A neural circuit for male sexual behavior and reward

Abstract

Male sexual behavior is innate and rewarding. Despite its centrality to reproduction, a molecularly specified neural circuit governing innate male sexual behavior and reward remains to be characterized. We have discovered a developmentally wired neural circuit necessary and sufficient for male mating. This circuit connects chemosensory input to BNSTpr^Tac1 neurons, which innervate POA^Tacr1 neurons that project to centers regulating motor output and reward. Epistasis studies demonstrate that BNSTpr^Tac1 neurons are upstream of POA^Tacr1 neurons, and BNSTpr^Tac1-released substance P following mate recognition potentiates activation of POA^Tacr1 neurons through Tacr1 to initiate mating. Experimental activation of POA^Tacr1 neurons triggers mating, even in sexually satiated males, and it is rewarding, eliciting dopamine release and self-stimulation of these cells. Together, we have uncovered a neural circuit that governs the key aspects of innate male sexual behavior: motor displays, drive, and reward.

Keywords: BNST; LTP; POA; Tac1; Tacr1; aggression; bed nucleus of stria terminalis; brain stimulation reward; dopamine; drive; hypothalamic LTP; hypothalamus; libido; male sexual behavior; mating; motivation; preoptic hypothalamus; reinforcement; reward; social behavior network; substance P.

Figure 1:. Activity of male BNSTpr^Tac1 neurons identifies sex of conspecifics and promotes mating.

A. Schematic of section through the adult mouse brain showing that BNSTpr^Tac1 neurons are a subset of BNSTpr^Aro neurons, which are a subset of BNSTpr^Esr1 neurons. B-E. Schematic of fiber photometry of BNSTpr^Tac1 neurons in males investigating swabs wetted with female urine, male urine, or saline (B). Peri-event time plot (PETP) of normalized GCaMP6s fluorescence (ΔF/F; dark line, mean, and shaded area, SEM for all Figure panels with fiber photometry; dashed vertical line marks insertion of swab into the cage) (C). BNSTpr^Tac1 neurons are activated by urine (D), with larger response to female than male urine (E). F-J. GRIN lens imaging of BSNTpr^Tac1 GCaMP6s fluorescence with segmented neurons (F). Schematic of miniscope imaging of BNSTpr^Tac1 neurons in males investigating swabs as in panel B (G). Heatmaps of activation of individual neurons during presentation of urine or saline; row numbers correspond to the same neuron across panels, with rows sorted by activation to female urine (H-J). K-N. Traces of GCaMP6s fluorescence (ΔF/F) of individual neurons in response to stimulus presentation (dashed lines) (K). Percent neurons activated by female or male urine or both (L). Percent co-activated neurons with differential response to female or male urine (M). Among co-activated neurons, the response to female urine was greater than that to male urine (N). Neurons were classified as activated if the peak z-score during the 10s period following swab insertion was >2σ of the peak z-score during the 10s period preceding swab insertion. O-R. Schematic of optogenetic activation of BNSTpr^Tac1 neurons of a resident male interacting with an intruder male (O). Activation during the first 90s eliminates attacks and promotes mating (P). Raster plot of a male showing aggression toward the intruder without optogenetic activation (Q). Raster plot of behavior of male showing mating with the intruder following optogenetic activation (R). S-V. Schematic of optogenetic activation of BNSTpr^Tac1 neurons of a resident male interacting with intruder female (S). Optogenetic activation of these cells during the first 90s does not alter the probability (T), number (U), or latency (V) of sniffing or mounting. Mean ± SEM. n = 6 mice (B-E), 105 neurons from 3 mice (F-N), and 5 mice (O-V). * p < 0.05, ** p < 0.01. Scale bar = 100 μm (F). See also Fig. S1; Tables S1-3.

Figure 2:. Innervation of POA^Tacr1 neurons by BNSTpr^Tac1 neurons is essential for male mating.

