Social behaviour shapes hypothalamic neural ensemble representations of conspecific sex

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1 Letter doi:.38/nture23885 Soil ehviour shpes hypothlmi neurl ensemle representtions of onspeifi sex Ryn Remedios *, Ann Kennedy *, Moriel Zelikowsky, Benjmin F. Grewe 2,3, Mrk J. Shnitzer 2,3,4 & Dvid J. Anderson,4 All nimls possess repertoire of innte (or instintive,2 ) ehviours, whih n e performed without trining. Whether suh ehviours re medited y ntomilly distint nd/or genetilly speified neurl pthwys remins unknown 3 5. Here we report tht neurl representtions within the mouse hypothlmus, tht underlie innte soil ehviours, re shped y soil experiene. Oestrogen reeptor -expressing (Esr + ) neurons in the ventrolterl sudivision of the ventromedil hypothlmus (VMHvl) ontrol mting nd fighting in rodents 6 8. We used miroendosopy 9 to imge Esr + neuronl tivity in the VMHvl of mle mie engged in these soil ehviours. In sexully nd soilly experiened dult mles, divergent nd hrteristi neurl ensemles represented mle versus femle onspeifis. However, in inexperiened dult mles, mle nd femle intruders tivted overlpping neuronl popultions. Sex-speifi neuronl ensemles grdully seprted s the mie quired soil nd sexul experiene. In mie permitted to investigte ut not to mount or ttk onspeifis, ensemle divergene did not our. However, 3 minutes of sexul experiene with femle ws suffiient to promote the seprtion of mle nd femle ensemles nd to indue n ttk response 24 h lter. These oservtions unover n unexpeted soil experiene-dependent omponent to the formtion of hypothlmi neurl ssemlies ontrolling innte soil ehviours. More generlly, they revel plstiity nd dynmi oding in n evolutionrily nient deep suortil struture tht is trditionlly viewed s hrd-wired system. We performed miroendosopi lium imging 9, (Insopix, In.; Fig. ) of VMHvl Esr + neurons expressing GCMP6s (Fig., ; see Methods) during resident intruder ssys 2 tht were onduted in the home ge of the resident. On given dy, the implnted resident mouse engged in five trils with (different) femle intruders, shuffled with five trils with (different) mle intruders (Fig. d). We oserved no impirment in soil ehviour in nimls hituted to the miniture mirosope (Fig. e, f). Behviourl dt from synhronized videos were mnully sored t 3 Hz. We reorded etween 75 nd 3 Esr + ells per dy per mouse (n = 7; pproximtely 3,4 ells were imged; see Methods). In soilly nd sexully experiened residents, VMHvl Esr + neurl tivity ws low on seline trils ut inresed y onspeifis of either sex (Fig. e g nd Extended Dt Fig., ). Cellulr response kinetis following introdution of n intruder vried ontinuously, from trnsient to persistent (Extended Dt Fig. d f). The numer of ells tivted (>2σ ove seline; Extended Dt Fig. ) y onspeifis (pproximtely 2 3%) ws signifintly higher thn y toy mouse (less thn 5%; Extended Dt Fig. ). Repeted trils with different intruders of the sme sex tivted similr neurl ensemles (Fig. g), refleted y high Person s orreltion oeffiient (PCC) etween ensemle responses to the sme sex (Fig. h). The popultion tivity vetor during soil enounter, in the prinipl omponent spe (Fig. k nd Extended Dt Fig. 2, ), or in the prtil lest squres regression 3 spe (Extended Dt Fig. 2), ws remrkly similr ross trils with different intruders of the sme sex. By ontrst, mles versus femles tivted different ensemles (Fig. g) nd the PCC etween sexes ws lose to zero (Fig. h), independent of inter-niml distne (Extended Dt Fig. 3). Mleversus femle-preferring ells (defined s tivted >2σ y one ut not the other sex during soil intertions) were sptilly intermingled (Fig. i nd Extended Dt Fig. g), onsistent with prior Fos-tFISH (ellulr omprtment nlysis of temporl tivity y fluoresent in situ hyridiztion of Fos) studies 4. The numer of mle-preferring ells ws round 5% higher thn the numer of femle-preferring ells 7,4 (Fig. j nd Extended Dt Fig. ). Approximtely 2% of ells tivted y one sex were preferentilly inhiited y the opposite sex (Fig. j nd