Limbic Neurons Shape Sex Recognition and Social Behavior in Sexually Naive Males

Abstract

Sexually naive animals have to distinguish between the sexes because they show species-typical interactions with males and females without meaningful prior experience. However, central neural pathways in naive mammals that recognize sex of other individuals remain poorly characterized. We examined the role of the principal component of the bed nucleus of stria terminalis (BNSTpr), a limbic center, in social interactions in mice. We find that activity of aromatase-expressing BNSTpr (AB) neurons appears to encode sex of other animals and subsequent displays of mating in sexually naive males. Silencing these neurons in males eliminates preference for female pheromones and abrogates mating success, whereas activating them even transiently promotes male-male mating. Surprisingly, female AB neurons do not appear to control sex recognition, mating, or maternal aggression. In summary, AB neurons represent sex of other animals and govern ensuing social behaviors in sexually naive males.

Keywords: aggression; aromatase; bed nucleus of the stria terminalis; emotional behavior; gender recognition; innate behavior; mating; sexual behavior; sexual dimorphism; social behavior.

Figure 1:. AB neurons are transiently activated in aggressive encounters

(A) Strategy to express GCaMP6s in AB neurons. Coronal section through adult male BNST (middle panel) expressing GCaMP6s and nuclear β-galactosidase (scale bar = 100 μm). Midline is on right, and tract of optic fiber implant is outlined dorsal to BNST. Insets show higher magnification of gray box in the middle panel (scale bar = 10 μm). (B) Fiber photometry setup to image GCaMP6s in a freely behaving mouse. (C-Q) Fiber photometry imaging of AB neurons in singly housed Aro^Cre/IPIN male (C-G) or mother (H-Q) interacting with WT intruder for 15 min. (C) A WT male is inserted into the cage of a sexually naïve male. (D,E) Peri-event time plot (PETP) of GCaMP6s signal around entry of male and onset of attack episodes. For PETPs shown in this and other Figures, dark colored line and paler shaded region represent mean fluorescence and SEM, respectively. Vertical dashed black line indicates beginning of event or behavior; Fn on y-axis of PETP represents fold change from baseline preceding the event. (F) GCaMP6s signal increases upon entry of male. Peak Fn represents maximal fold change from baseline preceding the event. Tests of significance were performed, unless otherwise mentioned, by comparing peak Fn of various events to baseline GCaMP6s fluorescence preceding the event and correcting for multiple comparisons. (G) Number of attacks by resident male. (H) Male is inserted into the cage of a mother. (I-K) GCaMP6s signal increases in mothers upon entry of male but not when she attacks him. (L) Number of attacks by mothers. (M) Female is inserted into the cage of a mother. (N-P) GCaMP6s signal increases in mothers upon entry of female but not when she attacks her. (Q) Number of attacks by mothers. Mean ± SEM; hollow circles represent individual mice in these and subsequent figures. n = 7 (C-G), 11 (H-Q). **p<0.01, ***p<0.001. See also Figure S1, Table S1, and Movie S1.

Figure 2:. Male AB neurons are active during interactions with females

Fiber photometry imaging of AB neurons in sexually naive Aro^Cre/IPIN males (A-G) or females (H-N) during 30 min test of mating behavior. (A) Female is inserted into cage of a singly housed male. (B-F) GCaMP6s signal increases upon entry of female, mounting, intromission, and ejaculation. Dashed pink line in (C) shows mean time at which mounting proceeded to intromission. (G) Number of mount and intromission events displayed by each male. (H) A receptive female is inserted into the cage of a WT male. (I-M) No change in GCaMP6s signal following entry into the male’s cage, mounting, intromission, and ejaculation. (N) Number of mounts and intromissions by WT males toward Aro^Cre/IPIN females. Mean ± SEM. n = 7 (A-G), 10 (H-N). *p<0.05, **p<0.01. See also Figure S2, Table S1, and Movie S2.

Figure 3:. Male AB neurons respond differently to the two sexes and regulate sexual preference

(A-N) Fiber photometry of AB neurons in singly housed Aro^Cre/IPIN male. (A-I) A WT male, receptive female, or novel object was inserted for 3 min into the cage of a sexually naïve male. (B,C) Larger change in activity of AB neurons upon entry of female compared to male. Males or females elicited larger response compared to object. (D, E) AB neurons responded more to a female irrespective of whether they were exposed first to a female or male. (F,G) Larger change in activity of AB neurons upon entry of female compared to male. Males and females elicited larger response compared to object. (H,I) Larger change in activity of AB neurons in sexually naïve males prior to and after Cx upon entry of female compared to male. Males and females elicited larger response compared to object. (J-N) A cotton swab wetted with female or male urine or saline was inserted for 3 min each into cage of a Aro^Cre/IPIN male. (K,L) AB neurons responded more to swab wetted with female compared to male urine. Urine wetted swabs elicited larger response compared to saline. (M,N) Female urine elicited larger response irrespective of whether the male was exposed first to female or male urine. (O-S) Cotton swabs wetted with male or female urine were simultaneously inserted into the cage of Aro^Cre/IPIN males expressing DREADDi (O,P,Q) or engineered caspase-3 (O,R,S) to enable inhibition or ablation of AB neurons. Aro^IPIN males injected with caspase-3 payload served as controls (R,S). (P) CNO eliminates preference for female urine. (Q) Males given saline or CNO sniffed both swabs for similar total duration. (R) Ablation of AB neurons eliminates preference for female urine. (S) Both groups of males sniffed both swabs for similar total duration. Mean ± SEM. n = 14 (A-E), 4 (F-I), 10 (J-N), 12 (P,Q), 5 (R,S, Aro^IPIN), 4 (R,S, Aro^Cre/IPIN). *p<0.05, **p<0.01, ***p<0.001. See also Figure S3, Table S1, and Movie S3.

