WO2023122254A2 - Procédés de déclenchement du désir d'accouplement et du comportement d'accouplement - Google Patents

Procédés de déclenchement du désir d'accouplement et du comportement d'accouplement Download PDF

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WO2023122254A2
WO2023122254A2 PCT/US2022/053779 US2022053779W WO2023122254A2 WO 2023122254 A2 WO2023122254 A2 WO 2023122254A2 US 2022053779 W US2022053779 W US 2022053779W WO 2023122254 A2 WO2023122254 A2 WO 2023122254A2
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neurons
poa
tacr1
male
mating
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WO2023122254A3 (fr
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Nirao SHAH
Joseph KNOEDLER
Daniel BAYLESS
Sayaka Inoue
Chung-Ha DAVIS
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The Board Of Trustees Of The Leland Stanford Junior University
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    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B30/00—Methods of screening libraries
    • C40B30/06—Methods of screening libraries by measuring effects on living organisms, tissues or cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • Sex differences in behavior reflect the action of a sexually differentiated brain.
  • neural circuits underlying sex-typical physiology or social interactions such as mating and aggression are under the control of SHs.
  • SHs bind to cognate nuclear receptors in SH-sensitive neuronal populations within these neural circuits to influence gene expression and signaling cascades, thereby influencing physiology and behavior (McCarthy and Arnold, 2011 ; Yang and Shah, 2014).
  • SHs bind to cognate nuclear receptors in SH-sensitive neuronal populations within these neural circuits to influence gene expression and signaling cascades, thereby influencing physiology and behavior.
  • VMHvl Cckar/Esr1 tCT is essential for female sexual behavior whereas VMHvl Cckar-/Esr1 tCTs are required for maternal aggression, male sexual behavior, and male territorial aggression.
  • VMHvl Cckar/Esr1 but not VMHvl Cckar-/Esr1 , projections to the AVPV exhibit structural plasticity with peak connectivity at estrus.
  • the BNSTpr Tac1/Esr1 tCT mediates the exclusively male role of BNSTpr Aro neurons in social behaviors.
  • a virally encoded, Cre-dependent, inhibitory chemogenetic actuator (AAV- flex-DREADDi:mCherry) was delivered to the BNSTpr of adult males (Taclcre mice) bearing a Cre recombinase inserted in a gene-conserving manner into the Tad locus.
  • BNSTpr Tac1 neurons perform and are sufficient for sex recognition, shown with fiber photometry analysis of activity, and in optogenetic models where stimulation of BNSTpr Tac1 neurons leads males to mis-recognize other males as mating targets and to attempt to mate with them.
  • BNSTpr Tac1 neurons project to a brain region called the medial preoptic area (POA). It is shown herein that POA neurons expressing the substance P receptor Tacrl (POA Tacr1 ) are responsible for initiating mating.
  • POA Tacr1 the substance P receptor Tacrl
  • the BNST Tac1 neurons are monosynaptically connected to POA Tacr1 neurons.
  • PR expressing neurons are equivalently active during both estrus and diestrus, including Cckar neurons.
  • AVPV kisspeptin expressing neurons receive monosynaptic inputs from Cckar neurons, and are significantly more active when the female is in estrus.
  • Monosynaptic tracing data as well as fiber photometry demonstrate that AVPV KISS1 are directly downstream in the functional circuit for female sexual behavior. While inhibition of VMHvl PFt neurons suppresses female sexual behavior, simultaneous activation of AVPV KISS1 overrides the upstream inhibition and elicits female sexual behavior.
  • methods are provided for screening male or female neurons for agents that modulate specific sex-typical behaviours.
  • agents may be small molecule agents, peptides, genetic agents, etc., particularly small molecule agents, e.g. provided in a compound library. Screening can be performed for agents that inhibit, or activate, the desired behaviour.
  • screening for female sex-typical behaviours the method comprising contacting a VMHvl Cckar/Esr1 tCT with a candidate agent, where activation of the tCt elicits sexual responsiveness and inhibition decreases sexual responsiveness.
  • screening for female sex-typical behaviors comprising contacting AVPV Kiss1 neurons with a candidate agent, where activation to inhibit or increase female sexual behaviors in a sex hormone independent manner.
  • screening comprises contacting male or female VMHvl Cckar-/Esr1 tCTs, where activation of the tCt elicits maternal aggression, male sexual behavior, and male territorial aggression.
  • the neurons for screening are present in a slice culture, where activation is detected.
  • the neurons are provided on a microelectrode array.
  • the cells are mammalian cells. Insome embodiments the cells are primate cells, e.g. non-human primates or humans.
  • the cells are mammals in which it is desirable to modulate mating behavior, including, without limitation, livestock, e.g. horses, cattle, sheep, goats, etc.; pet animals, e.g. dogs, cats, etc.; zoo animals; and the like.
  • livestock e.g. horses, cattle, sheep, goats, etc.
  • pet animals e.g. dogs, cats, etc.
  • zoo animals e.g. dogs, cats, etc.
  • an optogenetic animal model is provided.
  • the models provided herein are useful in the design and testing of therapeutic modulation, e.g. surgery, pharmacologic intervention, and the like, where the effect of a modulation on behaviors can be determined.
  • the models are also useful in the design of drugs, for animal husbandry including endangered species, and the like. Development of small molecules that can modulate neurons in the male that can induce the motivation to mate, or induce mating on command, or bypass the refractory period. Inhibitory modulation of these neurons may also be useful in preventing premature ejaculation or suppressing sexual behavior.
  • FIGS. 1A-1 C TRAPseq identification of sDEGs.
  • A Schematic of TRAPseq workflow.
  • C TRAPseq identification of sDEGs.
  • FIGS. 2A-2D TRAPseq 820 identification of eDEGs.
  • C Venn diagram illustrating that the majority of DEGs is restricted to one Esr1+ population.
  • D Venn diagram illustrating that the majority of DEGs within an Esr1 + population is specific to one comparison between sexes or estrous states. See also Fig. 13.
  • FIGS. 3A-3C ASD-association and intra-regional distribution of DEGs.
  • FIG. 4A Categorizing Esr1 + populations into tCTs with snRNAseq.
  • A-D Violin plots classifying tCTs (rows) by virtue of expression of SH receptors, major neuronal neurotransmitter type (excitatory and inhibitory), and enriched genes in Esr1 + cells in the BNSTpr (A), MeA (B), POA (C), and VMHvI (D).
  • the BNSTprTaci/Esn tCT is essential for sex recognition, mating, and aggression in males.
  • A. Violin plots of a subset of DEGs in the BNSTprTaci/Esri .
  • B. Salt and pepper distribution of Tac1 in Esr1 + BNSTpr neurons visualized by HCR-ISH. Scale bar 100 gm.
  • C. Schematic of intersectional chemogenetic strategy to inhibit BNSTprTaci/Esn (top) or 920 BNSTprTaci /Esri tCTs. D.
  • F. Inhibition of the BNSTprTaci/Esri tCT reduces the probability of resident males attacking intruder males as well as the number of attacks per test.
  • G. Inhibition of BNSTprTaci /Esri tCTs does not alter mating of resident males with receptive females.
  • FIGS. 7A-7K The VMHvIcckar/Esn tCT and VMHvlcckar-/Esri tCTs are required for female mating and maternal aggression, respectively.
  • B. Cckar expression is restricted to the lateral component of the Esr1 + VMHvI population, in agreement with previous work (Hashikawa et al., 2017; Xu et al., 2012). Scale bar 100 gm.
  • VMHvIcckar/Esn tCT 16C for quantification of cell number in these tCTs in M, FR, and FNR.
  • D eDEGs significantly enriched in VMHvIcckar/Esn tCT compared to the VMHvlTrim36/Esri tCT.
  • E Schematic of intersectional chemogenetic strategy to inhibit VMHvIcckar/Esn (top) or VMHvlcckar-/Esri (bottom) tCTs.
  • FIGS. 8A-8C BNSTpr TAC1 activity encodes conspecific sex and promotes mating in male mice.
  • A. BNST Tac1 neuron activity identifies the sex of interaction partners.
  • B. Transient BNST Tac1 neuron stimulation elicits mating behavior toward males.
  • C. BNST Tac1 neurons are presynaptic to POA Tacr1 neurons.
  • FIGS. 9A-9D POA Tacr1 neurons in males.
  • A. POA Tacr1 neurons are active during mating behavior.
  • B. POA Tacr1 neuron stimulation elicits mating behavior in an optogenetic model.
  • C. POA Tacr1 neuron stimulation overcomes the refractory period in an optogenetic model.
  • FIGS. 10A-10B A. POA Tacr1 neuron inhibition disrupts mating. B. POA Tacr1 neuron inhibition is neither aversive nor anhedonic.
  • FIGS. 1 1 A-1 1 C Kisspeptin+ neurons in a primed female are active during mating.
  • FIGS. 12A-12F Characterization of sDEGs. Related to Fig. 1 .
  • A Schematic of workflow to generate FR and FNR mice.
  • B Enrichment of RNA obtained following immunoprecipitation (IP) of the L22 ribosomal subunit encoded by the RiboTag allele from the BNSTpr of Esr1cre;RiboTag (experimental) mice compared to RiboTag (control) mice, y-axis, Bioanalyzer fluorescence signal in arbitrary units (AU); x-axis, RNA size in nucleotides (nt).
  • IP immunoprecipitation
  • sDEGs are distributed across all autosomes and the X chromosome. M>F, sDEGs upregulated in M compared to FR or FNR in any Esr1 + population; F>M, sDEGs upregulated in FR or FNR compared to M in any Esr1 + population; Mixed, sDEGs upregulated in M compared to FR or FNR in >1 Esr1 + population and upregulated in FR or FNR compared to M in >1 Esr1 + population.
  • FIGS. 13A-13D Characterization of eDEGs and all DEGs. Related to Fig. 2.
  • A. eDEGs are distributed across all autosomes and the X chromosome. FR > FNR, eDEGs upregulated in FR compared to FNR in >1 Esr1 + populations; FNR > FR, eDEGs upregulated in FNR compared to FR in >1 Esr1 + populations; Mixed, eDEGs upregulated in FR and FNR each in > Esr1 + population.
  • FIGS. 14A-14C Genomic context and functional or disease association of DEGs. Related to Fig. 3 and Table S4. A.
  • FIGS. 15A-15G snRNAseq analysis of Esr1 + populations.
  • A Schematic of snRNAseq workflow.
  • B Enrichment of GFP+ nuclei with FANS (left) and total yield of GFP+ nuclei (right) per condition.
  • Horizontal gray bar Mean.
  • C Enrichment of Esr1 and GFP and depletion of GFAP mRNA in GFP+ nuclei.
  • D Violin plots with distribution of cell number in tCTs from each of the four Esr1 + populations.
  • E Graph-based clustering shows that Esr1 + cells cluster by region but not by batch or condition (sex or estrous states).
  • F is
  • VMHvI coronal section through VMHvI (left) and Vgat mRNA expressing cells labeled in the VMHvI (right).
  • Scale bar 100 gm.
  • G. Violin plots classifying tCTs (rows) into major neuronal types: excitatory (Slc17a6, Slc17a7), inhibitory (Gad1 ), aromatase-expressing, estrogen receptor b (ERb or Esr2), and tyrosine hydroxylase (TH).
  • FIGS. 16A-16C Characterization of shared or sex or estrous-state specific tCTs. Related to Fig. 5.
  • Bar graph shows percent cells in individual tCTs for each condition (M, FR, FNR). Violin plots to the right of each bar graph show whether the tCT is excitatory or inhibitory and highlight the marker gene enriched in that tCT.
  • FIGS 17A-17J Molecular and behavioral characterization of the BNSTprTaci/Esn tCT. Related to Fig. 6.
  • A. Violin plots showing enriched sex and estrous state-shared genes in the Tac1 tCT (highlighted in yellow).
  • B. Violin plots showing expression of genes in panel A in M, FR, and FNR conditions.
  • C. Expression of mCherry+ (DREADDi) in BNSTpr of Taclcre and Tad Cre;Esr1 Fipo mice after delivery of AAV-flex-DREADDi and AAV-Coff/Fon-DREADDi to this region, respectively.
  • PCR of INTRSECT hM4Di- mCherry plasmid DNA generates an amplicon larger than WT, while PCR of cDNA from cells co-transfected with same plasmids and activating recombinases generates an amplicon equivalent to WT; a smaller PCR product is noted in all formats. cDNA amplicon sequences are seamless across the exon junctions. H.