A-B. Syp:mRuby expression in BNSTpr^Tac1 neurons (A). Syp:mRuby+ termini of BNSTpr^Tac1 neurons in the POA; inset shows area boxed in gray (B). C-D. mCherry+ BNSTpr^Tac1 neurons (C) and EGFP+ POA^Tacr1 neurons (D) visualized in coronal sections. E-I. TVA (mCherry) and Rabies (EGFP) expression in POA^Tacr1 starter neurons (E). EGFP+ and TVA–BNSTpr neurons innervating POA^Tacr1 neurons (F). Co-labeling for Tac1 mRNA and EGFP in BNSTpr neurons innervating POA^Tacr1 neurons; arrows show Tac1+, EGFP+ cells (G). Most BNSTpr neurons presynaptic to POA^Tacr1 neurons are BNSTpr^Tac1 neurons (H), and nearly half of BNSTpr^Tac1 neurons innervate POA^Tacr1 neurons (I). J-L. Strategy to activate BNSTpr^Tac1→POA projections (J). Schematic of optogenetic activation of BNSTpr^Tac1→POA projections of a resident male interacting with intruder male (K). Activation during the first 90s eliminates attacks and promotes mating (L). M-Q. Strategy to inhibit BNSTpr^Tac1→POA projections (M). Schematic of optogenetic inhibition of BNSTpr^Tac1→POA projections of a resident male interacting with intruder female (N) or male (P). Inhibition suppresses mating with females (O) but does not alter aggression toward males (Q). Mean ± SEM. n = 4 (A-I), 7 (J-L), and 9 (M-Q) mice. * p < 0.05. Scale bars = 1 mm (C,D), 200 μm (A,B,E,F), 20 μm (B inset, G). See also Fig. S2; Tables S1,3.

Figure 3:. POA^Tacr1 neurons are active during male mating and drive male sexual behavior.

A-C. Fiber photometry of POA^Tacr1 neurons in males interacting with intruder female. PETP of GCaMP6s fluorescence (ΔF/F) during sniffing (A) and mounting (B). BNSTpr^Tac1 neurons are activated during sniffs and mounts (C). D-F. Fiber photometry of POA^Tacr1 neurons in males interacting with intruder male. PETP of GCaMP6s fluorescence (ΔF/F) during sniffing (D) and attacks (E). No discernible activation of BNSTpr^Tac1 neurons during sniffs or attacks (F). G-J. Schematic of optogenetic activation of POA^Tacr1 neurons of males interacting with females (G). Activation (15/45 s on/off) does not alter percent males mounting females (H) but reduces mating latency ~100-fold (I). Raster plot shows male mounting time-locked to light. K-N. Schematic of optogenetic activation of POA^Tacr1 neurons of males interacting with intruder males (K). Activation (15/45 s on/off) abrogates aggression and elicits mating (L). Fight latency is prolonged whereas mating latency is reduced by ≥100-fold (M). Raster plot shows male mounting time-locked to light (N). O-V. Schematic of optogenetic activation (30/30 s on/off; 2 min encounter) of POA^Tacr1 neurons of males interacting with inanimate objects (O). Activation progressively increased investigation of a beaker (P), wood block (Q), and tube (R) and elicited mounting toward the tube outfitted with a toy mouse tail (S), toy mouse (T), male (U), and an unreceptive female (V). Mean (dark trace) ± SEM (lighter shading) of GCaMP6s activity shown in PETPs of all Figures. Mean ± SEM. n = 8 (A-F), 6 (G-N), and 4 (O-V) mice. * p < 0.05, *** p < 0.001. See also Fig. S3; Tables S1,3,4; Movie S1.

Figure 4:. POA^Tacr1 neurons are functionally downstream of BNSTpr^Esr1 neurons for male mating.

A-C. Schematic of optogenetic inhibition of POA^Tacr1 neurons in males interacting with intruder females (A). Silencing eliminates mounting without altering sniffing (B,C). D-F. Schematic of closed-loop optogenetic inhibition of POA^Tacr1 neurons during mounting (D). Silencing reduced transitions from mount to intromission (E,F). n = 44 mounts (F, laser off), 29 mounts (F, laser on). G-K. Schematic of optogenetic inhibition of POA^Tacr1 neurons in males interacting with intruder males (G). Silencing did not alter the probability (H), number (I), latency (J), or duration (K) of attacks. L-P. Strategy to activate BNSTpr^Esr1 neurons while inhibiting POA^Tacr1 neurons (L). Schematic of chemogenetic inhibition of POA^Tacr1 neurons in males (red cross over POA^Tacr1 neurons in circuit models above M-P) interacting with females (M). Inhibition of POA^Tacr1 neurons eliminates mating (N). Schematic of optogenetic activation of BNSTpr^Esr1 neurons (shaded blue in circuit model above O-P) in males given CNO and interacting with females (O). Inhibition of POA^Tacr1 neurons eliminated mating even when BNSTpr^Esr1 neurons were activated (P). Q-U. Strategy to activate POA^Tacr1 neurons while inhibiting BNSTpr^Esr1 neurons (Q). Schematic of chemogenetic inhibition of BNSTpr^Esr1 neurons (red cross over BNSTpr neurons in circuit models above R-U) in males interacting with females (R). Inhibition of BNSTpr^Esr1 neurons suppressed mating (S). Schematic of optogenetic activation of POA^Tacr1 neurons (shaded blue in circuit model above TU) in males given CNO and interacting with females (T). Activation of POA^Tacr1 neurons induced mating even when BNSTpr^Esr1 neurons were inhibited (U). Mean ± SEM. n = 11 (A-C), 3 (D-F), 11 (G-K), 6 (L-P) and 7 (Q-U) mice. * p < 0.05, ** p < 0.01. See also Fig. S3,4; Tables S1,3.