Extended Dt Fig., g, h). Intruder sex ounted for 56 ± 5.2% of the oserved vrine (Extended Dt Fig. 2), inditing tht popultion tivity is dominted y the representtion of onspeifi sex. Refleting this, liner support vetor mhine (SVM) deoder ould orretly lssify n intruder s sex identity with lmost % ury, within 3 s fter its introdution (Fig. l). SVM deoders trined on dt from residents seprted from intruders y mesh rrier lso performed urtely, suggesting tht sex-speifi ensemles represent sensory ues, not sex-speifi ehviours (see elow). Optogeneti mnipultion of Esr + neurons in the VMHvl evoked speifi soil ehviours, suh s mounting or ttk 7, ut ehvi ourtriggered verge nlysis of lium trnsients reveled few timeloked responses during suh tions 4 6. We therefore investigted whether VMHvl ensemle responses ould t lest predit epohs when these ehviours ourred (Fig. 2). Liner SVM deoders showed signifintly higher thn hne performne in prediting episodes of ttk, sniffing or mounting (Fig. 2, ), with errors typilly ffeting their preise timing. Suh errors my reflet inonsistenies in the mnul soring of ehviour onset/offset or iologil ftors. Additionlly, we exmined whether VMHvl neurons might e proilistilly tuned to prtiulr ehviours, y lulting the hoie proility 7 for eh ell, defined s the hnes of orretly prediting whih of two lterntive ehviours is ourring sed on tht neuron s tivity (Extended Dt Fig. 4). Approximtely hlf of the ells (52 53%) were tuned for periods when soil ehviour ourred (Fig. 2h, drk grey). Within tht tegory, there were ells tuned to sniff versus ttk for mles or to sniff versus mount for femles (Fig. 2d h nd Extended Dt Fig. 4). Surprisingly, the ehviourl tuning of mny ells ws independent of their sex-preferene (Fig. g): for exmple, sniff mle -tuned units were eqully represented mong mle- versus femle-preferring ells (Fig. 2i). Therefore, individul Esr + neurons n prtiipte in ensemles Division of Biology nd Biologil Engineering 56-29, Tinqio nd Chrissy Chen Institute for Neurosiene, Cliforni Institute of Tehnology, Psden, Cliforni 925, USA. 2 Jmes H. Clrk Center for Biomedil Engineering & Sienes, Stnford University, Stnford, Cliforni 9435, USA. 3 CNC Progrm, Stnford University, Stnford, Cliforni 9435, USA. 4 Howrd Hughes Medil Institute, Cliforni Institute of Tehnology, 2 Est Cliforni Boulevrd, Psden, Cliforni 925, USA. Present ddress: Institute of Neuroinformtis, ETH Zurih, CH-857 Zurih, Switzerlnd. *These uthors ontriuted eqully to this work. 388 NATURE VOL 55 9 otoer Mmilln Pulishers Limited, prt of Springer Nture. All rights reserved.

2 Letter RESEARCH Esr > GCMP6s ΔF/F d Ƃ ƃ T Ƃ Mirosope Intruder in ƃ Ƃ Time Dt olleted Intruder out Soil intertions 2 3 min GRIN lens 3 s DAQ μm Ƃ In f Ƃ Out Soil intertions ƃ In i intruder 35 intruder ƃ vs ƃ Ƃ vs Ƃ 27% z-sored ΔF/F (V) 5 μm 25 Ƃ intruder (V) oth.25 k Ƃ 4% 35 2V Exited (>2V) Inhiited ( 2V) Other Ƃ intruder Both 8% 2V ƃ ƃ intruder (V) 35 9% Exited (>2V) Inhiited ( 2V) Other ƃ T 6 Ƃ ƃ l Bseline (n = 2) Toy (n = 2) Ƃ Trils (n = 5) ƃ Trils (n = 5) 6 ƃ intruder ƃ vs Ƃ j Ƃ min min z-sored ΔF/F (V) 6 2 First prinipl omponent (AU) Intruder sex deoder ury (%) All trils on dy 3 (n = 5 ƃ, 5 Ƃ) 7 Seond prinipl omponent (AU) Correltion oeffiient 5 min g Intruder in -preferring ΔF/F V ΔF/F V h Soil ƃ Out intertions Mle intruder -preferring Femle intruder Behviourl video 5 μm VMHvl e 3 s Home ge C2+ imging dt VMHvl Bseline 2 Toy mouse (T) Pseudo5 Femle mouse rndomized 5 Mle mouse Bseline Ƃ 2 ƃ Intruder in 3s 5 Chne 5 s Time reltive to introdution Figure VMHvl Esr+ neurons represent intruder sex., Shemti of preprtion. Redrwn from Allen Mouse Brin Atls, version (28)., Approximte imging plne (dshed line)., Smple frme showing tive neurons. d, Experimentl design. DAQ, dt quisition ox. e, f, Smple video frmes nd F/F tres. g, Rster of responses of mlepreferring vs. femle-preferring ells (n = 35) rnked y response strength. h, Averge PCC etween ell responses to mles nd femles, eh dtpoint is single mouse. i, Sptil mps of verged