  • Flow cytometry of cells transfected with INTRSECT DREADDi-mCherry and activating Flpo recombinase shows expression comparable to WT (constitutive DREADDi-mCherry) and more than that observed in control conditions (negative, no transfection; alone, INTRSECT transfection without recombinase; +Cre +Flpo, INTRSECT transfection with Cre and Flpo). Diminished, but residual, expression is observed post-transfection when co-transfected with Cre and Flpo. I.
  • Chemogenetic inhibition of BNSTprTaci/Esn tCT increases latency of resident males in initiating mounts or intromission with receptive females and attacks toward intruder males.
  • FIGS. 18A-18K VMHvlcckar-/Esri , but not VMHvIcckar/Esri , tCTs are required for male sexual behavior and aggression.
  • Fig. 7. A. VMHvIcckar/Esn and VMHvlTrim36/Esri tCTs are restricted to females. B. Shared expression of genes between VMHvIcckar/Esn and VMHvlTrim36/Esri tCTs (see also Table S7).
  • FIGS. 19A-19V Activity of male BNSTpr Tac1 neurons identifies sex of conspecifics and promotes mating.
  • A Schematic of coronal section through the adult mouse brain showing that BNSTpr Tac1 neurons are a subset of BNSTpr Aro neurons, which are themselves a subset of BNSTpr Esr1 neurons.
  • B-E Schematic of fiber photometry of male BNSTpr Tac1 neurons in mice investigating cotton swabs wetted with female urine, male urine, or saline (B).
  • PTP Peri-event time plot
  • AF/F normalized GCaMP6s fluorescence
  • SEM shaded area, SEM; dashed vertical line marks insertion of swab into the cage
  • C Peri-event time plot
  • BNSTpr Tac1 neurons are activated by urine (D), with a greater maximal response to female compared to male urine (E).
  • F Schematic of miniscope imaging of male BNSTpr Tac1 neurons investigating cotton swabs wetted with female urine, male urine, or saline
  • K-N. Representative traces of normalized GCaMP6s fluorescence (AF/F) of individual neurons in response to male urine, saline, and female urine (K). Dashed vertical lines mark insertion of swabs into the cage. Pie chart illustrating percent neurons activated by female or male urine or both (L). Pie chart illustrating percent co-activated neurons with a greater response to female or male urine (M). Among co-activated neurons, the response to female urine was greater than that to male urine (N).
  • O-R Schematic of optogenetic activation paradigm for BNSTpr Tac1 neurons of a resident male interacting with an intruder male (O). Activation of these cells during the first 90s of the encounter eliminates attacks and promotes mating (P). Raster plot of behavior of a male showing aggression toward an intruder male in the absence of laser illumination (Q). Raster plot of behavior of experimental male showing mating toward an intruder male following transient activation of BNSTpr Tac1 neurons (R). S-V.
  • FIGS. 20A-20U Male BNSTpr Tac1 neurons are more active during interactions with females than males and forced activation of these cells promotes male-male mating.
  • A-E Schematic of fiber photometry of BNSTpr Tac1 neurons in males following insertion of a receptive female, male, or toy mouse (A). PETP of normalized GCaMP6s fluorescence (AF/F) during insertion of mice or a toy mouse (B). BNSTpr Tac1 neurons are activated by mice (C), with greater activation by females (D). Activation perdures longer following encounter with females than males (E). F. GCaMP6s expression in BNSTpr Tac1 neurons.
  • G-J Schematic of fiber photometry of BNSTpr Tac1 neurons during male sexual behavior (G). PETP of normalized GCaMP6s fluorescence during mounts (H) and ejaculation (I) shows significant increase in activity above baseline (J).
  • K-M Schematic of fiber photometry of BNSTpr Tac1 neurons in males attacking an intruder male (K). PETP of normalized GCaMP6s fluorescence during attacks (L) reveals no discernible activation of BNSTpr Tac1 neurons (M).
  • N-O GCaMP6s expression in BNSTpr Tac1 neurons (N). GRIN, GRIN lens tract.
  • More neurons are activated by female compared to male urine (O).
  • P Laser illumination of ChR2+ BNSTpr Tac1 neurons induces Fos expression.
  • Q-R Optogenetic activation of ChR2+ BNSTpr Tac1 neurons in males for the first 90s of an encounter with an intruder male eliminates attacks and induces mounts.
  • S-U Schematic of optogenetic activation of BNSTpr Tac1 neurons in males interacting with a receptive female (S). Optogenetic activation (30/30 s on/off ) does not alter mounting behavior (T). Raster plot of behavior of male mounting under this optogenetic activation paradigm (U).
  • FIGS. 21 A-21 Q Innervation of POA Tacr1 neurons by BNSTpr Tac1 neurons is essential for male mating.
  • A-B Syp:mRuby expression in soma of BNSTpr Tac1 neurons (A). Syp:mRuby+ termini of BNSTpr Tac1 neurons in the POA, with inset panel showing area outlined in gray at higher magnification (B).
  • C-D mCherry+ BNSTpr Tac1 neurons (C) and EGFP+ POA Tacr1 neurons (D) visualized in coronal sections.
  • E-l TVA (mCherry) and Rabies (EGFP) expression in POA Tacr1 starter neurons (E).
  • EGFP+ and TVA- BNSTpr neurons presynaptic to POA Tacr1 neurons
  • F Go-labeling for Tac1 mRNA and EGFP in BNSTpr neurons presynaptic to POA Tacr1 neurons, with arrows showing cells co-expressing Tac1 and EGFP (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).
  • FIGS. 22A-22R Activity of BNSTpr Tac1 ->POA projections is necessary and sufficient for male sexual behavior.
  • A Laser illumination of ChR2+ BNSTpr Tac1 neurons induces Fos expression.
  • B-C Optogenetic activation of BNSTpr Tac1 -»POA projections during first 90s of encounter with an intruder male eliminates attacks and induces mounting during the rest of the assay.
  • D-H Schematic of optogenetic activation of BNSTpr Tac1 -»POA projections in males interacting with a receptive female (D).
  • FIGS 23A-23V 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 a receptive female.
  • PETP of normalized GCaMP6s fluorescence (AF/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 an intruder male.
  • PETP of normalized GCaMP6s fluorescence (AF/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 paradigm for POA Tacr1 neurons of males interacting with receptive females (G). Activation of these cells (15/45 s on/off) does not alter percent males mounting females (H), but it reduces latency to initiate mating ⁇ 100-fold (I). Raster plot of behavior of a male shows mounting time- locked to laser illumination epochs.
  • K-N Schematic of optogenetic activation paradigm for POA Tacr1 neurons of males interacting with intruder males (K). Activation of these cells (15/45 s on/off) abrogates aggression and elicits mating behavior toward the intruder (L).
  • Latency to fight extends to the end of the assay whereas that to mate is reduced by >100-fold (M).
  • Raster plot of behavior of a male shows mounting time-locked to laser illumination epochs (N).
  • O-V Schematic of optogenetic activation paradigm for POA Tacr1 neurons of males interacting with inanimate objects (O). These cells were activated (30/30 s on/off) during 2 min encounters.
  • FIGS. 24A-24U Activity of male POA Tacr1 neurons during mating is necessary and sufficient for the behavior.
  • A-D Schematic of coronal section through the adult mouse brain showing that POA Tacr1 neurons are a subset of POA ESFt1 neurons.
  • Co-expression of Tacrl (visualized with AAV-DI0-GCaMP6s delivered to POA of Tacr1 Cre males) and Esr1 in the POA (B).
  • Vast majority ofe POA Tacr1 neurons express Esr1 (C) whereas a small subset of POA Esr1 neurons express Tacrl (D).
  • F-l Schematic of coronal section through the adult mouse brain showing that POA Tacr1 neurons are a subset of POA ESFt1 neurons.
  • Co-expression of Tacrl visualized with AAV-DI0-GCaMP6s delivered to POA of Tacr1 Cre males
  • P-R Schematic of optogenetic inhibition of POA Tacr1 neurons in males interacting with a receptive female (P). Inhibition increases latency to initiate mating (Q) without altering locomotor activity of the male (R).
  • S-U Schematic of optogenetic inhibition of POA Tacr1 neurons in males interacting with an intruder male (S). Inhibition does not alter the latency to start fighting nor does it alter locomotor activity of the experimental male. Mean ⁇ SEM.
  • FIGS. 25A-25S The requirement of POA Tacr1 neurons for male mating is functionally downstream of BNSTpr Tac1 neurons.
  • A-C Schematic of optogenetic inhibition paradigm for POA Tacr1 neurons in males interacting with a receptive female (A). Silencing these cells eliminates mounting without altering sniffing (B,C).
  • FIGS. 26A-26Y POA Tacr1 neurons are functionally downstream of BNSTpr Tac1 neurons.
  • A-C Strategy to activate BNSTpr Tac1 neurons while inhibiting POA Tacr1 neurons (A).
  • DREADDi expression in POA Tacr1 neurons B).
  • ChR2 expression in BNSTpr Esr1 neurons B).
  • Laser illumination of BNSTpr Esr1 neurons induces Fos expression.
  • F-G F-G.
  • BNSTpr Tac1 neurons Optogenetic activation of ChR2+ BNSTpr Tac1 neurons in males given vehicle (rather than ONO) interacting with an intruder male increases mounts (F) and latency to fight (G), and reduces latency to mate (G). BNSTpr Tac1 neurons were optogenetically activated only for the first 90 s of the assay.
  • H-L. Chemogenetic inhibition of male POA Tacr1 neurons does not alter number of (H), or latency to (I), attack, nor does it alter locomotor activity (J), motivation to find hidden food (K), or performance on an elevated plus maze (L).
  • M-P Chemogenetic inhibition of male POA Tacr1 neurons does not alter number of (H), or latency to (I), attack, nor does it alter locomotor activity (J), motivation to find hidden food (K), or performance on an elevated plus maze (L).
  • ONO eliminates mounting behavior even when BNSTpr Tac1 neurons have been activated (M-N).
  • Optogenetic activation of BNSTpr Tac1 neurons tends to reduce aggression but it cannot induce mating in the presence of CNO (O- P).
  • Q-S. Strategy to activate POA Tacr1 neurons while inhibiting BNSTpr Tac1 neurons (Q).
  • Substance P induces excitatory LTP in the example POA Tacr1 neuron.
  • J Summary plot of normalized EPSP Amp and Rin as a function of time before and after perfusion of Substance P. Inset: Representative average EPSP traces of the baseline (1 ) and the last 5 min (2). Substance P induces excitatory LTP in POA Tacr1 neurons.
  • K Strategy for optogenetic activation of BNSTpr axons and electrophysiological recording from POA Tacr1 neurons.
  • L Representative trace showing light-evoke action potentials in BNSTpr neurons. Laser illumination elicits reliable action potentials in ChR2 + BNSTpr neurons.
  • M Summary plot of normalized EPSP Amp and Rin as a function of time before and after perfusion of Substance P. Inset: Representative average EPSP traces of the baseline (1 ) and the last 5 min (2). Substance P induces excitatory LTP in POA Tacr1 neurons.
  • K Strategy for optogenetic activation of BN
  • n 8 mice (A-F), 6 neurons from 4 mice (J), 8 neurons from 3 mice (M), 7 neurons from 3 mice (N), 7 mice (O-S), and 6 mice (T-X). * p ⁇ 0.05, ** p ⁇ 0.01 .
  • FIGS 28A-28S Substance P-Tacr1 signaling in the BNSTpr Tac1 ->POA Tacr1 pathway regulates male sexual behavior.
  • A Dual cannula placement above the POA.
  • B-E Infusion of L-703,606 into the POA increases latency to start mating with a female (B) without altering latency to attack an intruder male (D) or locomotor activity (C,E).
  • F-L Strategy to optogenetically activate BNSTpr Tac1 -»POA projections (F).
  • Optogenetic activation of BNSTpr Tac1 neurons cannot overcome suppression of male mating induced by inhibition of Tacrl signaling in the POA with L-703,606 (K,L).
  • M-S Strategy to optogenetically activate POA Tacr1 neurons (M). Schematic of optogenetic activation of POA Tacr1 neurons in males given L-703,606 and interacting with a receptive female (N). Activation of POA Tacr1 neurons overrides L-703,606-induced inhibition of male sexual behavior, leading to more mounts and reduced latency to mate (O-P).
  • FIGS 29A-29P 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 a receptive female (A). Activation (30/30s on/off) of these cells re-ignites mating drive, increasing the probability (B) and number (C) of mating routines. Raster plot of behavior of sexually satiated male subsequent to activation of POA Tacr1 neurons (D).
