Distribution and chemical composition of estrogen receptor β neurons in the paraventricular nucleus of the female and male mouse hypothalamus

Abstract

Activation of estrogen receptor beta (ERβ)-expressing neurons regulates the mammalian stress response via the hypothalamic-pituitary-adrenal (HPA) axis. These neurons densely populate the paraventricular nucleus of the hypothalamus (PVN). Recent research has revealed striking differences between rat and mouse PVN cytochemistry, but careful exploration of PVN ERβ neurons in mice has been hindered by a lack of specific ERβ antisera. Therefore, we used male and female transgenic mice expressing EGFP under the control of the mouse ERβ promoter (ERβ-EGFP) to examine the chemical architecture of PVN ERβ cells. Using immunohistochemistry, we found that 90% of ERβ-immunoreactivity (-ir) colocalized with EGFP. Cellular colocalization of EGFP with neuropeptides, transcription modulators, and neuronal tracers was examined throughout the PVN. ERβ-EGFP cells expressed oxytocin more abundantly in the rostral (71 ± 3%) than caudal (33 ± 8%) PVN. Arginine vasopressin colocalized with EGFP more often in females (18 ± 3%) than males (4 ± 1%). Moreover, estrogen receptor α-ir colocalized with ERβ-EGFP at low levels (15 ± 3%). Using a corticotropin releasing hormone-cre driver X tdTomato reporter mouse, we found a moderate colocalization with ERβ-ir (48 ± 16%) in the middle PVN. Peripheral injection of fluorogold revealed that the rostral PVN ERβ-EGFP cells are neuroendocrine neurons whereas non-neuroendocrine (presumably pre-autonomic) ERβ-EGFP neurons predominated in the posterior PVN. These data demonstrate chemoarchitectural differences in ERβ neurons of the mouse PVN that are different from that previously described for the rat, thus, elucidating potential neuronal pathways involved in the regulation of the HPA axis in mice.

Keywords: ESR2; RRID:IMSR_JAX:007908; RRID:MMRRC_036904-UC; cytochemistry; estrogen receptor beta; paraventricular nucleus of the hypothalamus.

FIGURE 1

ERβ-EGFP PVN rostrocaudal partition. Photomicrographs showing bright-field Nissl (a–c) and confocal (d–f) images demonstrate the rostral (a, d), middle (b, e) and caudal (c, f) regions of the PVN, which are delineated using a white dashed line. Brain sections are presented rostrocaudally using the Nissl-stained edges (a–c) as the defining borders of the PVN at each level examined. Representative ERβ-EGFP (green) signal images matching the respective rostrocaudal level are shown in panels (d–f). Nuclear ir (To-Pro-3) for contrast is represented in magenta. Scale bars: (a–c) = 200 μm, (d–f) = 100 μm. 3V, Third ventricle. fx, fornix. ot, optic tract [Color figure can be viewed at wileyonlinelibrary.com]

FIGURE 2

Validation of the ERβ-EGFP mouse model for studying PVN ERβ expression. Representative confocal photomicrographs comparing the distribution of ERβ-EGFP in a female mouse (similar pattern is observed in males) (a) with ERβ-ir (c), using the Z8P antibody in the middle-caudal PVN. Enlarged images (b, d, f) shows a higher power view demonstrating the distribution of ERβ-EGFP (green) and ERβ-ir cells (magenta). And their colocalization (e, f). For all high power insets (dotted line box) the scale bar = 10 μm. Scale bars for all other images = 50 μm. 3V, Third ventricle [Color figure can be viewed at wileyonlinelibrary.com]

FIGURE 3

ERβ Z8P antibody validation using the global ERβ knockout mouse model. Photomicrographs showing ERβ-ir in PVN of wildtype (WT; a) and global ERβ knockout (βERKO; b) following IHC using the Z8P antibody (magenta). Scale bars = 50 μm. 3V, Third ventricle [Color figure can be viewed at wileyonlinelibrary.com]

FIGURE 4

OT colocalizes with PVN ERβ-EGFP cells in an PVN region-specific manner. Representative confocal photomicrographs of a female ERβ-EGFP mouse showing rostral (a–d), middle (e–h), and caudal (i–l) PVN oxytocin (OT) immunoreactivity and ERβ-EGFP (n = 3/sex). Bar graph (m) show the mean percentage colocalization of OT in ERβ-EGFP neurons in the rPVN, mPVP, and rPVN of male and female mice (m). % colocalization = number of OT-ir neurons/total number of ERβ neurons in selected region × 100. Data are expressed as mean percentage ± SEM. For all higher power (dotted line box) images (d, h, l) the scale bar = 10 μm. Scale bars for all other images = 50 μm. 3V, Third ventricle. Arrowheads show examples of double labeled cells [Color figure can be viewed at wileyonlinelibrary.com]