neuronl responses (top); intruder sex-preferring ells (>2σ ove seline; ottom left); stterplot of response intensities (ottom right). j, Frtion of ells tivted or inhiited (±2σ from seline) y onspeifis for trils (n = 5 mie; 2,379 ells imged). k, Popultion tivity vetors in prinipl omponent spe. AU, ritrry units. l, Deoder performne during the introdution of n intruder (n = 5 mie tht mounted nd fought, trils eh). e g, i, k, Pnels re from one exmple mouse. Dt re men ± s.e.m in ll figures nd extended dt figures. representing prtiulr sex, s well s in ensemles for speifi ppetitive or onsummtory ehviours8 (Fig. 2k (right) nd Extended Dt Fig. 4). An exeption ws tht ttk mle - nd sniff femle -tuned ells were enrihed for mle- versus femle-preferring neurons, respetively (Fig. 2i). Attk-tuned ells were more strongly tivted thn other ehviour-tuned neurons (Fig. 2j), suggesting higher threshold for this tion nd onsistent with optogeneti results7,5. The ove-mentioned dt were otined from the 3rd onseutive dy of soil enounter testing. Before dy, mie were nive to nonlittermte onspeifis, sexully inexperiened nd were individully housed following surgery for t lest four weeks (see Methods). During initil trils on dy, we oserved little mounting or ttk (Fig. 3j (dy ) nd Extended Dt Fig. 5). Notly, imging reveled mny ells tht responded to oth mles nd femles (Fig. 3 (dy )). Over the next two dys, more mle- or femle-preferring ells were oserved, while the overll perentge of tive ells remined firly onstnt (Fig. 3 (dy 2 nd 3)). Aordingly, the PCC etween the mle- nd femle-response popultion vetors deresed to nerly zero y dy 3 (Fig. 3e (gold urve), f). Similr hnges were oserved in deoder performne or the Mhlnois distne rtio9 etween sme- versus opposite-sex trils (Fig. 3g i). Sex-speifi ensemles were stle for months in nimls isolted following these trils. Interestingly, two out of seven nimls imged filed to exhiit ny seprtion of representtions, nd showed neither mounting nor ttk (for exmple, Extended Dt Fig. 6, d). These nomlous mie were oserved t similr frequenies (round 5 2%) mong unoperted ontrol nimls (dt not shown). The origin of these individul differenes mong inred mie is unler. To trk the origins of the sex-speifi ells oserved on dy 3, we registered 455 ells ross three dys of imging (Extended Dt Fig. 7; round 4% of ll ells ould e registered). Twenty-five per ent of dy 3 sex-preferring ells were i-responsive on dy, while lmost hlf derived from ells tht were initilly unresponsive (<2σ ove seline) to either sex (Extended Dt Fig. 8). A smll numer (%) rose from swithing sex-speifiity. Conversely, 42% of ells tht were tive on dy were no longer tive on dy 3. These dt revel omplex nd dynmil hnges in the response properties of VMHvl Esr+ neurons during the quisition of soil experiene. The progressive seprtion of mle versus femle representtions ross the three dys of testing ws ompnied y n inrese in mounting nd ttk (Fig. 3j nd Extended Dt Fig. 5). To quntify this orreltion, we omputed tril-y-tril hnges in the mle versus femle PCC, nd plotted the PCC for eh mouse ginst the umultive minutes of different soil ehviours (Fig. 4 nd Extended Dt Fig. 9). The strongest orreltion ws with mounting plus nogenitl investigtion (Fig. 4) nd the wekest with sniffing (Fig. 4). The wek orreltion with sniffing suggested tht olftory ues lone might not suffie to promote the seprtion of mle versus femle representtions. To test this, we suspended intruders y the til (llowing sniffing, ut preventing mounting or ttk) throughout eh 2-min tril of stndrd three-dy shedule (Fig. 4d). Under these onditions, the mle versus femle PCC remined high t dy 3 in ll three mie (Fig. 4e (tel rs)). However, when two of these mie ( third eme ill nd ws euthnized) were llowed free intertions with femles nd mles over two dditionl dys, sex-speifi representtions eme well-seprted, nd mounting nd ttks were oserved (Fig. 4f, g). 9 o to er 2 7 V O L 5 5 N A T U R E Mmilln Pulishers Limited, prt of Springer Nture. All rights reserved.

