Molecular regulation of sexual preference revealed by genetic studies of 5-HT in the brains of male mice

Abstract

Although the question of to whom a male directs his mating attempts is a critical one in social interactions, little is known about the molecular and cellular mechanisms controlling mammalian sexual preference. Here we report that the neurotransmitter 5-hydroxytryptamine (5-HT) is required for male sexual preference. Wild-type male mice preferred females over males, but males lacking central serotonergic neurons lost sexual preference although they were not generally defective in olfaction or in pheromone sensing. A role for 5-HT was demonstrated by the phenotype of mice lacking tryptophan hydroxylase 2 (Tph2), which is required for the first step of 5-HT synthesis in the brain. Thirty-five minutes after the injection of the intermediate 5-hydroxytryptophan (5-HTP), which circumvented Tph2 to restore 5-HT to the wild-type level, adult Tph2 knockout mice also preferred females over males. These results indicate that 5-HT and serotonergic neurons in the adult brain regulate mammalian sexual preference.

Figure 1. Male-male mounting and USV by mice lacking central serotonergic neurons

The numbers of animals used and statistical analysis are all included in Supplementary Data 1. In all figures, * indicates p<0,05, ** p<0.01 and *** p<0.001. a–d a test male was presented in its home cage with an adult wt male and its behavior was recorded for 30 min (all data shown as mean±SEM). Compared with Lmx1b^+/+, Lmx1b^+/− or ePet1-Cre, Lmx1b^−/− males mounted males at a higher percentage (a), lower latency (b), higher frequency (c) and longer duration (d). e, Typical USV patterns emitted by males when presented with female or male intruders. The two left panels show USVs in 2 min, while the two right panels show parts of USV graphs at higher magnifications. The X axis shows time in seconds and the Y axis shows frequency. f, Female intruders elicited USV from almost all males of ePet1-Cre, Lmx1b^−/− , Lmx1b^+/+ ,or Lmx1b^+/−. Male intruders elicited USVs more from Lmx1b^−/− males than from ePet1-Cre, Lmx1b^+/+ or Lmx1b^+/− males. g, The number of USVs emitted by Lmx1b^−/− males towards males is higher than those by ePet1-Cre, Lmx1b^+/+ or Lmx1b^+/− males, whereas ePet1-Cre, Lmx1b^+/+, Lmx1b^+/− and Lmx1b^−/− males were similar in USVs towards females.

Figure 2. Lack of sexual preference by mice without central serotonergic neurons

Each test male was presented with a male and an estrous female, and its mating choice was analyzed for 15 mins. a, more ePet1-Cre, Lmx1b^+/+ and Lmx1b^+/− males mounted female than male targets. A similar percentage of Lmx1b^−/− males mounted females and males. b, ePet1-Cre, Lmx1b^+/+ and Lmx1b^+/− males mounted female targets faster than male targets. Mounting latencies of Lmx1b^−/− males for females and males were similar. c, more than 40% Lmx1b^−/− but none of the ePet1-Cre, Lmx1b^+/+ or Lmx1b^+/− males chose the male as their first mounting target. d, ePet1-Cre males mounted females significantly more often than males as did Lmx1b^+/+ and Lmx1b^+/− males. Lmx1b^−/− males mounted females as often as males (p>0.05, t test). e, ePet1-Cre males spent more time mounting females than males, as did Lmx1b^+/+ and Lmx1b^+/− males. Lmx1b^−/− males did not show differences in mounting males or females. f, The mounting frequency ratio of Lmx1b^−/− was different from those of ePet1-Cre, Lmx1b^+/+ and Lmx1b^+/−.

Figure 3. Loss of sexual preference for genital odor and bedding by males without central serotonergic neurons

a, Lmx1b^+/+ males spent more time sniffing female than male genital odor as did Lmx1b^+/− males. Lmx1b^−/− males spent a similar amount of time on female and male genital odor. Three groups were not significantly different in male genital odor sniffing time but Lmx1b^−/− males spent less time in sniffing female genital odor than the other 2 groups. b, Sniffing ratio of Lmx1b^−/− males was significantly different from Lmx1b^+/+ and Lmx1b^−/− males (p<0.05 for Lmx1b^+/+ vs. Lmx1b^−/−, p<0.05 for Lmx1b^+/− vs. Lmx1b^−/−, p>0.05 for Lmx1b^+/+ vs. Lmx1b^+/−, one-way ANOVA). c, Compared with Lmx1b^+/+ and Lmx1b^+/−, a higher percentage of Lmx1b^−/− males spent more time sniffing male than female genital odor. d, ePet1-Cre males spent more time above female bedding than male bedding as did Lmx1b^+/+ and Lmx1b^+/− males. Lmx1b^−/− males spent a similar amount of time above female and male bedding. Compared with ePet1-Cre,Lmx1b^+/− and Lmx1b^+/+, Lmx1b^−/− males spent less time above female bedding but more time above male bedding. e, The bedding time ratio of Lmx1b^−/− was different from ePet1-Cre and Lmx1b^+/+. f, Compared with ePet1-Cre, Lmx1b^+/+ and Lmx1b^+/−, a significantly higher percentage of Lmx1b^−/− males spent more time above male bedding.

Figure 4. Odor discrimination

a, Both Lmx1b^+/+ and Lmx1b^−/− males showed habituation and dishabituation in sniffing time. No statistic difference was found between Lmx1b^+/+ and Lmx1b^−/− males at any point. b, After 7 training sessions with male and female urine, No significant difference was detected between Lmx1b^+/+ and Lmx1b^−/− males at any point.

Figure 5. Brain chemistry and behaviors of Tph2 knockout males

Compared with Tph2^+/+ and Tph2^+/−, Tph2^−/− males showed a shorter latency (a) and higher frequency in mounting males (b). c, Both Tph2^+/+ and Tph2^+/− males significantly preferred female over male bedding, whereas Tph2^−/− males did not show preference between male and female bedding. d, Both Tph2^+/+ and Tph2^+/− males significantly preferred female over male genital odor, whereas Tph2^−/− males did not show preference between male and female genital odor.

Figure 6. 5-HTP rescue of chemical and behavioral deficits in Tph2 knockout mice

a, Levels of 5-HT and 5-HIAA were analyzed in Tph2^+/+ and Tph2^−/− males 35 min after injection of either 5-HTP (40 mg/kg body weight) or control saline. b–c Male-male mounting in Tph2^−/− mice was significantly rescued by 5-HTP: the latency was lengthened and frequency reduced. d, Bedding preference was monitored between 35 and 40 min after injection. 5-HTP could significantly restore the preference of female over male bedding by Tph2^−/− males.