FIGURE 5

PVN ERβ-EGFP cells express AVP. Photomicrographs of a female ERβ-EGFP mouse showing rostral (a–d), middle (e–h), and caudal (i–l) AVP immunoreactivity in the PVN of ERβ-EGFP brain slices (n = 3–4/sex). Bar graphs (m) show the mean percentage colocalization of AVP in ERβ-EGFP neurons in the rPVN, mPVP, and rPVN of male and female mice. Representative male and female photomicrographs showing distribution of colocalized and non-colocalized cells in the mPVN. % colocalization = number of AVP-ir neurons/total number of EGFP neurons in each selected region. Data are expressed as mean percentage ± SEM. For all high power (dotted line box) images (d, h, l) the scale bar = 10 μm. Scale bars for all other images = 50 μm. 3V, Third ventricle. Arrowheads show examples of colocalized cells [Color figure can be viewed at wileyonlinelibrary.com]

FIGURE 6

ERα/ERβ-EGFP colocalized cells are present in low levels in the male and female mouse PVN. Photomicrographs of a female ERβ-EGFP mouse showing ERα immunoreactivity (magenta) in the rostral (a–d), middle (e–h), and caudal (i–l) parts of the PVN of ERβ-EGFP animals (n = 3–4/sex). Bar graphs (panel m) represent the mean percentage colocalization of ERα in ERβ-EGFP neurons of male and female mice. % colocalization = number of colocalized ERα/total number of ERβ neurons in selected region. Data are expressed as mean percentage ± SEM. For all high-power images (dotted line box) (d, h, l) the scale bar = 10 μm. Scale bars for all other images = 50 μm. 3V, Third ventricle. Arrowheads show examples of dual labeled cells [Color figure can be viewed at wileyonli-nelibrary.com]

FIGURE 7

ERβ-EGFP neurons do not colocalize with TH-ir neurons in the mouse PVN. Photomicrographs showing overlayed confocal images of the PVN through the rostral (a), middle (b), and caudal (c) regions of an ERβ-EGFP female mouse. Scale bars = 50 μm. 3V, Third ventricle [Color figure can be viewed at wileyonlinelibrary.com]

FIGURE 8

CRH-tdTomato cells co-express ERβ-ir in the male and female mouse PVN. Photomicrographs of a male CRH-cre; TdTomato mouse showing confocal images of the PVN through the rostral (ad), middle (e–h), and caudal (i–l) regions of CRH-cre;TdTomato animals. ERβ immunoreactivity was detected using the Z8P antibody (n = 3/sex). Bar graphs (m) representing the mean percentage colocalization of CRH-TdTomato expressing neurons and ERβ-ir neurons in the rPVN, mPVP, and rPVN of male and female mice. % colocalization = number of TdTom positive neurons/total number of ERβ-ir neurons in selected region. Data are expressed as the mean percentage ± SEM. All higher power images (dotted line box) (d, h, l) have a scale bar = 10 μm. Scale bars for all other images = 50 μm. 3V, Third ventricle. Arrowheads show examples of a dual labeled cell [Color figure can be viewed at wileyonlinelibrary.com]

FIGURE 9

ERβ-EGFP neurons are neuroendocrine neurons in the rostral but not caudal portions of the PVN. Confocal photomicrographs of a female ERβ-EGFP mouse showing the distribution of fluorogold (neuroendocrine) neurons in the rostral (a–d), middle (e–h), and caudal (i–l) portions of the PVN of ERβ-EGFP brain slices (n = 3–4/sex). EGFP was detected using an anti-GFP antibody. Fluorogold neurons were filled via subcutaneous injection of FG 5 days prior to euthanasia and harvesting the brain. Bar graph (m) represent the mean percentage colocalization of FG in ERβ-EGFP-ir neurons in the rPVN, mPVP, and rPVN of male and female mice. % colocalization = number of FG positive cells/total number of ERβ-EGFP-ir × 100. Data are expressed as mean percentage ± SEM. All high-power images (dotted line box) (d, h, l) have a scale bar = 10 μm. Scale bars for all other images = 50 μm. 3V, Third ventricle. Arrowheads show examples of dual labeled cells [Color figure can be viewed at wileyonlinelibrary.com]