Roles for gamma-aminobutyric acid in the development of the paraventricular nucleus of the hypothalamus

Abstract

The development of the hypothalamic paraventricular nucleus (PVN) involves several factors that work together to establish a cell group that regulates neuroendocrine functions and behaviors. Several molecular markers were noted within the developing PVN, including estrogen receptors (ER), neuronal nitric oxide synthase (nNOS), and brain-derived neurotrophic factor (BDNF). By contrast, immunoreactive gamma-aminobutyric acid (GABA) was found in cells and fibers surrounding the PVN. Two animal models were used to test the hypothesis that GABA works through GABA(A) and GABA(B) receptors to influence the development of the PVN. Treatment with bicuculline to decrease GABA(A) receptor signaling from embryonic day (E) 10 to E17 resulted in fewer cells containing immunoreactive (ir) ERalpha in the region of the PVN vs. control. GABA(B)R1 receptor subunit knockout mice were used to examine the PVN at P0 without GABA(B) signaling. In female but not male GABA(B)R1 subunit knockout mice, the positions of cells containing ir ERalpha shifted from medial to lateral compared with wild-type controls, whereas the total number of ir ERalpha-containing cells was unchanged. In E17 knockout mice, ir nNOS cells and fibers were spread over a greater area. There was also a significant decrease in ir BDNF in the knockout mice in a region-dependent manner. Changes in cell position and protein expression subsequent to disruption of GABA signaling may be due, in part, to changes in nNOS and BDNF signaling. Based on the current study, the PVN can be added as another site where GABA exerts morphogenetic actions in development.

Figure 1

Digital images show 50μm thick coronal sections taken in the region of the PVN at E17. Sections contain immunoreactive ERα and were counterstained for Nissl substance to delineate the boundaries of the paraventricular nucleus (dotted line). This series of sections shows the rostral (A) to caudal (D) extent of the PVN. All sections used for analysis came from the central region of the PVN (B, C). PVN= paraventricular nucleus, V= third ventricle, scale bars= 100μm.

Figure 2

Digital images show several immunoreactive peptides, receptors, and signaling molecules that populate the paraventricular nucleus (PVN) by embryonic day 15. Corticotropin releasing hormone (A), vasopressin (B), calbindin (C), neuronal nitric oxide synthase (D), estrogen receptor (ER)β (E), ERα (F), galanin (G), and neuropeptide Y (H) delineated distinct populations of cells in the PVN at early ages (scale bars = 50μm). The third ventricle is the white region on the left of each image. As the PVN developed, becoming larger in volume, the numbers of cells expressing these markers increased but with similar patterns of distribution.

Figure 3

Digital images show immunoreactive GAD 67 (A) or GABA (B-C) surrounded the region of the developing paraventricular nucleus of the hypothalamus (PVN) at embryonic ages. At E13 (A), E15 (B), and E17 (C) in the mouse, the PVN region is one of two hypothalamic regions where GABA is found surrounding the nucleus in development. The ventromedial nucleus of the hypothalamus (VMN), the other nuclear group where this has been found, is shown within the images of panels A and B. By adulthood GABAergic fibers have moved into the region of the PVN and VMN (data not shown). At the center of each image is the third ventricle (scale bars = 100μm).

Figure 4

Digital images show ERα immunoreactivity at E17 in female mice exposed to bicuculline between days E10 and E17. Bicuculline treatment (panel B, n=3) as compared to saline controls (panel A, n=3) caused about a 30% decrease in total ERα immunoreactivity (C). Immunoreactive ERα levels were lower both within the paraventricular nucleus (PVN; third ventricle is to the right in each image) and outside following 1mg/kg bicuculline treatment. There was a significant interaction between subjects in location by treatment (p < 0.01; scale bars =100μm).

Figure 5

Digital images of sections containing immunoreactive ERα that were counterstained with thionin to show the boundaries of the paraventricular nucleus (PVN) and the lateral group of cells containing immunoreactive ERα outside of these boundaries (E15 in A, E17 in B, scale bars = 50μm). In the adult (C), this lateral group of ERα cells is still visible outside of the PVN and appears to be just dorsal to the descending columns of the fornix (Fx). The third ventricle is the white area to the right in each image (scale bars =250μm).

Figure 6

Digital mages show ERα immunoreactivity at P0 in GABA_BR1 subunit wildtype (A,D) and knockout (B,E) mice in the plane of section corresponding to figure 4B. Seven 100μm wide columns were placed over images with the edge of the third ventricle and the top of the paraventricular nucleus as the boundaries for the grid. Female GABA_BR1 subunit knockouts (n=6) had an approximately 80% decrease in immunoreactivity in column 1, as measured by mean pixels², the first 100μm from the ventricle (Nguyen-Ba-Charvet et al., 2004), and an approximately 70% increase in immunoreactive area in column 5 (400-500μm from the ventricle), versus wildtype littermates (n=6). There was no difference in levels of immunoreactivity in male GABA_BR1 subunit knockout mice (D-F; n=6) as compared to wildtype (n=6) (scale bars =100μm).

Figure 7

Digital images show immunoreactive GABA receptor subunits and their relationship to ERα in the paraventricular nucleus (PVN). Adjacent sections with ERα (A) and GABA_BR1 subunit (B) immunoreactivity at E17. Black arrows point to immunoreactivity within the region of the paraventricular nucleus (PVN) and white arrows point to immunoreactivity lateral to the classical boundaries of the PVN. Based on the locations of the immunoreactive elements in adjacent sections, these proteins may be expressed in some of the same cells. γ2 (C) and α1 (D) are two GABA_A receptor subunits expressed in the PVN at E17 (V= third ventricle; scale bars =100μm).

Figure 8

Digital images show nNOS immunoreactive cells and fibers in the paraventricular nucleus (PVN) at E17 (A, B). Immunoreactive elements are more dispersed in the GABA_BR1 knockout (B,D n=5) in comparison to wildtype littermates (A,C n=5). NNOS immunoreactivity in the central PVN is found near the lateral boundaries of the PVN. To visualize the spread of immunoreactivity, a grid was placed over the image of the PVN (using the top of the PVN as the dorsal boundary and the third ventricle as the medial boundary). In the knockout, there was an approximate 20% increase in the number of boxes containing immunoreactive elements (E). The overall amount of immunoreactivity did not change (F) (scale bars =100μm). *Asterisk represents p<0.03.

Figure 9

GABA_BR1 subunit knockout mice (B, n=5) had a 32% decrease in immunoreactive BDNF in the PVN compared to wildtype mice (A, n=7) with a significant difference between genotypes (p<0.05). Analyzing the immunoreactivity by location revealed that the largest decrease in protein levels was between 300 and 500μm from the edge of the third ventricle (C, scale bars =100μm). *Asterisks represent p<0.05.