  • E-l Schematic of optogenetic activation of POA Tacr1 neurons in the SPP test (E).
  • Activation of POA Tacr1 neurons governs behavioral preference of males (F). Raster plots (each row is a male) showing that optogenetic activation governs investigatory behavior of males (H-l).
  • 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 (magenta) and sexually experienced (teal) males show more cumulative nose pokes to the active (cyan) compared to the inactive (gray) port (K,L), sexual experience enabling even more nose pokes (L).
  • M-N Schematic of optogenetic inhibition of POA Tacr1 neurons during the SPP test (M).
  • FIGS. 30A-30J Forced activation of POA Tacr1 neurons overcomes the post-ejaculatory refractory period and is rewarding.
  • A Raster plot of mating behavior in all males tested during the post-ejaculatory refractory period. Optogenetic activation following ejaculation rejuvenates mating drive such that all males display mounting, intromission, and some also ejaculate again.
  • B-C Schematic of laser illumination of mCherry+ POA Tacr1 neurons in post- ejaculatory males interacting with a receptive female (B). Without ChR2, laser illumination does not drive mating in the post-ejaculatory period (C).
  • D-E Schematic of laser illumination of mCherry+ POA Tacr1 neurons in post- ejaculatory males interacting with a receptive female
  • FIGS. 31A-31S The BNSTpr Tac1 -POA Tacr1 pathway is embedded in a neural circuit linking pheromone sensing to motor output.
  • A-D Strategy to activate POA Tacr1 neurons following ablation of AVPV/PVpo TH neurons with 6OHDA (A). Schematic of optogenetic activation of POA Tacr1 neurons in post-ejaculatory males interacting with a receptive female
  • E Schematic of optogenetic activation of POA Tacr1 neurons in males lacking AVPV/PVpo TH neurons in the SPP test (left). Activation of POA Tacr1 cells governs behavioral preference of males (right).
  • F Schematic of optogenetic selfstimulation (0.5 s, 40 Hz) of POA Tacr1 neurons in males lacking AVPV/PVpo TH neurons (left). Males show more cumulative nose pokes to the active compared to inactive port (right).
  • G-l Syp:mRuby expression in soma of POA Tacr1 neurons (G).
  • J-N Strategy for optogenetic activation of VTA termini of POA Tacr1 neurons (J). Males show more cumulative nose pokes to the active compared to inactive port (K,L). More mounts (M) but not intromissions (N) in epochs with optogenetic activation of VTA termini in males interacting with a receptive female.
  • O-S Strategy for optogenetic activation of PAG termini of POA Tacr1 neurons (O). Males show more cumulative nose pokes to the active compared to inactive port (P,Q).
  • Scale bars 100 pm (G-l), 20 pm (H,l inset).
  • FIGS. 32A-32T Relation of the BNSTpr Tac1 -POA Tacr1 pathway to sensory input, motivational centers, motor output, and reward.
  • A-D TH expression in the AVPV and VTA of 6OHDA or vehicle injected males (same mice as shown in Fig. 7A-F) (A). Fewer TH neurons in AVPV and VTA in mice given 6OHDA (B). 6OHDA injected males mated and the probability of mating routines (C) or number of mating events were not altered (D) even upon optogenetic activation of ChR2+ POA Tacr1 neurons. E-J.
  • Scale bar 100 pm (A,B,E,F,H,I).
  • FIG. 33 Analysis of mounts toward objects elicited by optogenetic activation of POA Tacr1 neurons. Forced activation of POA Tacr1 neurons elicited significantly more mounting as objects became more mouse-like in shape and size.
  • FIGS. 34A-34B Post-ejaculatory mating behavior, and CTB-mediated retrograde labeling of VTA and PAG projecting POA Tacr1 neurons.
  • control experiments in which both CTB-555 and CTB-647 were co-injected into either the VTA or PAG resulted in complete overlap of dually CTB+ neurons in the POA.
  • active agent refers to a chemical material or compound which, when administered to an organism (human or animal) induces a desired pharmacologic and/or physiologic effect by local and/or systemic action.
  • treatment refers to obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a behavior.
  • Treatment covers any treatment of a behavior in a mammal, particularly in a human.
  • a “therapeutically effective amount” or “efficacious amount” or “effective dose” means the amount of a compound that, when administered to a mammal or other subject for modulating a behavior, is sufficient to effect such modulation.
  • the “therapeutically effective amount” will vary depending on the compound, the disease and its severity and the age, weight, etc., of the subject to be treated.
  • unit dosage form refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of a compound calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle.
  • the specifications for unit dosage forms depend on the particular compound employed and the effect to be achieved, and the pharmacodynamics associated with each compound in the host.
  • a "pharmaceutically acceptable excipient,” “pharmaceutically acceptable diluent,” “pharmaceutically acceptable carrier,” and “pharmaceutically acceptable adjuvant” means an excipient, diluent, carrier, and adjuvant that are useful in preparing a pharmaceutical composition that are generally safe, non-toxic and neither biologically nor otherwise undesirable, and include an excipient, diluent, carrier, and adjuvant that are acceptable for veterinary use as well as human pharmaceutical use.
  • “A pharmaceutically acceptable excipient, diluent, carrier and adjuvant” as used in the specification and claims includes both one and more than one such excipient, diluent, carrier, and adjuvant.
  • a “pharmaceutical composition” is meant to encompass a composition suitable for administration to a subject, such as a mammal, especially a human.
  • a “pharmaceutical composition” is sterile, and preferably free of contaminants that are capable of eliciting an undesirable response within the subject (e.g., the compound(s) in the pharmaceutical composition is pharmaceutical grade).
  • Pharmaceutical compositions can be designed for administration to subjects or patients in need thereof via a number of different routes of administration including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, intracheal, intramuscular, subcutaneous, and the like.
  • the terms “individual,” “host,” “subject,” and “patient” are used interchangeably herein, and refer to an animal, including, but not limited to, human and non-human primates, including simians and humans; rodents, including rats and mice; bovines; equines; ovines; felines; canines; avians, and the like.
  • "Mammal” means a member or members of any mammalian species, and includes, byway of example, canines; felines; equines; bovines; ovines; rodentia, etc. and primates, e.g., non-human primates, and humans.
  • Non-human animal models e.g., mammals, e.g. non-human primates, murines, lagomorpha, etc. may be used for experimental investigations. Suitable animal models include particularly rodents, e.g. rats and mice.
  • determining As used herein, the terms “determining,” “measuring,” “assessing,” and “assaying” are used interchangeably and include both quantitative and qualitative determinations.
  • Embodiments of the present invention are directed to methods, systems and devices for assessing activity of a neural region.
  • a neural region is targeted for assessment of neural activity.
  • the neural region has subfields or local networks consisting of a relatively small number of neural cell groups.
  • Neuron may refer to electrical activity of a neuron (e.g., changes in membrane potential of the neuron), as well as indirect measures of the electrical activity of one or more neurons.
  • neural activity may refer to changes in field potential, changes in intracellular ion concentration (e.g., intracellular calcium concentration), and changes in magnetic resonance induced by electrical activity of neurons, as measured by, e.g., blood oxygenation level dependent (BOLD) signals in functional magnetic resonance imaging.
  • BOLD blood oxygenation level dependent
  • a slice of brain is obtained which comprises at least the region of interest for screening.
  • This block of tissue may be treated with an ion sensitive dye, e.g. a calcium dye.
  • a calcium dye are able to resolve ion flux on a cellular scale with microsecond precision.
  • candidate drugs are assessed with respect to their differential effect on these regions within the sample. Ideal drugs, for example, can serve single targeted neurophysiological roles, without creating additional neurophysiological effects which are competitive to the therapeutic goal.
  • Readout or image capture of the physiological activity of the neural network can be accomplished by a variety of techniques including calcium imaging, biochemical imaging and infrared imaging. PET and fMRI may also be applicable, albeit at lower spatial and temporal resolutions.
  • Stimulating the circuit may be accomplished with a variety of means that influence cellular activity, including application of drugs, magnetic fields, electrical current, optical (including opto-genetic) stimulation, ultrasound thermal and radiation methods as are known in the art.
  • data may be captured for example with a CCD camera, or as a digital matrix of sensor readings obtained from a sensor grid, or serially positioned sensors.
  • Processing resultant readout data by correlating activity level of cell types may be accomplished with computer software, for image analysis applications such as Image J. Providing processed correlation results to the user may be provided through screen displays, printed and transmitted data.
  • the microengineered physiological system comprises tissue explants seeded on a micropatterned platform.
  • the micropatterned platform can be configured to permit the formation of a neural architecture.
  • the microelectrode array comprises an area with a configuration that is complementary to that of the neural architecture.
  • the method can further comprise introducing one or more stimuli to the neural tissue; and measuring one or more responses from the neural tissue to the one or more stimuli.
  • the one or more responses comprise compound action potential amplitude, conduction velocity, waveform shape, histomorphological parameters, or combination thereof.
  • introducing the one or more stimuli comprises contacting the neural tissue with at least one pharmacologically active compound, electrical stimulus, chemical stimulus, optical stimuli, physical stimuli, or a combination thereof.
  • a method of evaluating the activity of a candidate agent can comprise exposing at least one agent to the neural tissue; measuring or observing changes in compound action potential amplitude, conduction velocity, waveform shape, histomorphological parameters, or combination thereof and correlating any measured or observed changes of the neural tissue with the agent.
  • a specific region of a brain of an individual is stimulated, in conjunction with combined electrophysiology, e.g. local field potentials (LFP) and functional magnetic resonance imaging (fMRI) scanning of different regions of the brain to determine functional connections.
  • Suitable protocols for analysis include electrophysiology; light-induced modulation of neural activity; electroencephalography (EEG) recordings; functional imaging and behavioral analysis.
  • Electrophysiology may include single electrode, multi electrode, and/or field potential recordings.
  • Light-induced modulation of neural activity may include any suitable optogenetic method, as described further herein.
  • Functional imaging may include fMRI, and any functional imaging protocols using genetically encoded indicators (e.g., calcium indicators, voltage indicators, etc.).
  • Behavioral analysis may include any suitable behavioral assays, such as behavioral assays for sextypical behaviors.
  • Some protocols such as fMRI, provide a non-invasive, brain-wide measure representative of neural activity.
  • Some protocols such as electrophysiology, provide cellular resolution and rapid measures of neural activity as well as cellular resolution and rapid control of neural activity.
  • Some protocols such as optogenetics, provide spatially-targeted and temporally-defined control of action potential firing in defined groups of neurons.
  • Suitable neuron-specific control sequences include, without limitation, an alpha subunit of Ca ++ -calmodulin-dependent protein kinase II (CaMKIla) promoter (see, e.g., Mayford et al. (1996) Proc. Natl. Acad. Sci. USA 93:13250) to target ventral hippocampal CAMKII neurons.
  • CaMKIla Ca ++ -calmodulin-dependent protein kinase II
  • Methods are provided for determining the effect of an agent on sex-typical behaviors, by analyzing the effects of stimulating specific neurons identified herein, and based on that information, selecting appropriate drug candidates and therapeutic modalities that are optimal for addressing these behaviors, while minimizing undesirable toxicity.
  • the treatment is optimized by selection for a treatment that minimizes undesirable toxicity, while providing for effective activity.
  • compositions comprising a pharmaceutically acceptable excipient.
  • the preferred form depends on the intended mode of administration and therapeutic application.
  • the compositions can also include, depending on the formulation desired, pharmaceutically-acceptable, non-toxic carriers or diluents, which are defined as vehicles commonly used to formulate pharmaceutical compositions for animal or human administration.
  • the diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, physiological phosphate-buffered saline, Ringer's solutions, dextrose solution, and Hank's solution.
  • the pharmaceutical composition or formulation may also include other carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenic stabilizers and the like.
  • the agents can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents.
  • conventional additives such as lactose, mannitol, corn starch or potato starch
  • binders such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins
  • disintegrators such as corn starch, potato starch or sodium carboxymethylcellulose
  • lubricants such as talc or magnesium stearate
  • auxiliary substances such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are commercially available.
  • Any compound useful in the methods and compositions of the invention can be provided as a pharmaceutically acceptable base addition salt.
  • “Pharmaceutically acceptable base addition salt” refers to those salts which retain the biological effectiveness and properties of the free acids, which are not biologically or otherwise undesirable. These salts are prepared from addition of an inorganic base or an organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, the sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like.
  • Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyidimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine,
  • Toxicity of the active agents can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the LD50 (the dose lethal to 50% of the population) or the LD100 (the dose lethal to 100% of the population). The dose ratio between toxic and therapeutic effect is the therapeutic index.
