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. 2020 Jan;206(1):15-31.
doi: 10.1007/s00359-019-01382-w. Epub 2019 Nov 28.

Acute neuroestrogen blockade attenuates song-induced immediate early gene expression in auditory regions of male and female zebra finches

Amanda A Krentzel  1   2   3   4 , Maaya Z Ikeda  5   6   7 , Tessa J Oliver  6 , Era Koroveshi  6 , Luke Remage-Healey  8   5   6   7
Affiliations

Acute neuroestrogen blockade attenuates song-induced immediate early gene expression in auditory regions of male and female zebra finches

Amanda A Krentzel et al. J Comp Physiol A Neuroethol Sens Neural Behav Physiol. 2020 Jan.

Abstract

Neuron-derived estrogens are synthesized by aromatase and act through membrane receptors to modulate neuronal physiology. In many systems, long-lasting hormone treatments can alter sensory-evoked neuronal activation. However, the significance of acute neuroestrogen production is less understood. Both sexes of zebra finches can synthesize estrogens rapidly in the auditory cortex, yet it is unclear how this modulates neuronal cell signaling. We examined whether acute estrogen synthesis blockade attenuates auditory-induced expression of early growth response 1 (Egr-1) in the auditory cortex of both sexes. cAMP response element-binding protein phosphorylation (pCREB) induction by song stimuli and acute estrogen synthesis was also examined. We administered the aromatase inhibitor fadrozole prior to song exposure and measured Egr-1 across several auditory regions. Fadrozole attenuated Egr-1 in the auditory cortex greater in males than females. Females had greater expression and clustering of aromatase cells than males in high vocal center (HVC) shelf. Auditory-induced Egr-1 expression exhibited a large sex difference following fadrozole treatment. We did not observe changes in pCREB expression with song presentation or aromatase blockade. These findings are consistent with the hypothesis that acute neuroestrogen synthesis can drive downstream transcriptional responses in several cortical auditory regions, and that this mechanism is more prominent in males.

Keywords: Aromatase; Egr-1; Estrogens; Sex differences; Zebra finch.

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Conflict of interest statement

Conflict of Interest: The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1
Anatomical landmarks and study designs. a) Sagittal schematic of zebra finch brain ~1mm from midline. Red boxes indicate regions of interest that were analyzed across studies. A=dNCM, B=vNCM, C=CMM. b) Sagittal schematic of zebra finch brain ~1.7mm from midline. Red boxes indicate regions of interest that were analyzed across studies. A=pHVCS, B=aHVCS. Red boxes are not representative of size of images. Grey zone is primary auditory cortical region Field L. Abbreviations: cerebellum (Cb) and hippocampus (Hp). c) Experimental design for song exposure. Animals from all studies were exposed to various designs of song exposure. White boxes indicate periods of silence and black boxes indicate periods of triplicate song. At the end of each timeline the protein analyzed is included for that study.
Fig. 2
Fig. 2
Fadrozole treatment decreases auditory-induced Egr-1 expression in secondary auditory regions more robustly in males compared to females. All bar graphs represent means and error bars standard error of the mean across each group (n=4/sex/treatment) per region a) aHVCS, b) pHVCS, c) dNCM, d) vNCM, and e) CMM. Data from Study 1. p<0.05*. f) Egr-1 expression decreases in dorsal NCM of males administered fadrozole but not female zebra finches. Images were taken with a bright field microscope at 20× magnification. Top row includes example images from females exposed to saline (left) and fadrozole (right). Bottom row includes example images from males exposed to saline (left) and fadrozole (right). Images are from Study 1. g) Asterisks next to effect sizes indicate mean differences with 95% confidence intervals (C.I.) that do not contain 0. Dotted-line boxes are around effect sizes that pass the C.I. threshold in males but not females.
Fig. 3
Fig. 3
Egr-1 expression does not degrade with fadrozole in either sex. Bar graph represents means and error bars standard error of the mean for each group (n=3/sex/treatment). Data is from Study 2 testing whether fadrozole administration degrades Egr-1 expression. No statistical significance detected.
Fig. 4
Fig. 4
Fadrozole has no effect on pCREB expression in secondary auditory regions of either sex. All bar graphs represent means and error bars standard error of the mean across each group (n=4/sex/treatment). a) aHVCS, b) pHVCS, c) dNCM, and d) vNCM. Data from Study 1. No significance detected. d) pCREB expression does not decrease in dorsal NCM of either sex administered fadrozole. Images were taken with a brightfield microscope at 20× magnification. Top row includes example images from females exposed to saline (left) and fadrozole (right). Bottom row includes example images from males exposed to saline (left) and fadrozole (right). Images are from Study 1.
Fig. 5
Fig. 5
pCREB expression does not change with song exposure. All bar graphs represent means and error bars standard error of the mean across each group (n=3 silence and 85 seconds, n=4 for 15 minutes). Data for females of dNCM (a) and vNCM (b) and males of dNCM (c) and vNCM (d) from Study 3. White bars are silence group, gray bars short exposure (85 seconds), and black bars longer exposure (15 minutes). No significance detected.
Fig. 6
Fig. 6
Females express more somatic aromatase in pHVCS than males. a) All bar graphs represent means and error bars standard error of the mean across each group (n=6 for males and n=3 for females. b) Example images of aromatase (red) and DAPI (blue) in male (left) and female (right) pHVCS. Dotted white circle shows an example of a 2-cell cluster in the female. Images taken with a confocal microscope at 60× magnification as z-stacks (~9–15um thick). Images are maximal projection images. p<0.05*
Fig. 7
Fig. 7
Aromatase organization into clusters differs by sex in pHVCS. Percentage of aromatase cells found in increasing cluster sizes are normalized to the total amount of aromatase quantified for individual images. All bar graphs represent means and error bars standard error of the mean for each sex (dNCM: n=6 males and n=5 females; anterior and posterior HVCS: n=6 males and n=3 females). Letters indicate posthoc tests for a significant main effect of cluster size. Black letters indicate there is no sex*cluster size interaction and red and blue letters indicate posthocs within each sex for cluster size. Asterisks in b) are between subject post hoc comparisons comparing male and female subjects for each cluster size. p<0.05*, p<0.0001*****.
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