Figure 5:. Substance P-Tacr1 signaling potentiates activation of POA^Tacr1 neurons and promotes male mating

A-D. Substance P induces LTP in POA^Tacr1 neurons. Strategy to record POA^Tacr1 neurons with local stimulation of glutamatergic inputs in the presence of picrotoxin (100 μM) (A). tdTomato+ POA^Tacr1 neurons (upper) and recording pipette and theta stimulation electrode (lower) (B). Summary plot of normalized EPSP amplitudes (Amp) and input resistance (R_in) before and after 5 min (green line) bath application of Substance P (50nM). Inset: Representative EPSP traces of baseline (1) and the last 5 min (2) (C-D). Substance P induces LTP in POA^Tacr1 neurons (C) and the Tacr1 antagonist L-703,606 (10 μM for slice recordings) blocks it (D). Black line (D) represents average trace with vehicle. E-H. Activation of BNSTpr projections induces LTP onto POA^Tacr1 neurons. Strategy to record POA^Tacr1 neurons while optogenetically activating BNSTpr→POA projections in the presence of picrotoxin (E). Trace of light-evoked firing of BNSTpr neurons (F, upper). Light elicits reliable firing early as well as later (F, lower). Summary plot of normalized EPSP Amp and R_in before and after transient activation (90 s, blue arrow) of BNSTpr→POA projections (G-H). Inset: Representative EPSP traces of baseline (1) and the last 5 min (2) (G-H). Light induces LTP in POA^Tacr1 neurons (G) and L-703,606 blocks it (H). Gray line (H) represents the average trace with vehicle. I-M. Female encounter strengthens excitatory synapses onto POA^Tacr1 neurons in a Substance P-dependent manner. Schematic of assay in which singly-housed males were administered vehicle or L-703,606 (10 mg/kg for in vivo studies) 30 min prior to initiating testing (I-K, upper panels), and strategy to record POA^Tacr1 neurons with local stimulation of glutamatergic inputs in the presence of picrotoxin (L). Darker and lighter traces show example excitatory postsynaptic currents recorded at −70 mV and +40 mV, respectively (I-K, lower panels). MPA/NMDA ratio is increased only in males given vehicle prior to encountering a female (M). N-P. Schematic of paradigm to infuse Substance P into the POA of WT males interacting with females. Substance P (10 ng in 200 nL) or vehicle was infused 15 min prior to female entry. Substance P did not alter probability of (O) but reduced latency to initiate (P) mating. Q-S. Schematic of infusion of L-703,606 (500 pmol in 200 nL) into the POA of WT males interacting with a receptive female (Q). L-703,606 reduced the probability of (R) and increased latency to initiate (S) mating. T-X. Strategy to activate BNSTpr^Tac1→POA projections (T). Schematic of optogenetic activation of BNSTpr^Tac1→POA projections in males given L-703,606 and interacting with females (U). L-703,606 suppresses mating even upon activation of BNSTpr^Tac1→POA projections (V). Schematic of optogenetic activation of BNSTpr^Tac1→POA projections in males given L-703,606 and interacting with males (W). L-703,606 eliminates the male-male mating promoted by activation of BNSTpr^Tac1→POA projections (X). Mean ± SEM. n = 7 neurons from 4 mice (C), 5 neurons from 3 mice (D), 8 neurons from 4 mice (G), 7 neurons from 3 mice (H), 30 neurons from 4 mice (Control), 33 neurons from 5 mice (Encounter + Vehicle), 31 neurons from 4 mice (Encounter + L-703,606) (M), 8 mice (N-S), 7 mice (T-X). * p < 0.05, ** p < 0.01, **** p < 0.0001. See also Fig. S5,6; Tables S1,3.