  • the data obtained from these cell culture assays and animal studies can be used in further optimizing and/or defining a therapeutic dosage range and/or a sub-therapeutic dosage range (e.g., for use in humans). The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition.
  • the term “isolated” refers to a molecule that is substantially free of its natural environment.
  • sample with reference to a patient encompasses blood and other liquid samples of biological origin, solid tissue samples such as a biopsy specimen or tissue cultures or cells derived therefrom and the progeny thereof.
  • the term also encompasses samples that have been manipulated in any way after their procurement, such as by treatment with reagents; washed; or enrichment for certain cell populations, such as diseased cells.
  • the definition also includes samples that have been enriched for particular types of molecules, e.g., nucleic acids, polypeptides, etc.
  • a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount different from the first dose amount. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount same as the first dose amount. In some embodiments, a dosing regimen is correlated with a desired or beneficial outcome when administered across a relevant population (i.e., is a therapeutic dosing regimen).
  • a computational system may be used in the methods of the present disclosure to control and/or coordinate stimulus through the one or more controllers, and to analyze data from scanning of the regions of the brain.
  • a computational unit may include any suitable components to analyze the measured images.
  • the computational unit may include one or more of the following: a processor; a non-transient, computer-readable memory, such as a computer-readable medium; an input device, such as a keyboard, mouse, touchscreen, etc.; an output device, such as a monitor, screen, speaker, etc.; a network interface, such as a wired or wireless network interface; and the like.
  • Each program can be implemented in a high level procedural or object oriented programming language to communicate with a computer system.
  • the programs can be implemented in assembly or machine language, if desired. In any case, the language may be a compiled or interpreted language.
  • Each such computer program can be stored on a storage media or device (e.g., ROM or magnetic diskette) readable by a general or special purpose programmable computer, for configuring and operating the computer when the storage media or device is read by the computer to perform the procedures described herein.
  • the system may also be considered to be implemented as a computer-readable storage medium, configured with a computer program, where the storage medium so configured causes a computer to operate in a specific and predefined manner to perform the functions described herein.
  • a variety of structural formats for the input and output means can be used to input and output the information in the computer-based systems of the present invention.
  • Data can also be sent from a computer to another computer or data collection point via methods well known in the art (e.g., the internet, ground mail, air mail).
  • methods well known in the art e.g., the internet, ground mail, air mail.
  • data collected by the methods described herein can be collected at any point or geographical location and sent to any other geographical location.
  • Sex differences in behavior reflect the action of a sexually differentiated brain.
  • neural circuits underlying sex-typical physiology or social interactions such as mating and aggression are under the control of SHs.
  • SHs bind to cognate nuclear receptors in SHsensitive neuronal populations within these neural circuits to influence gene expression and signaling cascades, thereby influencing physiology and behavior (McCarthy and Arnold, 2011 ; Yang and Shah, 2014).
  • SHs bind to cognate nuclear receptors in SHsensitive neuronal populations within these neural circuits to influence gene expression and signaling cascades, thereby influencing physiology and behavior.
  • Estrogen receptor alpha (ERa or Esr1 ) is essential for sex typical behaviors in both sexes and is expressed in subsets of neurons within the BNSTpr (BNSTprEsn neurons), POA (POAEsn neurons), MeA (MeAEsn neurons), and VMHvI (VMHvlEsn neurons, equivalent to VMHVIPR neurons, progesterone receptor expressing 70 VMHvI neurons).
  • BNSTprEsn neurons POA (POAEsn neurons), MeA (MeAEsn neurons), and VMHvI (VMHvlEsn neurons, equivalent to VMHVIPR neurons, progesterone receptor expressing 70 VMHvI neurons).
  • TRIPseq deep seq
  • TRAPseq In preparing our TRAPseq libraries, we had loaded equivalent starting material at each step so as to preclude biases introduced by cell number differences between sexes. Accordingly, TRAPseq identified numerous sDEGs that were upregulated in females even in regions such as the BNSTpr and POA that have more neurons in males (FIG. 12E) (Table S2). We next used hybridization chain reaction based in situ hybridization (HCR-ISH) to visualize differential expression of sDEGs in males and females (Fig 1 C, S1 F) (Choi et al., 2018).
  • HCR-ISH hybridization chain reaction based in situ hybridization
  • Estrogen receptor (Esr1 ) and progesterone receptor (PR) are generally thought to be transcriptional activators. Nevertheless, a large fraction of eDEGs was upregulated in FNR mice, suggesting de-repression as a mode of gene regulation in SH-sensitive neurons. Irrespective of the underlying mechanisms, our data reveal large scale genome-wide regulation of gene expression by estrous state.
  • the POA and MeA contain DEGs transcribed at low levels or in rare cellular subsets that could be identified with more sensitive profiling approaches in the future.
  • the large number of sDEGs in the BNSTpr supports the recent finding that BNSTpr neurons are functionally extremely sexually dimorphic as they regulate male but not female social interactions.
  • the estrous state exerts a profound influence on the morphology and function of VMHvI neurons, a feature that may explain the large number of eDEGs we observe in this region (Inoue et al., 2019).
  • Each Esr1 + population expresses a unique complement of DEGs such that only a subset of DEGs is common to more than 2 regions, and only 2 sDEGs and 11 eDEGs are common to all four regions (Fig. 20; Tables S2, S3).
  • This limited commonality in DGE patterns suggests that sex or estrous state impact different SH- sensitive populations differently such that there is not a generic signature of sex or estrous state across the brain.
  • That most DEGs are expressed in >1 tCT likely represents the fact that sexual differentiation of the brain, which is initiated perinatally and continues into adulthood, is superimposed upon the program of neuronal cell fate specification, which is completed earlier.
  • the expression of aromatase and androgen receptor (AR) is not sexually dimorphic at birth and it only resolves into its adult dimorphic pattern following the perinatal window of sexual differentiation (Juntti et al., 2010; Wu et al., 2009). It is possible that seq studies with higher depth of coverage, for example with well-based scRNAseq or studies focused on individual tCTs, will identify additional DEGs that individually label most tCTs uniquely. Alternatively, broad distribution of DEGs in >1 tCT may endow such tCTs with shared properties and functionality.
  • the remaining 7 tCTs contained fewer cells in one or the other estrous state and also exhibited a sex difference in cell number; of these, the Cckar tCT in the VMHvI (VMHvIcckar/Esri tCT) and the VMHvlTrim36/Esri tCT were only observed in FR and FNR mice, respectively.
  • VMHvIcckar/Esri tCT VMHvIcckar/Esri tCT
  • VMHvlTrim36/Esri tCT were only observed in FR and FNR mice, respectively.
  • BNSTprTaci/Esn tCT mediates the exclusively male role of BNSTprAro neurons in social behaviors.
  • the sex-shared BNSTprTaci/Esn tCT displays the highest enrichment for DEGs, with most DEGs upregulated in M compared to FR and FNR (Fig. 6A, S6A,B).
  • This tCT is also one of the few tCTs to express aromatase (encoded by Cypl9al) (Fig.
  • BNSTprAro neurons are critical for male but not female social interactions.
  • Male BNSTATO neurons are essential for sex recognition and the ensuing targeted display of mating with females and aggression toward males.
  • female BNSTATM neurons are not active during social interactions nor are they essential for mating, maternal care of pups, or maternal aggression.
  • Tac1 is an sDEG that is upregulated in males (Fig. 6A) and expressed in a salt-and- pepper fashion within the BNSTpr (Fig. 6B). Importantly, Tac1 signaling has been implicated in urine preference and social behaviors in males in diverse taxa. This suggested the interesting possibility that the BNSTTaci/Esri tCT is critical for male social interactions.
  • mice use pheromones to recognize the sexes, and male mice show a clear preference for urine, a rich source of pheromones, from females (Fig. 6D).
  • Chemogenetic inhibition of BNSTprTaci/Esri neurons in sexually naive males abrogated this preference (Fig. 6D, S6C).
  • BNSTprAro neurons there was no diminution in total time spent sniffing urine, indicating that inhibiting BNSTprTad/Esn neurons does not simply reduce motivation to explore pheromones (FIG. 17D).
  • VMHvlCckar/Esr1 tCT among all VMHvlEsrl tCTs, mediates female sexual behavior
  • VMHvIcckar/Esn tCT which expresses Cckar in a subset of VMHvlEsn neurons, is sexually dimorphic such that the constellation of genes that define this tCT does not cluster into an identifiable tCT in males (Fig. 7A,B, S7A,B).
  • VMHvIcckar/Esri tCT is highly enriched for eDEGs that are upregulated in FR mice and largely absent in FNR mice; by contrast, the VMHvlTrim36/Esri tCT is female-specific and restricted to FNR mice (Fig. 7C,D, S7A,B).
  • Cckar expression is dimorphic and restricted to FR mice (Fig. 7C), in accord with our previous findings.
  • VMHvlEsn neurons are essential for mating behavior in both sexes, maternal aggression, and male territorial aggression , and Esr1 knockdown in these cells reduces mating behavior in both sexes and male aggression.
  • CTB cholera toxin B
  • Cre-dependent Syp:mCherry to the VMHvI of Cckarcre females and enumerated the overlap between CTB and mCherry.
  • sDEGs and eDEGs are significantly enriched for sHREs, suggesting that their expression patterns may be directly regulated by binding of cognate nuclear hormone receptors. Because TRAPseq does not allow identification of non-coding RNA, we suspect that we have underestimated the number of sDEGs (and eDEGs) in these populations. In addition, it is likely that sex differences in post-transcriptional processes will further modulate differential cellular function between males and females.
  • VMHVIESM neurons whose projections to the AVPV exhibit structural plasticity across the cycle, also harbor the largest number of eDEGs. Many of these eDEGs label an Fn-restricted tCT (VMHvIcckar/Esn tCT) that is essential for female sexual behavior and whose projections peak when the female enters an estrus state.
  • VMHvIcckar/Esn tCT Fn-restricted tCT
  • Such functionality may arise from unique projection patterns, as is the case for the VMHvIcckar/Esn tCT, or from unique patterns of DEGs. That DEGs may indeed impart such specialization is in agreement with our finding that each of the four Esr1 + populations expresses unique complements of DEGs. In particular, our findings suggest that there is not a generic sex or estrous-state regulated signature of differential gene expression, but rather that such a signature varies between SH-sensitive populations and tCTs within individual populations. The specific set of connections, DEGs, and, potentially, post-transcriptional processes imbues each tCT with its modular role in sexually dimorphic social behaviors.
  • mice used for TRAPseq and snRNAseq were F1 Esr1 Cre/+;RiboTag/+ or Esr1 Cre/+;SunTag/+ and generated by mating homozygous male Esr1 Cre/Cre mice with homozygous female SunTag or RiboTag mice.
  • Adult mice were deeply anesthetized by injection with 2.5% Avertin and euthanized by decapitation. Brains were sectioned into 500 urn coronal slices using a brain matrix mold (BrainTree Scientific) chilled on ice.
  • Sections were floated in either chilled TRAPseq homogenization buffer (100 mM KOI, 50 mM Tris-HCL pH 7.4, 12 mM MgCL2) or nuclear homogenization buffer (snRNAseq; see below).
  • the BNSTpr, MeA, POA and VMHvI were identified using landmarks from the mouse brain atlas (Paxinos and Franklin, 2003) and dissected using a Zeiss microscope. Tissue was then either frozen on dry ice and stored at -80C until further processing (TRAPseq) or stored in cold homogenization buffer (snRNAseq) for subsequent nuclear extraction.
  • mice were deeply anesthetized by injection of 2.5% Avertin and then trans-cardially perfused with 20 mL ice-cold PBS followed by 20 mL ice-cold 4% paraformaldehyde (PFA). Following overnight fixation in 4% PFA at 4oC, brains were sectioned coronally on a vibrating microtome (Leica) at 50 iim (HCR-ISH) or 65 iim (immunohistochemistry).
  • Leica vibrating microtome
  • TRAPseq We followed previously published protocols to perform TRAPseq (Sanz et al., 2009). In brief, frozen brain regions were thawed in a pestle homogenizer in 1 mL of ice- cold buffer containing 100 mM KCI, 50 mM Tris-HCL pH 7.4, 12 mM MgCL2, 1% IGEPAL, 1 mM dithiothreitol (DTT), 1 mg/mL heparin, 100 mg/mL cycloheximide (CHX), complete mini protease inhibitor (Sigma- Aldrich), and 40 U/ml murine RNAse inhibitor (RNasin; NEB).
