Reduction in the density and expression, but not G-protein coupling, of serotonin receptors (5-HT1A) in 5-HT transporter knock-out mice: gender and brain region differences

Abstract

The aim of the present study was to investigate the mechanisms underlying the desensitization of 5-HT(1A) receptors in the dorsal raphe and hypothalamus of serotonin (5-HT) transporter knock-out mice (5-HTT -/-). The density of 5-HT(1A) receptors in the dorsal raphe was reduced in both male and female 5-HTT -/- mice. This reduction was more extensive in female than in male 5-HTT -/- mice. 8-OH-DPAT-induced hypothermia was absent in female 5-HTT -/- and markedly attenuated in 5-HTT +/- mice. The density of 5-HT(1A) receptors also was decreased significantly in several nuclei of the hypothalamus, amygdala, and septum of female 5-HTT -/- mice. 5-HT(1A) receptor mRNA was reduced significantly in the dorsal raphe region, but not in the hypothalamus or hippocampus, of female 5-HTT +/- and 5-HTT -/- mice. G-protein coupling to 5-HT(1A) receptors and G-protein levels in most brain regions were not reduced significantly, except that G(o) and G(i1) proteins were reduced modestly in the midbrain of 5-HTT -/- mice. These data suggest that the desensitization of 5-HT(1A) receptors in 5-HTT -/- mice may be attributable to a reduction in the density of 5-HT(1A) receptors. This reduction is brain region-specific and more extensive in the female mice. The reduction in the density of 5-HT(1A) receptors may be mediated partly by reduction in the gene expression of 5-HT(1A) receptors in the dorsal raphe, but also by other mechanisms in the hypothalamus of 5-HTT -/- female mice. Finally, alterations in G-protein coupling to 5-HT(1A) receptors are unlikely to be involved in the desensitization of 5-HT(1A) receptors in 5-HTT -/- mice.

Fig. 1.

Autoradiography of ¹²⁵I-MPPI binding in 5-HTT mutant mice. Left, Wild-type 5-HTT mice (5-HTT +/+). Middle, Heterozygous 5-HTT knock-out mice (5-HTT +/−). Right, Homozygous 5-HTT knock-out mice (5-HTT −/−). The brain sections from top to bottom are striatum (bregma 0.98–0.50 mm), medium hypothalamus (bregma 0.7 to −1.06 mm), caudal hypothalamus (bregma −1.34 to −1.94 mm), and midbrain (bregma −4.36 to −4.84 mm). ACo, Anterior cortical amygdaloid nucleus; AH, anterior hypothalamic nucleus;BLA, basolateral amygdaloid nucleus, anterior;BMA, basomedial amygdaloid, anterior;CA1–CA3, CA1–CA3 field of hippocampus;CeC, central amygdaloid nucleus, capsular division;CeMAD, central amygdaloid nucleus, medial anterodorsal;CeMAV, central amygdaloid nucleus, medial anteroventral;DEn, dorsal endopiriform nucleus; DG, dentate gyrus of hippocampus; DMN, dorsomedial hypothalamic nucleus; DRN, dorsal raphe nucleus;La, lateral amygdaloid nucleus; Ld, lambdoid septal zone; LH, lateral hypothalamic nucleus;LSI, lateral septal nucleus, intermediate;MeA, medial amygdaloid nucleus, anterodorsal;MRN, medial raphe nucleus; MS, medial septal nucleus; PVN, paraventricular hypothalamic nucleus; VMN, ventromedial hypothalamic nucleus.

Fig. 2.

Autoradiography of in situhybridization for 5-HT_1A receptor mRNA in 5-HTT mutant mice. The brain sections were hybridized with ³²P-riboprobe encoding the third intracellular loop, as described in Materials and Methods. The brain sections were adjacent to the medial hypothalamus and midbrain sections in the ¹²⁵I-MPPI binding.

Fig. 3.

An example of competitive RT-PCR for 5-HT_1A receptor mRNA in the dorsal raphe of 5-HTT mutant mice. A, Autoradiography of DNA sequence gel that resolves RT-PCR products from 5-HT_1A mRNA (5-HT_1A) and 5-HT_1A RNA standard (5-HT_1A st). Four concentrations of 5-HT_1A RNA standard were used to compete 5-HT_1A mRNA (10 ng of total RNA). B, Linear regression curve for calculation of concentration of 5-HT_1AmRNA. The intercept of the x-axis represents the concentration of 5-HT_1A mRNA.

Fig. 4.

8-OH-DPAT-stimulated ³⁵S-GTP-γ-S binding in the hippocampus of normal mice. Top,³⁵S-GTP-γ-S binding in the presence of 8-OH-DPAT.Middle, ³⁵S-GTP-γ-S binding in the absence of 8-OH-DPAT. Bottom, Nonspecific binding defined by the presence of 10⁻⁵mGTP-γ-S.

Fig. 5.

Autoradiography of 8-OH-DPAT-stimulated³⁵S-GTP-γ-S binding in the midbrain sections of 5-HTT mutant mice. Left, ³⁵S-GTP-γ-S binding in the presence of 8-OH-DPAT. Right,³⁵S-GTP-γ-S binding in the absence of 8-OH-DPAT.

Fig. 6.

G-protein concentrations in the midbrain (top) and hypothalamus (bottom) of female 5-HTT mutant mice. The data represent the mean ± SEM of eight mice per group. *Significant difference from 5-HTT +/+ mice;p < 0.05.

Fig. 7.

Hypothermic response to 8-OH-DPAT in female 5-HTT knock-out mice. The data represent the mean ± SEM of 10 mice per group. Two-way ANOVA: main effect of genotype,F_(2,147) = 31.07, p< 0.0001; main effect of time,F_(6,147) = 8.77, p< 0.0001; interaction between genotype and time,F_(12,147) = 1.49, p= 0.132. *Significant difference from those in the 0 time points of the same genotype of 5-HTT, p < 0.05 (Student–Newman–Keuls' multiple range test). #, Significant difference from the 5-HTT +/+ mice (at same time point),p < 0.05 (Student–Newman–Keuls' multiple range test).