Nucleus paragigantocellularis afferents in male and female rats: organization, gonadal steroid receptor expression, and activation during sexual behavior

Abstract

The supraspinal regulation of genital reflexes is poorly understood. The brainstem nucleus paragigantocellularis (nPGi) of rats is a well-established source of tonic inhibition of genital reflexes. However, the organization, gonadal steroid receptor expression, and activity of nPGi afferents during sex have not been fully characterized in male and female rats. To delineate the anatomical and physiological organization of nPGi afferents, the retrograde tracer Fluoro-Gold (FG) was injected into the nPGi of sexually experienced male and female rats. Animals engaged in sexual behavior 1 hour before sacrifice. Cells containing FG, estrogen receptor-alpha (ER(alpha)), androgen receptor (AR), and the immediate-early gene product Fos were identified immunocytochemically. Retrograde labeling from the nPGi was prominent in the bed nucleus of the stria terminalis, paraventricular nucleus (PVN), posterior hypothalamus, precommissural nucleus, deep mesencephalic nucleus, and periaqueductal gray (PAG) of both sexes. Sex differences were observed in the caudal medial preoptic area (MPO), with significantly more FG+ cells observed in males, and in the PAG and inferior colliculus, where significantly more FG+ cells were observed in females. The majority of regions that contained FG+ cells also contained ER(alpha) or AR, indicating sensitivity to gonadal steroids. The proportions of FG+ cells that co-localized with sex-induced Fos was high in the PVN of both sexes and high in the MPO of males but low in the PAG of both sexes despite the large number of PAG-nPGi output neurons and Fos+ cells in both sexes. The characterization of these afferents will lead to a further understanding of the neural regulation of genital reflexes.

Figure 1

Photomicrograph depicting a typical Fluorogold (FG) injection in the nucleus paragigantocellularis (nPGi) of the male (A) and female (B) rat. Note that FG injections are centered in the nPGi with little spread to surrounding regions. 4V=4^th ventricle, py=pyramidal tract.

Figure 2

Plots of FG+ cells retrogradely labeled from the nPGi (black: ipsilateral to injection site, gray: solely contralateral to injection site). Also plotted are FG+ cells containing estrogen receptor alpha (ER_α; pink), or androgen receptor (AR; blue) of a representative male (left side) and female (right side) rat in all sections examined, relative to Bregma.

Figure 2

Plots of FG+ cells retrogradely labeled from the nPGi (black: ipsilateral to injection site, gray: solely contralateral to injection site). Also plotted are FG+ cells containing estrogen receptor alpha (ER_α; pink), or androgen receptor (AR; blue) of a representative male (left side) and female (right side) rat in all sections examined, relative to Bregma.

Figure 2

Plots of FG+ cells retrogradely labeled from the nPGi (black: ipsilateral to injection site, gray: solely contralateral to injection site). Also plotted are FG+ cells containing estrogen receptor alpha (ER_α; pink), or androgen receptor (AR; blue) of a representative male (left side) and female (right side) rat in all sections examined, relative to Bregma.

Figure 3

Mean number (± standard error of the mean (SEM)) of FG+ cells retrogradely labeled from the nPGi for four rostrocaudal levels of the medial preoptic area (MPO; A). The mean percentage (± SEM) of FG+ cells that also contained ER_α (B) or AR (C) is also shown. Numbers below atlas images are millimeters relative to Bregma. Note that females have a higher number of FG+ cells in the rostral MPO, whereas males have a higher number of FG+ cells in the caudal MPO. Also note that females have a higher proportion of ER_α/FG+ in the rostral-most level, whereas males have a higher proportion of ER_α/FG+ cells in a caudal level. A higher proportion of AR/FG+ cells at all levels of the MPO were observed in males. * = significant male/female difference, p≤0.05; MPO=medial preoptic area.

Figure 4

Mean number (± SEM) of FG+ cells retrogradely labeled from the nPGi in all regions with where dense labeling was observed (A). Mean percentage (± SEM) of FG+ cells that also contained ER_α (B) or AR (C) are also shown. Note the regional specificity in sex differences in FG+ cells. Also note the similarity between the sexes in the proportion of ER_α/FG+ cells, but the male-bias at almost all levels with respect to the proportion of AR/FG+ cells. * = significant male/female difference, p≤0.0031; r/cBNST= rostral/caudal bed nucleus of the stria terminalis, CeM=central amygdala, PVN=paraventricular nucleus, PH=posterior hypothalamus, PeF=perifornical nucleus, PrC=precommissural nucleus, PF=parafascicular thalamic nucleus, DpMe=deep mesencephalic nucleus, InCo=intercollicular nucleus, rSC=rostral superior colliculus, DpG=deep gray layer of the superior colliculus, ECIC=external cortex of the inferior colliculus, m5=motor root of the trigeminal nerve, LRt=lateral reticular nucleus, Sp5I=interpolar spinal trigeminal nucleus