  • buffer RLT QIAgen RNeasy micro kit
  • IP sample 2.5 ml of rabbit anti-HA antibody (Cell Signaling) was added to the remaining homogenate (IP sample) and the sample was incubated in an end-ove rend rotator at 4°C for four hours. The sample was then added to 50 ml of pre-washed paramagnetic protein G Dynabeads (Invitrogen) and incubated overnight a 4°C on an end-over-end rotator. After incubation, beads were washed three times for ten minutes at 4°C with high-salt buffer (300 mM KCI, 50 mM Tris-HCL pH 7.4, 12 mM MgCL2, 1% 1080 IGEPAL, 1 mM DTT, 300
  • high-salt buffer 300 mM KCI, 50 mM Tris-HCL pH 7.4, 12 mM MgCL2, 1% 1080 IGEPAL, 1 mM DTT, 300
  • RNA samples were transferred to a new tube, collected by magnet and the supernatant removed.
  • RLT buffer (0.35 mL) was added directly to the beads and total RNA from Input and IP samples was extracted using the QIAgen RNeasy Micro kit according to manufacturer’s instructions, including on-column DNase digestion.
  • RNA was eluted in 20 ml RNAse-free water and stored at -80°C. RNA yield and quality was evaluated by Bioanalyzer 2000 (Agilent) using the Total RNA Pico kit.
  • RNA from each Input and IP sample was used for the Protoscript cDNA synthesis kit with random hexamer priming followed by real-time quantitative PCR (RT-qPCR) on either an ABI StepOne Plus or GeneQuant qPCR machine (Biorad).
  • RT-qPCR real-time quantitative PCR
  • Esr1 , Gfap and Gapdh oligonucleotide primers listed in Table S1
  • For each IP sample we calculated enrichment of Esr1 and depletion of Gfap relative to Input using the A/ACt method.
  • snRNAseq We modified the INTACT protocol (Mo et al., 2015) to isolate nuclei as follows. Microdissected brain regions were suspended in ice-cold nuclear homogenization buffer (HB) consisting of 0.25 M sucrose, 25 mM KCI, 5 mM MgCI2, and 20 mM Tricine-KOH pH 7.8 supplemented with 1 mM DTT, 0.15 mM spermine, 0.5 mM spermidine, complete mini protease inhibitor (EDTAfree)( Sigma-Aldrich,) and murine RNAse inhibitor.
  • HB nuclear homogenization buffer
  • the tissue was homogenized in a Dounce homogenizer by 5-10 strokes, 60 ml of HB/5% IGEPAL was added, and the suspension was homogenized with an additional 5-10 strokes before being passed through a 40 mm filter.
  • the resulting suspension was mixed with 1 mL (1 volume) of 50% Optiprep (Sigma-Aldrich) containing 25 mM KCI, 5 mM MgCI2, and 20 mM Tricine-KOH pH 7.8 and transferred to an ultracentrifuge tube.
  • the suspension was underlaid with a 30% Optiprep solution and spun at 10,000g in a swinging-bucket ultracentrifuge for 20 minutes at 4°C to pellet the nuclei.
  • the supernatant was removed and nuclei were resuspended in 1 mL HB supplemented with 1 mM DTT, 40 U/mL RNasin, and 0.1% IGEPAL.
  • Nuclei were pelleted by centrifugation at 2,000 g for 10 min at 4°C and resuspended in 60 j l of sterile PBS with 2% bovine serum albumin (BSA). They were again inspected and quantified by Trypan Blue staining prior to submission to the Stanford Functional Genomics Facility for generation of snRNA libraries using the 10X Genomics Chromium platform.
  • BSA bovine serum albumin
  • HCR-ISH Probes and fluorescently conjugated amplifiers were purchased from Molecular Instruments (Pasadena, CA). We designed as many probe pairs as possible for each gene, up to a maximum of 40 pairs/gene. Probes were designed to be detected with hairpin amplifiers conjugated to either AlexaFluor-488, 546 or 647 fluorophores for high-, medium- and low-expressing genes respectively (estimated from TRAPseq data). HCR hybridization, wash and amplification buffers were made following instructions manufacturer’s recommendations for mammalian cells on a slide (Choi et al., 2018).
  • Coronal sections were collected at 50 mm on a vibrating microtome (Leica), and the sections were washed with DEPC/PBS and incubated overnight in 70% ethanol/PBS at 4°C. Sections were washed twice in PBS and cleared by incubating for 45 minutes in 5% SDS/PBS at room temperature. After clearing, sections were washed twice in 2X SSC, then incubated twice in 2X SSC for 15 min at room temperature with gentle shaking. 1140 They were then incubated in hybridization buffer at 37°C for two hours. After prehybridization probes were added to a final concentration of 10 nM and sections were hybridized at 37°C overnight.
  • Transcript-length normalized counts were loaded into R using the tximport package and differential expression analyzed using DESeq2.
  • Y-chromosome genes, mitochondrial genes, and genes with fewer than 5 counts in at least 7 samples were excluded from analysis.
  • a generalized linear model was constructed for each Esr1+ population using libraries from all three conditions with default DESeq2 parameters.
  • Individual pairwise comparisons (M v FR, M v FNR, FR v FNR) were extracted using the ‘contrast’ function in DESeq2. Genes were considered differentially expressed if they passed a false discovery rate-adjusted p value ⁇ 0.05 in at least one pairwise comparison. Unless otherwise noted, for subsequent analyses we only considered genes with an absolute fold change > 1 .5 in either direction.
  • GO analysis GO analysis was conducted using topGO.
  • the union of all DEGs (M v FR, M v FNR, FR v FNR) from each Esr1 + population was used as input, with all genes expressed in that Esr1 + population used as background (based on TRAPseq data).
  • FIG. 14 For visualization (FIG. 14) we examined the top 100 most significant GO categories by p value in each Esr1 + population and selected those present in >1 region to highlight shared functions of sex and estrous-regulated genes. A complete list of enriched GO categories for each Esr1 + population is available in Table S4.
  • a cell x barcode matrix was loaded into R using the BusParse package and used as input to Seurat.
  • Each library (M, FR, FNR) from each Esr1 + population was initially analyzed individually for quality control. Cells were retained if at least 500 Unique Molecular Identifiers (UMIs) were detected, while a gene was retained if >1 UM I was detected for that gene in > 3 cells.
  • UMIs Unique Molecular Identifiers
  • Count data was variance stabilized in Seurat using the sctransform function.
  • the abundant non-coding RNA Malatl accounted for 1 - 4% of all UMIs/cell and showed systematic differences in relative abundance between libraries (not shown); we therefore calculated the percentage of reads mapping to this gene for each cell (%Malat1 ) to use as a regression variable for sctransform.
  • the top 3000 variable features were identified and used as input for principal component analysis (PCA) followed by identification of tCTs using the Jaccard- Louvain community detection algorithm. An initial round of clustering was performed using the top 30 PCs and a resolution of 1 .5 to identify non-neuronal tCTs and tCTs consisting of low- quality cells or probable doublets.
  • PCA principal component analysis
  • An initial round of clustering was performed using the top 30 PCs and a resolution of 1 .5 to identify non-neuronal tCTs and tCTs consisting of low- quality cells or probable doublets.
  • tCTs consisting of low-quality cells or probable doublets based on one or more of the following criteria: uniformly low UMI count (median >2-fold lower than that of all other tCTs), high mitochondrial content (any mitochondrial transcript detected in >25% of cells), and/or lack of significantly enriched markers relative to all other cells.
  • tCTs expressing Slc17a6 or Slc17a7 in >25% of cells as excitatory neurons and those expressing Gad1 in >25% as inhibitory neurons.
  • condition-biased tCTs To identify condition-biased tCTs in each Esr1 + population we examined the percentage of cells from each condition (M, FR, FNR) assigned to that tCT. We classified tCTs as condition-biased if they met the following criteria: 1 ) significant difference (p ⁇ 0.05) in counts of cells assigned between all libraries (Fisher’s exact
  • the BNSTTac1/Esr1 tCT also contained the largest set of sDEGs when each tCT within the BNSTEsrl population was examined for sDEGs between the two comparisons (M v FR and M v FNR).
  • PRFIpo, EsrIFIpo and CckarCre mice were generated using CRISPR/Cas9-mediated homologous recombination (Miura et al., 2018).
  • Guide RNAs gRNAs were designed using the Integrated DNA Technology ALT-R CRISPR HDR design tool. Targeting constructs were designed to contain 100 bp of 5’ and 3’ homology arms flanking the 2A-Flpo or 2A-Cre sequences. Homology arms were designed to target the 3’UTR of each gene (corresponding to exon 10 for Esr1 , exon 5 for Cckar and exon 8 for PR).
  • Single stranded DNA (ssDNA) doners were co-injected with trans-activating CRISPR RNA (tracrRNA) at the Gladstone Institute (San Francisco, CA).
  • ssDNA Single stranded DNA
  • tracrRNA trans-activating CRISPR RNA
  • Candidate founders were screened by PCR and backcrossed to C57BL6/J >3 generations prior to being used for behavioral experiments.
  • a derivative of the CMV Towne Variant intron B (Fenno et al., 2014) containing a cDIO cassette was inserted between exon 1 and exon 2, with the donor and acceptor sites fused directly to the 3’ terminus of exon 1 and 5’ terminus of exon 2, respectively.
  • a derivative of the mouse IgE intron 3 (Fenno et al., 2014) containing a cDIO cassette was inserted between exon 2 and exon 3, with the donor and acceptor sites fused directly to the 3’ terminus of exon 2 and 5’ terminus of exon 3, respectively.
  • Separate f DIO cassettes were added directly after the promoter (5’ to the entire coding sequence) and directly before the WPRE (3’ to the entire coding sequence).
  • the exon order is exon 3, exon 2, exon 1 , with all exons in the reverse complement orientation. All of these plasmids are constructed in an AAV-nEF backbone with a 3’ WPRE.
  • mRNA isolation 1355 and cDNA synthesis HEK293FT cells at 50% confluence were transfected with endotoxin-free DNA using Lipofectamine 3000 (Thermo Fisher) following the manufacturer’s protocol.
  • RNA extraction was performed using reagents from the RNeasy Mini Kit (Qiagen). Cells were disrupted with lysis buffer and homogenized using QiaShredder homogenizer columns.
  • Flow cytometry HEK293FT cells were grown in 24-well tissue culture plates to 50% confluence and transfected in duplicate with 500 ng total DNA with Lipofectamine 3000 (Thermo Fisher) following the manufacturer protocol. Five days post transfection, cells were removed by enzymatic dissociation (TrypLE, Gibco), resuspended in cold PBS, pelleted at 200g for 5 min and resuspended in 500 j L PBS supplemented with 5
  • DAPI 5
  • Flow cytometry was completed on a Novocyte Quanteon analyzer at the Stanford Shared FACS Facility using settings optimized for side scatter (SS), forward scatter (FS), vital dye (DAPI) and fluorophore (mCherry) acquisition using positive (non-recombinasedependent DREADDi-mCherry), negative (empty transfection) and dead (heat-killed; 95 °C for 3 min) conditions as controls.
  • Live-cell, singlet populations used in comparisons were isolated from debris and dead cells in post hoc analysis using FlowJo 10.7.2 (FlowJo) by (i) positively gating for the high-density population in plotting FS vs. SS, (ii) positively gating for singlets, and (iii) negatively gating for vital dye+ cells.
  • CNO Clozapine N-Oxide
  • Enzo Clozapine N-Oxide
  • sterile saline 5 mg/mL
  • CNO was freshly diluted in sterile saline to achieve the following doses: 1 mg/kg body weight (TacI Cre and Tac1 Cre;Esr1 Flpo), 15 mg/kg (female CckarCre and CckarCre;PRFIpo), 3 mg/kg (male CckarCre) and 5 mg/kg (male CckarCre;PRFIpo).
  • mice were injected intraperitoneally (IP) with CNO or sterile saline 30 min (experimental males) or 90 min (experimental females) prior to behavioral assays. Mice were tested on each behavioral assay once each with CNO and saline, with the order of CNO and saline administration counterbalanced across animals.
  • IP intraperitoneally
  • Viruses AAV-EF1 a-flex-DREAADi:mCherry was purchased from the UNC Vector Core.
  • AAV-EF1 a- DO-Syp:mCherry (serotype 8.2) was purchased from the MGH viral core.
  • AAV-EF1 a-DIO1400 Syp:mCherry (serotype 1) was custom packaged by Virovek (Hayward, CA).
  • AAV-hEF-CoffFon- DREAADi:mCherry was custom packaged by Stanford Virus Core (Stanford, CA).