3 RESEARCH Letter Time devoted to ehviour (%) ƃ-direted Oserved ehviour Attk Mount Sniff AG Sniff Other 9% 2% 3% ƃ Out ƃ In.8 Predited ttk NS F sore Ativity projeted onto SVM hyperplne (AU) 28% min % Ƃ-direted 4% Deoder performne ƃ-direted Oserved *** Chne * Attk Ƃ Out Ƃ In Sniff Sniff AG Ƃ-direted Oserved ehviour.8 Predited mounting F sore Ativity projeted onto SVM hyperplne (AU) *** * ** 36% e Sniff ƃ tuned..5 Choie proility i 32% Attk Sniff No pref 33% ƃ Ƃ Both Behviour outputs Dy 2 Averge response during intertion 4 D3 pref ƃ Ƃ Both Neither 5 4 ƃ Intruder (V) 4 5 ƃ Intruder (V) Mp of tive units d ƃ Trils Ƃ Trils Both PLS (AU) 4 Ƃ Intruder (V) 5 μm ƃ Intruder (V) Mouse Mouse 2 Mouse 3 Mouse 4 Mouse 5.4 Dy Dy 2 g j 2% <% Ƃ-direted 7% 7% 24% 28% 5 Dy 2 3% 4% Dy 3 Dy 2 2 Within-dy log(dopp / dsme) dsme ƃ Distriution Dy 2 Dy 8% 3% 9% 8% 9% 3% ƃ Test point Dy ƃ-direted Ƃ Distriution ΔF/F, unit 2 ** Time devoted to ehviour (%) 9% Dy i ΔF/F, unit NS.2 Dy 3 h dopp **** 4 Within-dy lssifier ury (%) Within-dy PCC, ƃ vs Ƃ. ƃ Trils vs dy 3 ƃ verge Ƃ Trils vs dy 3 Ƃ verge Avg(ƃ trils vs dy 3 Ƃ vg Ƃ trils vs dy 3 ƃ vg) 5 f e 4 Dy 3 Behviour outputs ƃ-preferring Ƃ-preferring Sniff ƃ tuned Sniff Ƃ tuned Attk ƃ tuned Mount Ƃ tuned Ƃ Intruder (V) Dy Ƃ Cues ƃ Cues Attk ƃ tuned Mount Ƃ tuned Sniff ƃ tuned Sniff Ƃ tuned 5% ƃ Cues *** 7 Ƃ Intruder (V) Ative units 2 min Ƃ Cues el l Mount Sniff No pref 56% V ƃ Ƃ Both NS Al j 2% 52%. ls 25% Behviourresponsive Cell hoie proilities Ƃ-direted ehviours k **** 75 Sniff Ƃ tuned 8 ells.5 Choie proility 2 min Mount tuned Correltion oeffiient 43% 85 ells V Cell hoie proilities ƃ-direted ehviours 53% Mount Sniff Ƃ No pref PLS 2 (AU) h 5 ells Sniff AG g Mount vs sniff Ƃ 4 Sniff M o e unt lls Sn iff e ƃ lls Sn i e ff Ƃ lls At t e k lls Attk tuned Ative ells (%) Cells (ount) 68 ells f Attk Sniff ƃ No pref Cells (ount) Attk vs sniff ƃ 4 Men ΔF/F (V) d Mount Figure 2 Popultion tivity predits soil ehviour., Behviourl responses to mles nd femles (n = 5 mie). AG, nogenitl., Exmple deoder ury., Deoder performne (n = 5 mie, trils eh; see Methods for signifine testing). d, e, Choie proilities of 29 neurons (d) nd tres from two exmple neurons (e) during three seprte mle intertions. f, g, Choie proilities for the sme 29 neurons during femle intertions (f) nd two exmple neurons (g). h, Proportion of ells exhiiting signifint (>2σ) hoie proilities (tht is, ehviourl tuning). i, Proportion of sex-preferring ells within eh tegory of ehviour-tuned ells (χ2 test, 3 degrees of freedom). j, Response strength of ehviourtuned popultions, during their preferred ehviour. (orreted for multiple omprisons). k, Two models of ensemle representtions of ehviour mong Esr+ VMHvl neurons. The dt rgue ginst the model on the left. *P <.5; **P <.; ***P <.; NS, not signifint y two-sided t-tests unless speified otherwise for ll figures nd extended dt figures. Speifi P vlues for Figs 2 4 re provided in Supplementry Tle. % Dy 3 Figure 3 Sex-speifi ensemles emerge with experiene. d, Ensemle representtions hnge over three dys of imging., Frtion of responsive ells ross pirs of onseutive trils (n = 455 ells from 5 mie)., Response strength ( F/F) for 35 ells identified ross three dys. Dy 3 preferene (D3 pref.) indites the response preferene of eh unit on dy 3 towrds mles (lue), femles (red) or oth (grey). Grey lines in Dy 2 nd Dy 3 plots indite shifts in preferene from Dy or Dy 2 position, respetively., Exmple sptil mps of intruder sex preferene. d, Popultion vetors in prtil lest squres (PLS) regression spe. e, PCC etween the tril-verged ensemle responses on dy 3 nd eh tril (points) for five mie. Signifint ensemle seprtion strts on dy (post ho Bonferroni test to orret for multiple omprisons). f, Averge PCC for eh mouse. g, SVM deoder ury ross dys. h, Shemti desription of Mhlnois distne (d) lultion. opp, etween opposite sexes; sme, within sme sex. i, Chnges in the log of the Mhlnois distne rtio. j, Perentge of time spent engging in eh soil ehviour (n = 5 mie). 