Figure 6:. Forced activation of POA^Tacr1 neuron overrides the post-ejaculatory refractory period and is self-reinforcing.

A-D. Schematic of optogenetic activation of POA^Tacr1 neurons in post-ejaculatory males interacting with females (A). Activation (30/30 s on/off) of these cells re-ignites mating drive, increasing the probability (B) and number (C) of mating routines. Raster plot of sexually satiated male subsequent to activating POA^Tacr1 neurons (D). E-I. Schematic of optogenetic activation of POA^Tacr1 neurons in the SPP test (E). Activation governs behavioral preference of males (F). Raster plots (each row is a male) showing that optogenetic activation governs investigation by males (H-I). J-L. Schematic of optogenetic self-stimulation (0.5 s, 40 Hz) of POA^Tacr1 neurons in virgin and sexually experienced males (J). Both virgin and experienced males show more nose pokes to the active port (K,L), with sexual experience increasing self-stimulation (L). M-N. Schematic of optogenetic inhibition of POA^Tacr1 neurons during the SPP test (M). Inhibition does not alter male preference for side containing the female (N). O-P. Schematic of optogenetic inhibition of POA^Tacr1 neurons during sucrose consumption test (O). Inhibition does not alter sucrose consumption (P). Mean ± SEM. n = 10 mice (A-D), n = 10 mice (E-I), n = 7 virgin mice and 10 experienced mice (J-L), n = 10 mice (M-N), n = 6 mice (O-P). * p < 0.05, ** p < 0.01, *** p < 0.001. See also Fig. S7; Tables S1,3; Data S1; Movie S2.

Figure 7:. The BNSTpr^Tac1→POA^Tacr1 pathway is embedded in a neural circuit linking pheromone sensing to motor output and reward.

A-D. Strategy to activate POA^Tacr1 neurons following bilateral ablation of AVPV/PVpo^TH neurons with 6OHDA (10 μg in 1 μL) (A). Schematic of optogenetic activation of POA^Tacr1 neurons in post-ejaculatory males interacting with females (B). Activation (30/30 s on/off) re-ignites mating drive, increasing the probability (C) and number (D) of mating routines. E. Schematic of optogenetic activation of POA^Tacr1 neurons in males lacking AVPV/PVpo^TH neurons in the SPP test (left). Activation governs behavioral preference of males (right). F. Schematic of optogenetic self-stimulation (0.5 s, 40 Hz) of POA^Tacr1 neurons in males lacking AVPV/PVpo^TH neurons (left). Males show more nose pokes to the active port (right). G-K. Strategy for optogenetic activation of VTA termini of male POA^Tacr1 neurons (G). Males show more nose pokes to the active port (H,I). More mounts (J) but not intromissions (K) in light epochs in encounters with females. L-P. Strategy for ptogenetic activation of PAG termini of POA^Tacr1 neurons (L). Males show more nose pokes to the active port (M,N). More mounts (O) and intromissions (P) in light epochs in encounters with females. Q-T. Optogenetic activation of POA^Tacr1→VTA projections induces DA release in NAc. Strategy for fiber photometry imaging of DA release in NAc during activation (Q). PETP (R) and change in fluorescence (S) of NAc neurons during activation. GRAB_DA signal trace (T) during light epochs (laser 10 s on, 30 s off) shows time-locked DA release. U. Schematic of the male mating circuit outlined in this study. BNSTpr^Tac1 neurons receive input from the AOB and release Substance P to potentiate activation of POA^Tacr1 neurons. Activation of POA^Tacr1 neurons is necessary and sufficient to elicit mating, and it re-motivates sexually satiated mice to mate and is rewarding. Projections of these cells to the VTA or PAG also drive mating, mating in satiated males, and reinforcement, and activation of VTA projections elicits DA release in NAc. Arrows indicate pathways established by prior work (black) or from our study (blue). Dashed arrows (purple) indicate pathways emanating from VTA and PAG that remain to be identified and connect with centers that drive mating-related motor programs, establish a motivational state for sexual behavior, and make sexual behavior rewarding. Mean ± SEM. n = 6 mice (A-F), n = 7 mice (H-I), n = 11 mice (J-K), n = 7 mice (M-N), n = 14 mice (O-P), n = 8 mice (Q-T). * p < 0.05, ** p < 0.01, *** p < 0.001. See also Fig. S8,9; Tables S1,3; Data S1.