  • AAV titers were 1 .5 x 10 12 - 2.5 x 10 13 genomic copies/mL.
  • Viruses were delivered into brains of mice at 10-16 weeks of age exactly as described previously (Bayless et al., 2019; Inoue et al., 2019; Yang et al., 2017). The following volumes of virus were injected bilaterally: 0.5 j l (BNSTpr), 1 pd (male VMHvI), 0.3
  • mice were allowed to recover individually over a heat pad and then returned to their home cages.
  • Females used for mating behavior assays or viral tracing studies were ovariectomized at the time of stereotaxic surgery. Animals were allowed at least 2 weeks of recovery following surgery prior to being tested in behavioral assays.
  • Behavioral Assays All behavioral assays were performed in the dark cycle (>1 hr after lights out) and recorded using a webcam under infrared illumination. Videos were played at 30 frames per second and manually annotated using custom software as described previously.
  • Urine preference assay Prior to testing, males were single housed for > 7 days. Males were tested on the pheromone preference assay once each with CNO and saline injected 30 min prior to the assay, with the order of CNO and saline administration counterbalanced across animals.
  • two 1 ”x1 ” cotton swabs (one wetted with 80 jil of undiluted, WT, group housed, hormonally primed C57BL/6J female urine and the other wetted with 80 jil of undiluted WT, group housed C57BL/6J male urine) were placed at opposite ends of their home cage. Males were given 5 min to investigate the urine swabs, and the duration, latency, and number of sniffs directed to each swab were scored. Following completion of this test, males were tested for performance in mating and aggression.
  • Female sexual behavior Female mice can exhibit low levels of sexual receptivity during the first mating experience, and to reduce variability in behavior, we subjected all experimental females to a round of mating experience with a WT sexually experienced male. In this setting, we primed the female, inserted her into home cage of a singly housed male for 30 min, and verified that mounting and intromission occurred. Following this, females were tested and analyzed for behavioral performance in mating tests with WT sexually experienced males. Lordosis was defined by the display of a dorsiflexed back while braced on all four legs during a mount or intromission.
  • the receptivity index measures whether or not the female allows mounts to proceed to intromission. Rejection behavior was defined by the display of postures (running or walking away and rearing up against male) during mounting that delayed or precluded subsequent mating events.
  • Maternal behaviors Following recovery from surgery, females used for tests of maternal behavior were housed with a WT male until visibly pregnant. Males were then removed and females allowed to deliver their litter in single housing. To test performance in pup retrieval, all pups were briefly removed from the cage 2 and 4 days following birth and 4 of them were subsequently placed separately in each cage corner. Pup retrieval was assayed for 5 min, allowing sufficient time for mothers to retrieve all pups. Following the assay, remaining pups were returned to the cage. To assay maternal aggression, pups at postnatal day 6 and 8 were removed, and a WT group housed male was inserted into the cage for 15 min. Pups were returned to the cage following completion of the assay. Maternal aggression was defined as physical attacks towards the intruder male (episodes of biting, wrestling, tumbling, chasing).
  • the number of presynaptic termini was enumerated using Image J software (Analyze Particle plugin) and divided by the area imaged to obtain the density of these termini in any given region. This estimate of the density of presynaptic termini was then normalized to the number of mCherry+ VMHvlCckar/Esr1 or VMHvlCckar-/Esr1 neurons to correct for subtle variations 1480 in infection in each animal.
  • AAV- EF1 a-Flex- Syp:mCherry or AAV-EF1 a-Coff-Syp:mCherry were injected into the VMHvI of CckarCre female mice.
  • AAV injection we injected 0.3 j L of the retrograde tracer CTB (conjugated to Alexa Fluor 488) into the AVPV. Mice were perfused 5 days after CTB injection.
  • the middle three sections through the VMHvI were imaged by confocal microscopy using a 63x objective, with image stacks containing 5 optical sections at 1 urn intervals.
  • the number of CTB/mCherry+ cells in the VMHvI was quantified using Photoshop (manual counting) and Imaged software (Analyze Particle plugin for automated enumeration).
  • CTB+ VMHvlCckar/Esr1 or VMHvlCckar-/Esr1 neurons are represented as a fraction of the CTB+ neuron counts in the mCherry+ neurons of WT females.
  • BNSTpr Tac1 Male Tac1 -expressing BNSTpr (also referred to as BNSTpr Tac1 ) neurons are more active upon encountering a female than a male. Shown in FIG. 8B, optogenetic activation of these male BNST pr Tac1 neurons, even transiently, at the beginning of an encounter with a male suppresses aggression and promotes mating with a male. Optogenetic activation of the projections of male BNSTpr Tac1 neurons to the POA (preoptic area of the hypothalamus), even transiently, at the beginning of an encounter with a male suppresses aggression and promotes mating with the male.
  • POA preoptic area of the hypothalamus
  • FIG. 8C Optogenetic activation of POA Tacr1 neurons elicits mating that is time-locked to the optogenetic activation.
  • Male POA Tacr1 neurons are required for mating with females but not aggression with males. Optogenetic inhibition of these neurons inhibits mating but not aggression.
  • Chemical antagonist of Tacrl profoundly reduces male mating with females but not aggression with males. Infusion of L703,606 into the POA inhibits mating with females but not aggression with males.
  • Female Kissi -expressing AVPV neurons can elicit mating even when the female has no circulating ovarian hormones and is sexually unreceptive
  • AVPV anteroventral periventricular nucleus of the hypothalamus
  • Female AVPV Kiss1 neurons are active when the female is mating.
  • Figure 1 1 A Inhibition of these neurons suppresses female mating even though she is in estrus (heat) and sexually receptive. In other words, activity of these neurons is reguired for female sexual behavior.
  • Figure 1 1 B Activation of female AVPV Kiss1 neurons enables the female to mate even though she is not in estrus and therefore sexually unreceptive. This dataset shows that we can elicit desire to mate in a female who would otherwise be sexually unreceptive.
  • AVPV Kiss1 Female Kissi - expressing AVPV (AVPV Kiss1 ) neurons are innervated by Cckar-expressing neurons of the VMHvI (ventrolateral sector of the ventromedial hypothalamus). Shown in FIG. 1 1 C, activity of AVPV Kiss1 neurons is crucial to drive female sexual behavior.
  • BNSTpr Tac1 neurons we have discovered a neural circuit for male sexual behavior emanating from chemosensory pathways to BNSTpr Tac1 neurons, which in turn innervate POA Tacr1 neurons that project to centers that regulate motor output and reinforcement. Functional interrogation of BNSTpr Tac1 and POA Tacr1 neurons reveals that this neural circuit is necessary and sufficient for mating. Neural circuit epistasis studies reveal that BNSTpr Tac1 neurons are upstream of POA Tacr1 cells and that substance P released by BNSTpr Tac1 neurons signals through its cognate receptor Tacrl in POA Tacr1 cells to promote mating.
  • POA Tacr1 neurons Activation of POA Tacr1 neurons is reinforcing and, remarkably, abrogates the postejaculatory refractory period to enable multiple mating bouts. Together, this neural circuit governs the key aspects of male sexual behavior, mating displays, drive, and reinforcement.
  • BNSTpr Tac1 neurons are preferentially activated by females and critical for sexual behavior.
  • BNSTpr Esr1 neurons consist of many molecularly distinct neuronal cell types that have been defined with snRNAseq 9 (Fig. 19A).
  • Several neuronal cell types within the Esr1 population express aromatase, and these cells (BNSTpr Aro or AB neurons) are essential for functionally distinguishing between the sexes in sexually naive and experienced males. They show higher activation upon encountering a female compared to a male and are active during various components of mating but not aggression.
  • female BNSTpr Aro neurons are not essential to distinguish between the sexes, mate, or exhibit maternal behavior.
  • BNSTpr Tac1 neurons of sexually naive Tac1 Cre males 35 and performed fiber photometry Fig. 19B-E, FIG. 20A-M.
  • BNSTpr Aro neurons we observed that BNSTpr Tac1 neurons were significantly more active upon presentation of female urine compared to male urine or saline (Fig. 19B-E).
  • BNSTpr Tac1 neurons were more active upon encountering a female compared to a male or inanimate object (FIG. 20A-E), activated during mating routines such as mounting and ejaculation, and quiescent when attacking a male (FIG. 20G-M).
  • BNSTpr Tac1 neurons like that of BNSTpr Aro neurons, of sexually naive males distinguishes between the sexes and reports mating but not aggressive displays.
  • the high level of activation of BNST pr Tac1 neurons detected by fiber photometry toward females compared to males could arise from higher activation of all cells or from more cells activated by female compared to male cues.
  • BNSTpr Tac1 The projection of BNSTpr Tac1 to POA Tacr1 neurons governs mating but not aggression. We reasoned that BNSTpr Tac1 neurons would transmit information regarding the presence of a potential mate to postsynaptic cells that would, in turn, regulate mating displays. In order to identify BNSTpr Tac1 -recipient targets, we expressed a Cre-dependent fusion of synaptophysin with mRuby (Syp:mRuby) in BNSTpr Tac1 neurons of Tac1 Cre males and visualized mRuby+ presynaptic termini.
  • BNSTpr Tac1 neurons projected to diverse regions, we noticed that the largest zone of innervation was the preoptic hypothalamus (POA) (Fig. 21 A-B), a center critical for male sexual behavior. Delivery of Cre-dependent EGFP to the POA of Tacr1 Cre males showed a collection of POA Tacr1 neurons that could, in principle, be postsynaptic to BNSTpr Tac1 cells (Fig. 21 C-D). To test this possibility, we employed engineered monosynaptic rabies vector for trans-synaptic retrograde labeling.
  • POA preoptic hypothalamus
  • BNSTpr Tac1 neurons are a small subset of the larger BNSTpr Aro and BNSTpr Esr1 populations 9 (Fig. 19A), and our data therefore show that these cells represent the major source of presynaptic input from the BNSTpr to POA Tacr1 neurons.
  • POA Tacr1 neurons are activated during and critical for mating but not aggression.
  • the activity of male BNSTpr Tac1 -POA Tacr1 projections regulates mating but not aggression, suggesting that the postsynaptic POA Tacr1 neurons regulate mating specifically.
  • This hypothesis makes the predictions that POA Tacr1 neurons are active during mating but not fighting and that experimental manipulation of their activity should bidirectionally regulate mating displays.
  • virtually all POA Tacr1 neurons express the estrogen receptor alpha (ERa or Esr1 ) (FIG. 24A-D), and POA Esr1 neurons have previously been shown to be active during and critical for male sexual behavior.
  • POA Tacr1 neurons show no discernible activation during aggression toward other males, indicating that they do not regulate this behavior. Indeed, laser illumination of eNpHR3.0+ POA Tacr1 neurons in Tacr1 Cre males did not alter aggressive displays or locomotor activity during encounters with male intruders (Fig. 25G-I, FIG. 24S- U). Taken together, our findings show that POA Tacr1 neurons are active during mating and this activity is acutely necessary and sufficient to induce mating. [00230] POA Tacr1 neurons function downstream of BNSTpr Tac1 neurons in promoting male mating.
  • Substance P released by BNSTpr Tac1 neurons acts on POA Tacr1 neurons to promote mating.
  • Substance P which is encoded by Tac1
  • Tacrl signaling is important for male sexual behavior in rodents.
  • Substance P and Tacrl are functionally relevant to diverse behavioral domains beyond social interactions and expressed widely in the brain. Accordingly, it is presently unclear where this neuropeptide-receptor pathway acts to regulate male sexual behavior.
  • Substance P activates POA Tacr1 neurons to promote mating then it should be possible to bypass L-703,606-imposed mating block by forced activation of POA Tacr1 neurons.
  • Cre-dependent ChR2 in POA Tacr1 neurons of Tacrl Cre males and asked whether optogenetic activation of these cells would elicit male sexual behavior even following provision of L-703,606 (Fig. 27T-U).
  • L-703,606 profoundly reduced male mating displays toward females but forced activation of POA Tacr1 neurons with laser illumination bypassed this chemically induced mating-block to restore sexual behavior (Fig. 27V, FIG. 28M-P).
  • POA Tacr1 neurons regulate mating drive and reinforcing aspects of sexual behavior. Subsequent to mating to ejaculation, males lose their mating drive and enter a refractory period during which they do not seek to mate with a potential sexual partner. C57BL/6J male mice have a post- ejaculatory refractory period of ⁇ 5 days (FIG. 34). Activation of periventricular dopaminergic AVPV TH neurons in the anterior hypothalamus re-ignites the mating drive in mice such that males will mate, albeit following variable delays, during timepoints in which they would otherwise be in their post-ejaculatory refractory period.