4% 2% Dy 3 3% 28%.5 Dy Dy 2 Dy 3 36% Attk Mount Sniff AG Sniff Other 3 9 N A T U R E V O L o to er Mmilln Pulishers Limited, prt of Springer Nture. All rights reserved.

4 Letter RESEARCH... d h 3 Tril numer Cumultive ehviour (min)4 PCC, vs.5 Cumultive tril 25 time (min) l Held intruder Dys 3: per dy Mount Attk Soil isoltion post implnttion 4+ weeks n. Within-sex vs R 2 =.66 e i PCC, vs..5 PCC, vs..25 Before first mount Proe trils s eh **** Dy 3, free ess mie Dy 3, ess-restrited **** ** After first mount, efore first ttk NS After first ttk Single-sex free ess 3 min Proe trils s eh min 24 h min Dy PCC, vs ** * **** R 2 = Cumultive time spent mounting + nogentil sniffing (min) j f Soil isoltion week Free ess Dys 4 5: per dy o. Mouse R 2 =.529 Mouse 2 Mouse 3 Mouse 4 Mouse 5 Mouse 6 Eh point is one tril.5 Cumultive time spent 45 sniffing (min) PCC, vs Group 2: mting experiene 3 min Group : nonmted Dy 2 Other sex free ess min Within-sex vs 24 h NS g..5 Group 2: R-I test min Dy 3, restrited Dy 4, free ess Dy 5, free ess **** * **** * Mouse 9 Mouse 3 Time ttking (%) 3 % Group Group 2 % No With experiene experiene NS PCC PCC PCC, vs Group : R-I test 3 min m Time ttking (%) k.25 Before 24 h fter Before Mouse 2 Mouse 3 24 h fter Figure 4 Soil experiene promotes ensemle seprtion., PCC for the nth tril versus umultive soil experiene (), umultive mounting nd nogenitl sniffing () or umultive ody/fe-direted sniffing (). R 2 vlues re for fit urve y = ( x) + (lk). d, Illustrtion of the restrited-ess experiment. e, Ensemle seprtion for five mie with free ess (grey) nd three mie with restrited ess (tel) were signifintly different (one-wy ANOVA). f, g, PCC deresed fter two restrited-ess mie reeived free ess (dy 4 nd dy 5;.25 Before 24 h fter Mouse 4 Before 24 h fter Mouse 5 one-wy ANOVA); dy 3 rs (tel) re duplited from e to filitte omprison. h, Cumultive time spent engging in eh ehviour (n = 5). i, Ensemles seprte fter mounting ommenes. j, Priming experiment. k, Mles primed with femles (group 2, n = 4) ttked onspeifi mles; unprimed mles (group, n = 8) did not. l, Priming experiment dpted for imging. m, Mles primed with femles fought. n, o, Mie primed with femles (n = 2) showed ensemle seprtion 24 h lter (n); mie primed with mles (n = 2) did not (o). R-I; resident intruder ssy. Similr results were otined using wire mesh to lok soil intertions with intruders (Extended Dt Fig. ). These dt suggested tht exposure to onspeifi odours is insuffiient to promote sex-speifi ensemle seprtion, implying requirement for soil ehviours. To identify these ehviours, we next investigted the temporl reltionship etween mounting, ttk nd ensemle seprtion. Mounting ppered efore ttk, in oth operted (Fig. 4h) nd unoperted mie (Extended Dt Fig. 5), onsistent with previous reports 2. Ensemle seprtion inresed signifintly fter the first mount, ut efore the first ttk (Fig. 4i). These oservtions suggested tht soil experiene with femles might suffie to promote oth ttk 22 nd sex-speifi ensemle seprtion. To test the former possiility, soilly inexperiened, unoperted mles