Figure 5

Photomicrograph of a representative section of the paraventricular nucleus (PVN) in a male (A and C) and female (B and D) rat. C and D are higher magnifications of the area indicated by the box. Neurons stained brown contain FG retrogradely labeled from the nPGi, nuclei stained black contain AR and neurons stained brown with nuclei stained black contain both FG and AR. Note that both males and females have a similar high density of retrogradely labeled cells, and that some of these cells contain AR.

Figure 6

Mean number (± SEM) of FG+ cells retrogradely labeled from the nPGi for six rostrocaudal levels of the periaqueductal gray (PAG; A). The mean percentage (± SEM) of FG+ cells that also contained ER_α (B) or AR (C) is also shown. Numbers below atlas images are millimeters relative to Bregma. Note that females have a higher number of FG labeled cells throughout the PAG, most notably in the dorsomedial subdivision of the PAG, and that females differed from males significantly at four rostrocaudal levels. Also note that males have a higher proportion of ER_α/FG+ or AR/FG+ cells at almost all levels. * = significant male/female difference, p≤0.05

Figure 7

Plots of FG+ cells retrogradely labeled from the nPGi (black: ipsilateral to injection site; gray: solely contralateral to injection site), and FG+ cells also containing Fos (green) of representative male (left sides) and female (right side) rat in all sections examined.

Figure 7

Plots of FG+ cells retrogradely labeled from the nPGi (black: ipsilateral to injection site; gray: solely contralateral to injection site), and FG+ cells also containing Fos (green) of representative male (left sides) and female (right side) rat in all sections examined.

Figure 7

Plots of FG+ cells retrogradely labeled from the nPGi (black: ipsilateral to injection site; gray: solely contralateral to injection site), and FG+ cells also containing Fos (green) of representative male (left sides) and female (right side) rat in all sections examined.

Figure 8

Mean proportion (± SEM) of FG+ cells retrogradely labeled from the nPGi that also contain sex-induced Fos for four rostrocaudal levels of the MPO. Numbers below atlas images are millimeters relative to Bregma. Note that females have a higher proportion of Fos/FG+ cells in rostral MPO levels, and that males have a higher proportion of Fos/FG+ cells in caudal MPO levels. * = significant male/female difference, p≤0.05.

Figure 9

Mean proportion (± SEM) of FG+ cells that also contain sex-induced Fos in all regions with dense input to the nPGi. Note the regional specificity in sex differences in the proportion of Fos/FG+, and that only a few regions show high sex-induced Fos in projections to the nPGi. No differences reached significance at the α ≤ 0.0031 level.

Figure 10

Photomicrograph of a representative section of the perifornical nucleus (PeF) of the hypothalamus in a male (A and C) and female (B and D) rat. C and D are higher magnifications of the area indicated by the box. Neurons stained brown contain FG retrogradely labeled from the nPGi, nuclei stained black contain sex-induced Fos, and neurons stained brown with nuclei stained black are retrogradely labeled FG cells that also contain sex-induced Fos. Note that females have a higher proportion of FG labeled cells that also contain sex-induced Fos.

Figure 11

Photomicrograph of a representative section of the PVN in a male (A and C) and female (B and D) rat. C and D are higher magnifications of the area indicated by the box. Neurons stained brown contain FG retrogradely labeled from the nPGi, nuclei stained black contain sex-induced Fos, and neurons stained brown with nuclei stained black contain both FG and Fos. Note that both males and females have a similar high density of retrogradely labeled cells, and that many of these cells contain sex-induced Fos.

Figure 12

Mean proportion (± SEM) of FG+ cells retrogradely labeled from the nPGi that also contain sex-induced Fos for six rostrocaudal levels of the PAG. Numbers below atlas images are millimeters relative to Bregma. Male, dark gray; female, light gray. Note that in both sexes the proportion of Fos/FG+ cells is quite low. No differences reached significance at the α ≤ 0.05 level.

Figure 13

Summary diagram of regions that provide dense input to the nPGi in male (A) and female (B) rats. The color and length of bars adjacent to labels represent the relative density of FG labeled cells that also contain ER_α (pink), AR (blue), or sex-induced Fos (green) in those regions.