  • the hypothalamus including the POA, is known to contain sites whose activation elicits self-stimulation. Given that mating is reinforcing, we tested whether activation of POA Tacr1 neurons would reinforce behavioral performance or elicit self-stimulation.
  • SPP social place preference
  • the BNSTpr Tac1 -POA Tacr1 circuit directly links to centers for sensory input, motor output, and motivated behaviors. Activation of dopaminergic AVPV TH neurons shortens the refractory period, but the resulting mating displays are not time-locked to optogenetic stimulation epochs and emerge after a variable delay following such activation. By contrast, activation of POA Tacr1 neurons leads to immediate resumption of complete mating bouts that can progress to ejaculation. Together, these findings suggest that POA Tacr1 neurons, which do not express dopaminergic receptors, are functionally downstream of AVPV TH neurons.
  • BNSTpr Tac1 -POA Tacr1 pathway was placed in relation to the centers that govern the key characteristics of male sexual behavior: pheromonal control, motivated behavior, and motoric elements of mating.
  • BNSTpr Tac1 or POA Tacr1 neurons received innervation from main or accessory olfactory bulbs (MOB and AOB, respectively), which are the sole recipients of pheromonal input from sensory neurons in the nose.
  • MOB and AOB main or accessory olfactory bulbs
  • TTX tetradotoxin
  • BNSTpr Tac1 neurons exhibit extreme sexual dimorphism in that they only regulate sexual behavior in males. Although BNSTpr Tac1 neurons also regulate male territorial aggression, their projection to POA Tacr1 neurons is necessary and sufficient for mating but not aggression. POA Tacr1 neurons in turn are necessary and sufficient for male sexual behavior but not aggression. Activation of POA Tacr1 neurons actuates mating drive because it enables sexually satiated males to mate again, and mice will perform arbitrary behaviors to stimulate their POA Tacr1 neurons, thereby indicating that it is a rewarding activity. Together, the neural circuit we have discovered implements these seemingly disparate, but essential, features that define male sexual behavior.
  • the “social behavior network” comprises six fully connected centers, the bed nucleus of the stria terminalis principalis (BNSTpr), medial amygdala (MeA), preoptic hypothalamus (POA), anterior hypothalamus, ventromedial hypothalamus ventrolateralis (VMHvI), and the PAG and adjacent tegmentum.
  • Pheromonal input from the VNO sends second order afferents to BNSTpr Tac1 , but not POA Tacr1 , neurons and enables distinguishing between males and females in naive males.
  • disrupting pheromone signal transduction in the VNO disables BNSTpr neurons from distinguishing between the sexes; together with our current neuroanatomical findings, this suggests that the input from the AOB may play a critical role in endowing BNSTpr Tac1 neurons with the ability to distinguish between the sexes.
  • Our neural circuit epistasis studies also establish an upstream-downstream functional connectivity between BNSTpr Tac1 and POA Tacr1 neurons. This directional transfer of information is in contrast to the functional equivalence of connections between BNSTpr and POA in the “social behavior network” model of mating behavior.
  • POA Tacr1 neurons regulate male mating but not aggression and therefore bestows this pathway with behavioral specificity. Recent studies have identified additional centers that also regulate male mating and aggression and could considered to be components of an extended “social behavior network”. Nevertheless, information flow even within this otherwise fully connected “social behavior network” cannot compensate for removal of POA Tacr1 cells from the network.
  • An attractive feature of a fully connected network is that it is robust to failure or drop-out of an individual node. This would also seem to be evolutionarily advantageous as it would render male reproductive behaviors impervious to functionally disabling mutations that are restricted to any single node. However, we find that loss-of-function of POA Tacr1 neurons eliminates male sexual behavior. We propose that reliance on individual populations such as POA Tacr1 neurons may serve a gatekeeping function to ensure reproductive fitness.
  • circuit model for mating provides a different architecture for behavioral output than the one proposed for another innate behavior, parenting 73 , in which distinct behavioral elements, including internal states, are broadcast uniquely to different brain regions to enable displays of different aspects of parental behavior. Together, this suggests that different innate social behaviors arise from distinct underlying circuit architectures, presumably reflecting unique evolutionary histories or needs.
  • POA Tacr1 neurons are also not essential for social approach, other general social interactions such as grooming, or male aggression. The activity of these cells is also not essential for reward associated with non-social behaviors. Together, our findings demonstrate that POA Tacr1 neurons are purposed for male sexual behavior and its associated reward.
  • mice Animals. Adult mice 10-24 weeks of age were used for all studies. All mice were bred in our colony (Ta Cre , Tacr1 Cre , Tac1 null, Esr1 F!p0 ' - 35 - 3a - a7 or purchased from Jax (C57BL/6J, used as WT resident males and stimulus females in mating assays) and Charles River (BALB/c, used as WT intruder males). Mice were housed under a reverse 12:12 hour light:dark cycle (lights off at 1 pm) with controlled air, temperature, and humidity, and food and water were provided ad libitum unless otherwise mentioned.
  • mice were group housed by sex after weaning at 3 weeks of age and were therefore sexually naive prior to initiation of behavioral testing. All animal studies were done in compliance with Institutional Animal Care and Use Committee guidelines and protocols approved by Stanford University’s Administrative Panel on Laboratory Animal Care and Administrative Panel of Biosafety.
  • AAV-hSyn-DIO-GCaMP6s (serotype 1 ), AAV-EF1 a-DIO-PPO:Venus (serotype 9), AAV-EF1 a-DIO-hChR2(H134R)-EYFP (serotype 1 ), AAV-hSyn-hChR2(H134R)- EYFP (serotype 1 ), AAV-CAG-DIO-EGFP (serotype 1 ), and AAV-CAG-DIO-tdTomato (serotype 1 ) were purchased from Addgene.
  • AAV-hSyn-DIO- mGFP-2A-Synaptophysin:mRuby (serotype DJ), and AAV-DIO-TVA:mCherry-2A-oG (serotype 8.2) were custom packaged by Virovek (Hayward, GA).
  • EnvA G-deleted Rabies- EGFP was purchased from Salk Institute.
  • Cholera Toxin Subunit B (CTB), Alexa Fluor 555 and 647 conjugates were purchased from ThermoFisher Scientific. All virus titers were > 10 12 genomic copies/mL.
  • Viruses were delivered into brains of male mice at 10-16 weeks of age, using a Kopf stereotaxic alignment system (model 1900), as described previously 18 . Viruses were injected into empirically determined coordinates for the BNSTpr and POA (BNSTpr: ⁇ 0.85 mm mediolateral (ML), -0.20 mm anteroposterior (AP), and -4.30 mm dorsoventral (DV) relative to bregma; POA: ⁇ 0.60 mm ML, +0.05 mm AP, and -5.20 mm DV).
  • BNSTpr ⁇ 0.85 mm mediolateral (ML), -0.20 mm anteroposterior (AP), and -4.30 mm dorsoventral (DV) relative to bregma
  • POA ⁇ 0.60 mm ML, +0.05 mm AP, and -5.20 mm DV.
  • Viruses were infused at a rate of 100 nl/min using a syringe pump (Harvard Apparatus), and the needle was left for an additional 5 min and withdrawn at 1 min/mm.
  • mice were implanted with an optic fiber (0.5 NA, 400 pm diameter and 0.39 NA, 200 pm diameter, respectively; RWD Life Sciences) placed 0.5 mm above the viral injection site of either the BNSTpr or POA depending upon the functional manipulation.
  • optic fibers were placed at empirically determined coordinates for the VTA and PAG (VTA: ⁇ 0.50 mm ML, -2.92 mm AP, and -4.50 mm DV; PAG: ⁇ 0.50 mm ML, -4.60 mm AP, and -2.50 mm DV).
  • mice were implanted with dual cannulas (RWD Life Sciences) placed above the POA ( ⁇ 0.60 mm ML, +0.05 mm AP, and -4.70 mm DV).
  • a 0.6 mm x 7.3 mm (diameter x length) GRIN lens (Inscopix) was implanted 3 weeks after viral injection.
  • the GRIN lens was connected to a miniscope imaging system (nVista, Inscopix), lowered at 0.5 mm/min while monitoring fluorescence, and placed between 150 and 250 pm dorsal to the coordinates used for viral delivery.
  • the GRIN lens was capped with a small piece of parafilm and silicon adhesive (Kwik-Sil, WPI) prior to closing the skin incision.
  • the silicon cover was removed 14 days after GRIN lens implantation and a baseplate (Inscopix) was installed above the GRIN lens.
  • the baseplate was connected to the miniscope, lowered until clear cellular morphology was detected across the imaging plane, anchored to the skull with adhesive, and covered with a baseplate cover (Inscopix).
  • Cannulas, GRIN lens, and baseplates were secured to the skull using adhesive dental cement (C&B Metabond, Parkell). Following surgery, mice were allowed to recover individually over a heat pad and then returned to their home cages. [00258] Histology.
  • mice were anesthetized with 2.5% avertin and perfused with HBS followed by 4% paraformaldehyde (PFA). Brains were dissected, postfixed in 4% PFA overnight, sectioned at 65pm thickness with a vibratome (Leica VT1000S), and immunolabeled and counter-stained with DAPI (0.2 pg/mL).
  • ONO solution was prepared as previously described. In brief, ONO (Enzo) was dissolved in sterile saline at 5 mg/mL, aliquots were frozen, and each aliquot was freshly diluted with sterile saline prior to intra-peritoneal (i.p.) administration. The final dose of ONO for chemogenetic studies was 1 mg/kg.
  • L-703,606 was chosen as the Tacrl antagonist for use in our studies as i.p. injection of 10 mg/kg of L-703,606 into male mice has previously been shown to disrupt recognition of female urine. Therefore, we used the same 10 mg/kg dose for our Tacrl antagonist studies with i.p. injections. As in published studies, L-703,606 was dissolved in 45% 2-hydroxypropyl-
  • 60HDA was used to ablate TH + neurons in the AVPV/PVpo.
  • the buffer solution was made, and 6OHDA was dissolved to 10 mg/ml in ascorbic acid saline (0.9 % NaCI and 0.1 % Ascorbic acid) and filter-sterilized.
  • Bilateral stereotaxic injection of 1 ul of 6OHDA solution was delivered to empirically determined coordinates for the AVPV/PVpo (+/- 0.30 mm ML, +0.25 mm AP, and -5.50 mm DV).
  • TTX tetrodotoxin
  • Fiber photometry Fiber photometry was conducted as described previously. Briefly, the implanted optic fiber cannula on the mouse was connected via a patch cable (RWD Life Science) to a previously described custom-built fiber photometry setup (Bayless et al., 2019).
  • Fluorescence emitted by GCaMP6s during the behavioral assay was then passed through a fiber collimator, a GFP emission filter (Thorlabs, MF525-39), and a dichroic mirror, and focused by a plano-convex lens (Thorlabs, LA1255-A) onto a femtowatt photoreceiver (Newport, 2151 ).
  • the signal from the photoreceiver was relayed to a lock-in amplifier (Stanford Research System, SR810), which also received a phase lock-in signal from the optic chopper.
  • the output signal from the amplifier was recorded on a computer via a data acquisition device (LabJack, U6-Pro) at a 250 Hz sampling rate.
  • mice Prior to any behavioral testing, mice were habituated to the weight and feel of the optic fiber cable. The cable was attached to the optic fiber implants on the mice, and mice were allowed to move freely in their home cages during 3 separate 15 min habituation sessions. Behavioral video files and fluorescence data were time-locked via a light flash present in both datasets that was initiated by a pulse generator (Doric OTPG-4). The raw fluorescence data was normalized to the median fluorescence of the 5 min baseline period before the entrance of any animal or object into the cage.
  • Doric OTPG-4 pulse generator
  • time zero was set to the start of a behavior or event of interest, and the average fluorescence during the time window 10 s prior to a behavioral event was used as the normalization factor to calculate change in fluorescence from baseline (AF/F).
  • AF/F the 95 percent peak fluorescence
  • Miniscope calcium imaging We used a miniaturized fluorescence microscopy setup (nVista, Inscopix) to perform miniscope calcium imaging. During imaging sessions, the baseplate cover was removed and a miniscope was mounted and secured with a side screw. To synchronize fluorescence signals with annotated behaviors, we aligned both the imaging data and behavior videos to the event of a LED flash captured by the camera and recorded in the imaging data by a LED-triggered TTL signal generated by a data acquisition device. We used identical LED power, lens focus, digital gain, exposure time, and recording frame rate for all sessions for the same animal.