Figure 4:. Sex-typical response of male AB neurons is dependent on pheromone sensing

(A-C) Sexually naïve Trpc2^−/− and Trpc2^+/− males were tested for urine preference. (B) Trpc2^−/− males sniff male and female urine equally. (C) Trpc2^−/− and Trpc2^+/− males sniff male and female urine for similar total duration. (D-K) Fiber photometry of AB neurons in singly housed males exposed to urine swab (D-G) or male, female, or novel object inserted into the cage for 3 min (H-K). (D) A cotton swab wetted with female or male urine or saline was inserted into the cage. (E,F) Response of AB neurons was similar to male or female urine and larger than that to saline. (G) Similar response to male or female urine. (H) A WT male, receptive female, or novel object was inserted into the cage. (I,J) Response of AB neurons was similar to male or female intruder and larger than that to the object. (K) Similar response to male or female intruder. Mean ± SEM. n = 5 males/genotype. *p<0.05, **p<0.01. See also Figure S4 and Movie S4.

Figure 5:. AB neurons play an essential role in male mating

(A-C) Behavior of singly housed male expressing DREADDi in AB neurons with receptive female intruder. (B) CNO reduced percent males mounting or intromitting and eliminated ejaculation. (C) CNO reduced mounts per assay. (D-F) Behavior of singly housed males with caspase-3 targeted to AB neurons with receptive female intruder. (E) Ablation of AB neurons reduced percent males mounting and precluded intromission or ejaculation. (F) Ablation of AB neurons reduced mounts and intromissions per assay. (G-J) Behavior of receptive female expressing DREADDi in AB neurons upon insertion into male cage. (H-J) Behavior of females given CNO or saline is comparable. (K-N) Behavior of receptive females with caspase-3 targeted to AB neurons upon insertion into male cage. (L-N) Behavior of both groups of females is comparable. Mean ± SEM. n = 14 (A-C), 8 (D-F, each genotype), 14 (G-J), 7 (K-N, Aro^IPIN) and 11 (K-N, Aro^Cre/IPIN). *p<0.05, **p<0.01, ***p<0.001. See also Figure S5, Table S1.

Figure 6:. AB neurons play an essential role in male aggression

(A-C) Behavior of resident male expressing DREADDi in AB neurons with WT male intruder. (B) CNO reduced percent residents attacking. (C) CNO reduced attacks per assay. (D-F) Behavior of resident males with caspase-3 targeted to AB neurons with WT male intruders. (E) Ablation of AB neurons reduced percent residents attacking. (F) Ablation of AB neurons reduced attacks per assay. (G-I) Behavior of mothers expressing DREADDi in AB neurons with male intruder. (H,I) Behavior of mothers given CNO or saline is comparable. (J-L) Behavior of mothers with caspase-3 targeted to AB neurons with male intruder. (K,L) Behavior of both groups of mothers is comparable. Mean ± SEM. n = 14 (A-C), 8 (D-F, each genotype), 13 (G-I), 7 (J-L, each genotype). *p<0.05, **p<0.01. See also Figure S6, Table S1.

Figure 7:. Early activity in male AB neurons promotes mating and inhibits fighting

(A-D) Laser illumination of ChR2+ AB neurons in resident males interacting with WT intruder male for 15 min. (B) Laser illumination reduces probability of attacks and increases that of mounts toward the intruder. (C) Laser illumination reduces attacks and increases mounts toward the intruder per assay. (D) Latency to mount male intruder is similar to latency to mount receptive female intruder (shown by horizontal dashed line). Data for males mating with females is from panel H. (E-H) Laser illumination of ChR2+ AB neurons in resident males interacting with receptive female intruder for 30 min. (F-H) Laser illumination does not alter mating with females. (I-L) Brief (90s) laser illumination of eNpHR3.0+ AB neurons in resident males interacting with receptive female intruders for 30 min. (J) No significant change in percent males mating with optogenetic inhibition of AB neurons. (K,L) Optogenetic inhibition of AB neurons reduces mounts per assay and increases latency to mount, intromit, and ejaculate. (M-P) Brief (90s) laser illumination of ChR2+ AB neurons in resident males interacting with WT intruder males for 15 min. (N) Laser illumination eliminates attacks and promotes mounting. (O) Laser illumination reduces attacks and increases mounts per assay. (P) Latency to mount intruder male is similar to latency to mount receptive female intruder (shown by horizontal dashed line). Data for males mating with females is from panel H. Mean ± SEM. n = 8 (A-H), 8 (I-L), 5 (M-P). *p<0.05, **p<0.01. See also Figure S7 and Movie S7.