were primed in single 3-min unrestrited intertion with femles, nd then tested 24 h lter in single, -min resident intruder ssy with mle (Fig. 4j). Notly, this priming with femles (during whih mounting ws oserved) promoted ggressiveness in % of mles (Fig. 4k (group 2)). By ontrst, ontrol mles with no priming showed no ttk towrds n intruder mle, even fter 3 min (Fig. 4k (group )). To determine whether sexul experiene n lso promote the pperne of sex-speifi neurl ensemles, we repeted this priming experiment while imging Esr + neurons in nive mie (Fig. 4l). Proe trils with intruders tht were suspended y their til (three mles, three femles, s eh) were performed min efore, nd 24 h fter, the 3-min priming intertion with femles. Notly, the PCC etween mle versus femle representtions ws signifintly redued, in proe trils performed fter (ut not efore) femle priming, ompred to the 9 otoer 27 VOL 55 NATURE Mmilln Pulishers Limited, prt of Springer Nture. All rights reserved.

5 RESEARCH Letter sme-sex PCC (Fig. 4n). These primed mie lso proeeded to ttk mles during the resident intruder test on dy 2 (Fig. 4m). By ontrst, the mle versus femle PCC ws not redued fter 24 h in mie primed with mle on dy (Fig. 4o). Therefore, single 3-min priming experiene with femle suffied to promote oth sex-speifi ensemle seprtion nd ggression. Whether ensemle seprtion itself is required for ttk remins unler. Here we report the imging nlysis of neuronl tivity in deep hypothlmi struture during soil ehviour in freely moving nimls. Our oservtions in experiened mie reveled distint nd reproduile VMHvl Esr + supopultions tivted y mles versus femles, suggestive of lelled lines 4. However, in soilly isolted nd sexully inexperiened nimls these divergent representtions were not present, ut emerged with sexul experiene. How n these findings e reoniled with the innte nture of mting nd ggression,2? One possiility is tht iruits downstrem of the VMH re hrd-wired, ut tht sexul experiene is neessry to diret these ehviours towrds pproprite soil stimuli 23. We nnot exlude tht plstiity in the VMHvl is inherited from upstrem regions Nevertheless, our findings suggest tht some omputtionl fetures hrteristi of the hippompus nd ortex inluding dynmi oding, mixed seletivity nd experiene-dependent plstiity 28 3 lso operte in deep suortil iruits. More generlly, our findings rise the possiility tht the neurl representtions underlying other instintive ehviours my lso inorporte lerned fetures. Online Content Methods, long with ny dditionl Extended Dt disply items nd Soure Dt, re ville in the online version of the pper; referenes unique to these setions pper only in the online pper. reeived 3 Jnury; epted 3 July 27.. Tinergen, N. The Study of Instint (Clrendon Press, 95). 2. Lorenz, K. On Aggression (Hrourt, Bre & World, 966). 3. Root, C. M., Denny, C. A., Hen, R. & Axel, R. The prtiiption of ortil mygdl in innte, odour-driven ehviour. Nture 55, (24). 4. Newmn, S. W. The medil extended mygdl in mle reprodutive ehvior. A node in the mmmlin soil ehvior network. Ann. NY Ad. Si. 877, (999). 5. Veening, J. G. et l. Do similr neurl systems suserve ggressive nd sexul ehviour in mle rts? Insights from -Fos nd phrmologil studies. Eur. J. Phrmol. 526, (25). 6. Yng, C. F. et l. Sexully dimorphi neurons in the ventromedil hypothlmus govern mting in oth sexes nd ggression in mles. Cell 53, (23). 7. Lee, H. et l. Slle ontrol of mounting nd ttk y Esr + neurons in the ventromedil hypothlmus. Nture 59, (24). 