  • Imaging data were loaded on Inscopix data processing software (IDPS, Inscopix) and the size of the image was cropped to the area of the GRIN lens.
  • the cropped data was processed to rectify defective pixels, spatially down-sampled by a factor of two to reduce data size, filtered with a spatial bandpass to remove low and high spatial frequency content, and corrected for motion so that each pixel corresponded to the same location across all frames.
  • Imaging data were then converted into AF/F values.
  • To identify the spatial locations of neurons ⁇ spatial masks of identified regions of interest (ROIs) ⁇ and its associated fluorescence signal from the processed imaging data, a constrained nonnegative matrix factorization- extended (CNMF-E) algorithm was applied in IDPS.
  • ROIs identified regions of interest
  • CNMF-E constrained nonnegative matrix factorization- extended
  • Identified ROIs were further screened based on all pixels being singly connected, morphology, location in imaging field, size, dynamics of associated raw calcium signal, and signaknoise of calcium signal.
  • Calcium signals associated with each identified ROI were synchronized with annotated behaviors and z-scored based on the mean and standard deviation of the entire imaging session.
  • the mean z-score between -10 s and 0 s to the onset of the behavioral event was compared with that between 0 s and 10 s following the onset of the behavioral event.
  • a ROI (neuron) was considered significantly activated if the mean z-score following the behavioral event (0-10s) was >2 o above the mean z-score prior to the behavioral event (-10- 0s).
  • Optogenetic manipulations were conducted as described previously. Briefly, the implanted optic fiber cannula on the mouse was connected via a patch cable (RWD Life Science) to a diode laser (Opto Engine). All optogenetic stimuli were produced by a pulse generator (Doric OTPG-4) that triggered a blue light (473 nm) laser for ChR2 and PPO studies and a yellow light (593.5 nm) laser for eNpHR3.0 studies. Laser illumination commenced as soon as an intruder was placed into the resident’s cage. Descriptions of the laser parameters are detailed below for each experiment. As with fiber photometry, prior to any behavioral testing, mice were habituated to the weight and feel of the optic fiber cable.
  • mice were given 3 separate 15 min habituation sessions. Mice were tested on each behavioral assay once each with laser illumination and no laser illumination, with the order of “Laser on” and “Laser off” assays counterbalanced across mice. [00269] Chemogenetic inhibition. Chemogenetic inhibition studies were performed as described previously. Briefly, experimental mice were i.p. injected with CNO at 1 mg/kg or sterile saline 30 min prior to behavioral assays. Mice were tested on each behavioral assay once each with CNO and saline, with the order of CNO and saline administration counterbalanced across animals.
  • Tacrl antagonist manipulations Fifteen minutes prior to behavioral testing, the bilateral cannula (RWD Life Science) implanted above the POA of experimental males was connected to two Hamilton syringes via polyethylene tubing, loaded with 1 pL of L-703,606 (500 pmol) or control vehicle per side and infused slowly over a period of 2 min using a syringe pump (Harvard Apparatus). Needles were left connected for an additional 2 min and then withdrawn. Prior to any behavioral testing, mice were habituated to the handling and the weight and feel of being connected to the Hamilton syringes via polyethylene tubing. Mice were given 3 separate “mock infusion” habituation sessions on separate days.
  • Electrophysiology Slice preparation. Brain slices (300 pm) were obtained using standard techniques. Briefly, animals were anesthetized with isoflurane and decapitated. The brain was exposed and chilled with ice-cold artificial CSF (ACSF) containing 125 mM NaCI, 2.5 mM KCI, 2 mM CaCI 2 , 1.25 mM NaH 2 PO4, 1 mM MgCI 2 , 25 mM NaHCOs, and 15 mM D- glucose. ACSF was saturated with 95% O 2 and 5% CO 2 . Osmolarity was adjusted to 300-305 mOsm.
  • ACSF ice-cold artificial CSF
  • Coronal brain slices containing POA were prepared with a vibrating microtome (Leica VT1200 S, Germany) and left to recover in ACSF for 30 min at 34°C and then at room temperature for an additional 30 min. Slices were then moved to a submerged recording chamber perfused with ACSF at a rate of 2-3 ml/min at 30-31 °C, and brain slices were recorded within 5 hours after recovery.
  • a vibrating microtome Leica VT1200 S, Germany
  • Perforated patch was performed with a borosilicate glass microelectrode (3-3.5 MQ), front-filled with 1 pl K + - based internal solution (135 mM KMeSOs, 8.1 mM KCI, 10 mM HEPES, 8 mM Na 2 - Phosphocreatine, 0.3 mM GTP-Na, 4 mM ATP-Mg, 0.1 mM CaCI 2 , 1 mM EGTA; pH 7.2-7.3; osmolarity 285-290 mOsm) and back-filled with 10 pl Gramicidin A-containing internal solution.
  • 1 pl K + -based internal solution (135 mM KMeSOs, 8.1 mM KCI, 10 mM HEPES, 8 mM Na 2 - Phosphocreatine, 0.3 mM GTP-Na, 4 mM ATP-Mg, 0.1 mM CaCI 2 , 1 mM EGTA; pH 7.2-
  • Gramicidin A-containing internal solution was made fresh before use: Gramicidin A (Sigma) was dissolved in dimethyl sulfoxide (DMSO, Sigma) to 20 mg/mL and then diluted in the K + -based internal solution yielding a final concentration of 200 pg/mL. The solution was thoroughly mixed by vortexing and then sonicated for 5 min and filtered with a centrifuge tube filter (0.22 gm, Spin-X, Costar). After the microelectrode formed a giga seal with the cell membrane, access resistance was continuously monitored during perforation by applying a - 5 mV pulse from a holding potential of -70 mV, under the voltage-clamp mode.
  • DMSO dimethyl sulfoxide
  • GABAA were blocked by the bath application of 100 pM picrotoxin throughout the recording, and presynaptic excitatory inputs, recorded as EPSPs, were evoked by focal extracellular stimulation at 0.05Hz with a small theta glass electrode positioned 50-100 pm from the recorded cell body.
  • Stimulation intensity (0.2 ms, 5-30 pA) was adjusted to evoke stable EPSPs with an amplitude of around 2-5 mV.
  • Basal EPSPs were recorded for at least 5 min, before LTP was induced by Substance P perfusion or BNSTpr terminal stimulation.
  • Substance P (1 pM) was applied in bath perfusion for 5 min to induce LTP, and EPSPs were continuously recorded for another 25 min to monitor the change of amplitudes.
  • BNSTpr terminal stimulation we injected AAV-ChR2:EYFP to the BNSTpr as well as AAV-DIO- tdTomato to the POA of Tacr1 Cre males, and slices were prepared 8-10 weeks after viral delivery.
  • BNSTpr terminals were stimulated with blue light train stimulation (450 nm, OptoEngine, 0.5 ms pulse, 5 Hz, 90 s), and EPSPs were recorded afterwards for 30 min.
  • L-703,606 (10 pM) was added in perfusion throughout the recordings.
  • Behavioral testing All behavioral testing was initiated > 1 hr after onset of the dark cycle and recorded using camcorders (Sony) under infrared illumination as described previously (Bayless et al., 2019). Videos were played at 30 frames per second and manually annotated using custom software described previously. This permitted analysis of multiple parameters (including number, duration, latency, probability, and inter-event interval) of different behavioral routines. In particular, anogenital investigation (sniff), mounting, repeated pelvic thrust (intromission), and ejaculation were scored for sexual behaviors. Aggression was scored as occurring when physical attacks (episodes of biting, wrestling, tumbling, chasing) were observed.
  • BNSTpr Tac1 neurons To examine the response of individual BNSTpr Tac1 neurons to pheromones (Fig. 19F- N, Fig. 20N-O), sexually naive males with GCaMP6s expressed in and GRIN lenses above BNSTpr Tac1 neurons were single housed for > 7 days prior to miniscope imaging studies. Males were exposed for 3 min each in their home cage to a 1 ”x1 ” cotton swab wetted with 80 .1 of undiluted urine from WT group housed primed C57BL/6J females, WT group housed C57BL/6J males, or saline.
  • the swabs were presented during the same imaging session with the order of the presentation of the swabs counterbalanced across mice and a 7 min interval between swab presentations. Urine was collected 3-6 hours prior to use and kept on ice until pipetted onto the swab.
  • laser illumination (473 nm, 5 Hz, 5 ms pulse width, 8 mW from the fiber tip) was provided for the first 90s of the assay, and then illumination was turned off for the rest of the assay.
  • laser illumination (473 nm, 5 Hz, 5 ms pulse width, 8 mW from the fiber tip) was provided in cycles of 30 s laser on and 30 s laser off throughout the entire assay.
  • the refractory period mating assay consisted of a 30 min assay with a WT female intruder.
  • Laser illumination (473 nm, 40 Hz, 10 ms pulse width, 5 mW from the fiber tip) was provided in cycles of 30 s laser on and 30 s laser off throughout the entire refractory period mating assay.
  • Laser illumination and no laser illumination assays were counterbalanced and performed at least one week apart.
  • the female that received the initial ejaculation was replaced by a new WT female. In these cases, the male interacted with a new female during both the laser illumination and no laser illumination assays.
  • Each experimental mouse underwent three 10 minute trials: “Laser off” in which there was no laser illumination, “Laser on, female side” in which laser illumination (473 nm, 40 Hz, 10 ms pulse width, 5 mW from the fiber tip) was provided only when the experimental mouse was located in the half of the behavior arena containing the stimulus female, and “Laser on, empty side” in which laser illumination (same as above) was provided only when the experimental mouse was located in the half of the behavior arena with the empty container.
  • the location of the experimental mouse was tracked using custom code (MATLAB). The 3 trials were conducted consecutively, with the “Laser off” trial always occurring first and the “Laser on, female side” and “Laser on, empty side” being counterbalanced across mice.
  • Female preference index was calculated as (investigate female container - investigate empty container) I (investigate female container + investigate empty container). Sniffing or placement of forepaws onto the female or empty container was manually classified as investigation behavior, and duration of investigation behavior towards the containers was quantified to construct the female preference index.
  • mice were acclimated to the arena 1 day before the first trial and gained experience drinking from a spout that dispensed 30% sucrose water. In a 10 min trial, the amount of times the mouse drank the liquid and crossed the midpoint of the arena was quantified.
  • “Laser on” trials the mouse received constant 5 mW 593.5 nm laser illumination, and during “Laser off” trials, no laser illumination was provided.
  • the trials were conducted in a block of two consecutive trials of “Laser on” and “Laser off” for each concentration of sucrose (0%, 15%, and 30%), and each block was performed twice, with one block performing the “Laser on” trial first and the other block performing the “Laser off” trial first.
  • MAST a flexible statistical framework for assessing transcriptional changes and characterizing heterogeneity in single-cell RNA sequencing data. Genome Biology 16, 278.
  • CD24 is a genetic modifier for risk and progression of multiple sclerosis. Proc Natl Acad Sci U S A 100, 15041-15046.

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Abstract

L'invention concerne une structure cellulaire fonctionnelle pour l'expression et le comportement du gène dépendant du cycle œstral et sexuel, dans laquelle des neurones sensibles aux hormones sexuelles (SH) sont présentés pour réguler des comportements typiquement sexuels. À l'aide d'approches de séquençage orthogonales, des événements d'expression différentielle entre sexes ou états œstraux sont classés dans quatre populations sensibles aux SH. Ces gènes exprimés de manière différentielle (DEG) sont distribués à travers des types de cellules définis par transcription (tCT). Deux tCT, BNSTTac1/Esr1 et VMHvlCckar/Esr1, sont essentiels pour la reconnaissance sexuelle chez les sujets mâles et l'accouplement chez les sujets femelles, respectivement. Les projections du tCT VMHvlCckar/Esr1 sont distinctes de celles d'autres tCT dans cette population sensible aux SH. Il est en outre montré que des neurones POA exprimant le récepteur de substance P Tacr1 (POATacr1) sont responsables de l'initiation de l'accouplement chez les sujets mâles. Une sous-population spécifique de neurones chez les sujets femelles, AVPVKISS1, sont nécessaires et suffisants pour le comportement sexuel féminin, et reçoivent des entrées monosynaptiques de neurones Cckar.
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CN113785052A (zh) * 2019-02-19 2021-12-10 维也纳兽医大学 新重组二胺氧化酶及其治疗以过量组胺为特征的疾病的用途
CN113785052B (zh) * 2019-02-19 2024-06-28 维也纳兽医大学 新重组二胺氧化酶及其治疗以过量组胺为特征的疾病的用途

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