  1 , Amanda Schneider, Lydia L DonCarlos, S Marc Breedlove, Cynthia L Jordan
Affiliations
Comparative Study

Astrocytes in the rat medial amygdala are responsive to adult androgens

Ryan T Johnson et al. J Comp Neurol. .

Abstract

The posterodorsal medial amygdala (MePD) exhibits numerous sex differences including differences in volume and in the number and morphology of neurons and astroctyes. In adulthood, gonadal hormones, including both androgens and estrogens, have been shown to play a role in maintaining the masculine character of many of these sex differences, but whether adult gonadal hormones maintain the increased number and complexity of astrocytes in the male MePD was unknown. To answer this question we examined astrocytes in the MePD of male and female Long Evans rats that were gonadectomized as adults and treated for 30 days with either testosterone or a control treatment. At the end of treatment brains were collected and immunostained for glial fibrillary acidic protein. Stereological analysis revealed that adult androgen levels influenced the number and complexity of astrocytes in the MePD of both sexes, but the specific effects of androgens were different in males and females. However, sex differences in the number and complexity of adult astrocytes persisted even in the absence of gonadal hormones in adulthood, suggesting that androgens also act earlier in life to determine these adult sex differences. Using immunofluorescence and confocal microscopy, we found robust androgen receptor immunostaining in a subpopulation of MePD astrocytes, suggesting that testosterone may act directly on MePD astrocytes to influence their structure and function.

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Figures

Figure 1
Figure 1
Representative photomicrographs of coronal sections through the adult MePD of a genetic male mouse with the testicular feminized mutation (Tfm) of the androgen receptor (AR) gene (A) and a normal wildtype male mouse (B) immunostained for androgen receptor (AR; Epitomics #1852-1 rabbit monoclonal anti-AR antiserum). Note the distinct and robust nuclear labeling (arrows) in the MePD of the normal adult male that is absent in the MePD of the Tfm male. Scale bar = 100 μm.
Figure 2
Figure 2
MePD volume is sexually dimorphic and maintained by androgens in adulthood especially in the right hemisphere. A: Volume of the adult MePD in rats is both sexually differentiated and asymmetric, as males have a larger MePD than females, and the right (R) MePD is larger than the left (L) in both sexes. B: T treatment increases MePD volume overall in both the left and right hemispheres of rats gonadectomized in adulthood (collapsed across sex). However, analyzing the hemispheres separately reveals that T treatment significantly increases the volume of the left MePD only in females (C) but increases the volume of the right MePD in both sexes (D). Values are means of n = 9 rats/group (±SEM).
Figure 3
Figure 3
Astrocyte numbers are lateralized, sexually dimorphic, and maintained by adult androgens in female rats. A: The number of astrocytes in the adult MePD is both sexually differentiated and asymmetric, with males having more MePD astrocytes overall than females, and with the right (R) MePD containing more astrocytes than the left (L) in both sexes. B: Testosterone (T) treatment increased the number of MePD astrocytes only in the right hemisphere (collapsed across sex). Examining the effect of androgens separately in each hemisphere revealed that in the left hemisphere, males have more MePD astrocytes than do females. (C). Males also have more astrocytes than females in the right MePD, where T treatment appears to result in more astrocytes in both sexes (D) (the difference in males is marginally significant, P = 0.056). Values are means of n = 9 rats/group (±SEM). Note that astrocyte numbers in this figure closely mirror the differences in MePD volume in Figure 1, indicating that the density of astrocytes per unit volume is maintained when androgens affect volume in the right MePD.
Figure 4
Figure 4
Astrocyte complexity is increased by androgens in adult male rats. Number of primary branches (A), branch points (B), branch endings (C), and average branch length (D) of astrocytes were measured in the MePD of adult gonadectomized male and female rats treated with testosterone (T) or blank capsules for 30 days. In males, astrocyte process complexity in both the left and right MePD is greater with T treatment, suggesting that adult androgens may normally maintain the elaborate processes of adult MePD astrocytes. The exception to this pattern is average branch length of MePD astrocytes (D), which was unaffected by androgens in males and was the only measure of MePD astrocyte complexity affected by T treatment in females, and only in the right hemisphere. Values are means of n = 5 rats/group, (±SEM).
Figure 5
Figure 5
Photomicrograph depicting a relatively simple astrocyte from a male rat gonadectomized for 30 days (A) and two relatively large and complex astrocytes from a male rat gonadectomized and treated with testosterone for 30 days (B). Testosteronetreated males generally had more complex astrocytes compared with blank-treated males, but simple and complex astrocytes could be found in the MePD of both treatment groups. Scale bar = 10 μm.
Figure 6
Figure 6
Confocal image showing nuclear AR immunoreactivity (magenta) within astrocytes of the arcuate nucleus (A) and CA1 hippocampus (B). Arrows point to AR+ immunoreactivity in nuclei of GFAP expressing cells (green). Scale bars = 10 μm.
Figure 7
Figure 7
Confocal images showing androgen receptor (AR) immunoreactivity (red) within nuclei of MePD astrocytes. In (A), DAPI, AR, and GFAP reactivity is shown for the same field of view in addition to a combined sequential scan for these three labels and indicates colocalization of DAPI and AR staining within the same nuclei (arrows) of GFAP+ astrocytes. The intensity of AR labeling in astrocytes varied and was often less intense than what was seen in presumptive neuronal nuclei. In (B), an MePD astrocyte nucleus with AR immunoreactivity is shown with orthogonal views (along the yellow and pink lines) demonstrating that AR immunoreactivity is within the same Z-plane as the astrocytic nucleus. Scale bars = 10 μm.
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