8. Sno, K., Tsud, M. C., Mustov, S., Skmoto, T. & Ogw, S. Differentil effets of site-speifi knokdown of estrogen reeptor α in the medil mygdl, medil pre-opti re, nd ventromedil nuleus of the hypothlmus on sexul nd ggressive ehvior of mle mie. Eur. J. Neurosi. 37, (23). 9. Ghosh, K. K. et l. Miniturized integrtion of fluoresene mirosope. Nt. Methods 8, (2).. Ziv, Y. et l. Long-term dynmis of CA hippompl ple odes. Nt. Neurosi. 6, (23).. Chen, T. W. et l. Ultrsensitive fluoresent proteins for imging neuronl tivity. Nture 499, (23). 2. Thurmond, J. B. Tehnique for produing nd mesuring territoril ggression using lortory mie. Physiol. Behv. 4, (975). 3. Geldi, P. & Kowlski, B. R. Prtil lest-squres regression: tutoril. Anl. Chim. At 85, 7 (986). 4. Lin, D. et l. Funtionl identifition of n ggression lous in the mouse hypothlmus. Nture 47, (2). 5. Flkner, A. L., Dollr, P., Peron, P., Anderson, D. J. & Lin, D. Deoding ventromedil hypothlmi neurl tivity during mle mouse ggression. J. Neurosi. 34, (24). 6. Flkner, A. L., Grosenik, L., Dvidson, T. J., Deisseroth, K. & Lin, D. Hypothlmi ontrol of mle ggression-seeking ehvior. Nt. Neurosi. 9, (26). 7. Shdlen, M. N., Britten, K. H., Newsome, W. T. & Movshon, J. A. A omputtionl nlysis of the reltionship etween neuronl nd ehviorl responses to visul motion. J. Neurosi. 6, (996). 8. Jennings, J. H. et l. Visulizing hypothlmi network dynmis for ppetitive nd onsummtory ehviors. Cell 6, (25). 9. Grewe, B. F. et l. Neurl ensemle dynmis underlying long-term ssoitive memory. Nture 543, (27). 2. Efron, B., Hstie, T., Johnstone, I. & Tishirni, R. Lest ngle regression. Ann. Sttist. 32, (24). 2. Ogw, S. et l. Survivl of reprodutive ehviors in estrogen reeptor β gene-defiient (βerko) mle nd femle mie. Pro. Ntl Ad. Si. USA 96, (999). 22. Gorogge, K. L., Liu, Y., Ji, X. & Wng, Z. Anterior hypothlmi neurl tivtion nd neurohemil ssoitions with ggression in pir-onded mle pririe voles. J. Comp. Neurol. 52, 9 22 (27). 23. Redondo, R. L. et l. Bidiretionl swith of the vlene ssoited with hippompl ontextul memory engrm. Nture 53, (24). 24. Xu, P. S., Lee, D. & Holy, T. E. Experiene-dependent plstiity drives individul differenes in pheromone-sensing neurons. Neuron 9, (26). 25. Stowers, L. & Lierles, S. D. Stte-dependent responses to sex pheromones in mouse. Curr. Opin. Neuroiol. 38, (26). 26. Gur, R., Tendler, A. & Wgner, S. Long-term soil reognition memory is medited y oxytoin-dependent synpti plstiity in the medil mygdl. Biol. Psyhitry 76, (24). 27. Cooke, B. M. Steroid-dependent plstiity in the medil mygdl. Neurosiene 38, (26). 28. Knierim, J. J. & Zhng, K. Attrtor dynmis of sptilly orrelted neurl tivity in the limi system. Annu. Rev. Neurosi. 35, (22). 29. Burk, Y. Sptil oding nd ttrtor dynmis of grid ells in the entorhinl ortex. Curr. Opin. Neuroiol. 25, (24). 3. Moser, E. I., Kropff, E. & Moser, M.-B. Ple ells, grid ells, nd the rin s sptil representtion system. Annu. Rev. Neurosi. 3, (28). Supplementry Informtion is ville in the online version of the pper. Aknowledgements We thnk X. Wng, J. S. Chng nd R. Roertson for tehnil help, H. Lee nd P. Kunwr for experimentl dvie, D. Senyuz for testing ehviour in wild-type mie, D.