Loss of TRPC2 function in mice alters sex differences in brain regions regulating social behaviors

Abstract

The transient receptor potential cation channel 2 (TRPC2) conveys pheromonal information from the vomeronasal organ (VNO) to the brain. Both male and female mice lacking this gene show altered sex-typical behavior as adults. We asked whether TRPC2, highly expressed in the VNO, normally participates in the development of VNO-recipient brain regions controlling mounting and aggression, two behaviors affected by TRPC2 loss. We now report significant effects of TRPC2 loss in both the posterodorsal aspect of the medial amygdala (MePD) and ventromedial nucleus of the hypothalamus (VMH) of male and female mice. In the MePD, a sex difference in neuron number was eliminated by the TRPC2 knockout (KO), but the effect was complex, with fewer neurons in the right MePD of females, and fewer neurons in the left MePD of males. In contrast, MePD astrocytes were unaffected by the KO. In the ventrolateral (vl) aspect of the VMH, KO females were like wildtype (WT) females, but TRPC2 loss had a dramatic effect in males, with fewer neurons than WT males and a smaller VMHvl overall. We also discovered a glial sex difference in VMHvl of WTs, with females having more astrocytes than males. Interestingly, TRPC2 loss increased astrocyte number in males in this region. We conclude that TRPC2 normally participates in the sexual differentiation of the mouse MePD and VMHvl. These changes in two key VNO-recipient regions may underlie the effects of the TRPC2 KO on behavior.

Keywords: amygdala; astrocyte; hypothalamus; laterality; neuron; sex difference; stereology.

Figure 1:. Loss of TRPC2 signaling alters posterodorsal aspect of the medial amygdala (MePD) regional volume in a sex- and hemisphere-specific manner.

(i) WT adult B6/129S males (M) have larger MePD than females (F) and the MePD of WT males is also lateralized, being larger in the right (R) hemisphere than the left (L) in Nissl-stained sections. MePD volume is not lateralized in WT females. In TRPC2 KO mice, the sex difference remains in the right hemisphere but is lost in the left. TRPC2 loss reduced MePD volume in both sexes, in a lateralized manner, significantly decreasing the left MePD in KO males, and significantly decreasing the right MePD in KO females. Consequently, the lateralization of MePD is lost in KO males, while the KO induces laterality in females that is absent in WT females. These complex effects of TRPC2 loss on the MePD suggest sex-specific and lateralized mechanisms regulate the sex-specific behaviors altered in KOs. Bar values are group means ± standard errors of the mean; numbers on bars indicate the n per group. Data points are individual values by hemisphere. The symbols > and < indicate significant main effects of sex or genotype and within-subject laterality with p<0.05.

Figure 2:. Sex differences and laterality of the adult posterodorsal aspect of the medial amygdala (MePD) are affected in TRPC2 KO mice.

A MePD in Nissl-stained sections from males (M) have more neurons than females (F), regardless of genotype. There was no significant effect of genotype on this measure (F_(1,29)=3.16, p=0.086). A(ii) Looking across hemispheres, the sex difference in the lateralized number of MePD neurons in WT mice is flipped by the loss in TRPC2, where only females display laterality. In short, the number of neurons shows sex differences in the left (L) hemisphere of WT mice, while KO mice show sex differences in number of neurons only on the right (R). B Neuronal somata are larger in male mice than females, regardless of genotype or hemisphere. C The number of glia in the MePD is greater in males than females, in both WT and KO mice, and is also reduced in KO mice compared to WTs. This count includes all glia types i.e., it does not differentiate between astrocytes, microglia, oligodendrocytes etc. While TRPC2 loss significantly reduced glial number the sex difference is retained, KO males have more glia than KO females. There were no hemisphere differences in overall glial number in Nissl-stained sections from either genotype in Nissl stain. D In glial fibrillary acidic protein-(GFAP)-stained tissue, the MePD males have more astrocytes than females in both WT and KO mice, and there was no main effect of genotype on this measure. In both WT and KO mice, females have more astrocytes in the left MePD than the right. These data show that TRPC2 has a significant influence in determining many cellular attributes of the adult MePD that may contribute to behavioral effects in KO mice. Bar values are group means ± standard errors of the mean, numbers on bars indicate the n per group. Data points are individual values by hemisphere. The symbols > and < indicate main effects of sex or genotype and within-subject laterality with p<0.05.

Figure 3:. Loss of TRPC2 introduces a sex difference in the regional volume of the ventromedial hypothalamus (VMH).

A) The volume of the ventrolateral (vl) subdivision of the VMH was reduced in TRPC2 KO mice, but only in males. B) Volume of the dorsomedial (dm) subdivision of the VMH was not affected by sex or genotype. There was no laterality detected in any measures of the VMH, in either genotype. Bar values are means ± standard error of the mean, data points are individual values averaged across hemisphere, bar numbers indicate the n per group, *=significant gene by sex interactions with p<0.05.

Figure 4:. TRPC2 KO affects neuronal size in the ventromedial hypothalamus (VMH).

A) There was no sex difference in the number of neurons in the VMH ventrolateral subdivision (vl) of WT mice, but TRPC2 KO reduced the number of neurons in males only. B) TRPC2 KO reduced the size of VMHvl neurons in both sexes. C) The number of overall glia in the VMHvl displayed neither a sex difference nor an effect of genotype in Nissl stained sections. D) The number of astrocytes seen in glial fibrillary acidic protein-stained sections was lower in males than in females for WT mice, but this sex difference was lost in TRPC2 KO mice due to an increase in astrocyte number in KO males. E, F) In contrast to measures of the vl subdivision of the VMH, there were no effects of sex or genotype on neither neuronal number nor number of glia in the dorsomedial (dm) subdivision. Bar values are means ± standard error of the mean, data points are individual values averaged across hemispheres, bar number represents n per group, *=significant gene by sex interactions with p<0.05.