-W. Kim for pilot experiments, M. MCrdle nd C. Chiu for genotyping, J.Costnz for mouse olony mngement, G. Stuer for dvie on GCMP6s expression, Insopix In. for tehnil support, P. Peron for mouse trking softwre, L. Aott for omments on the mnusript, R. Axel, D. Y. Tso nd M. Meister for ritil feedk, X. D nd C. Chiu for lortory mngement nd G. Mnuso for Administrtive Assistne. D.J.A. nd M.J.S. re Investigtors of the Howrd Hughes Medil Institute nd Pul G. Allen Distinguished Investigtors. This work ws supported in prt y NIH grnt no. RMH753, nd grnts from the Gordon Moore Foundtion, Ellison Medil Reserh Foundtion, Simons Foundtion nd Guggenheim Foundtion to D.J.A. A.K. is fellow of the Helen Hy Whitney Foundtion, M.Z. is reipient of fellowships from the NSF nd L Orél USA Women in Siene. Author Contriutions R.R. designed nd performed ll imging experiments, proessed the dt, ontriuted to nlysis nd o-wrote the mnusript; A.K. performed omputtionl nlysis, prepred figures nd o-wrote the mnusript; M.Z. designed nd performed ehviourl experiments; M.J.S. nd B.F.G. provided trining for R.R., guidne on experimentl design nd dt nlysis, nd ritil feedk; D.J.A. supervised the projet nd o-wrote the mnusript. Author Informtion Reprints nd permissions informtion is ville t The uthors delre ompeting finnil interests: detils re ville in the online version of the pper. Reders re welome to omment on the online version of the pper. Pulisher s note: Springer Nture remins neutrl with regrd to jurisditionl lims in pulished mps nd institutionl ffilitions. Correspondene nd requests for mterils should e ddressed to D.J.A. (wuwei@lteh.edu). 392 NATURE VOL 55 9 otoer Mmilln Pulishers Limited, prt of Springer Nture. All rights reserved.

6 Letter RESEARCH Methods Mie. All experimentl proedures involving the use of live nimls or their tissues were rried out in ordne with the NIH guidelines nd pproved y the Institutionl Animl Cre nd Use Committee nd the Institutionl Biosfety Committee t the Cliforni Institute of Tehnology (Clteh). Esr Cre/+ knok-in mie 7 krossed into the C57BL/6N kground (>N) were red t Clteh. The Esr Cre/+ knok-in line is ville from the Jkson Lortory (Stok no. 79). Heterozygous Esr Cre/+ mie were used for ll imging studies nd were genotyped y PCR nlysis of til DNA. Mie used s residents (see Methods: resident intruder ssy) were individully housed. All wild-type mie used s intruders in resident intruder ssys nd for ehviorl experiments without imging (unoperted ontrols) were of the C57BL/6N strin (Chrles River Lortories) purhsed t 2 4 weeks of ge, nd group-housed t Clteh efore use. Femle mie were purhsed ovrietomized nd reeived injetions of 7β-oestrdiol enzote (Sigm-Aldrih) in sesme oil ( μg t 48 h nd 5 μg t 24 h) nd of of progesterone (5 μg t 4 6 h) efore ehviourl testing 3. All mie were housed in ventilted miro- isoltor ges in temperture-ontrolled environment (medin temperture 23 C), under reversed 2 h drk light yle, nd hd d liitum ess to food nd wter. Mouse ges were hnged weekly on fixed dy on whih experiments were not performed. Surgery. Adult heterozygous Esr Cre/+ mles were single-housed for t lest five dys efore undergoing surgil proedures nd were operted on t 2 4 weeks of ge. Before this, mie were mintined with mle littermtes, following wening t three weeks, tht is, for 9 weeks. Mie were nesthetized using isoflurne (2.5% indution,.2.5% mintenne, in 95% oxygen) nd pled in stereotxi frme (Dvid Kopf Instruments). Body temperture ws mintined using heting pd. An inision ws mde to expose the skull for stereotxi lignment using the inferior ererl vein nd the Bregm s vertil referenes. We sed the oordintes for the rnio tomy nd stereotxi injetion of VMHvl on n ntomil mgneti resonne tls of the mouse rin (AP: 4.68 mm; ML: ±.78 mm; DV: 5.8 mm), s previously desried 7. Virus injetion nd GRIN endosope lens implnttion were performed during single surgery. Virus suspension ws injeted using pulled-glss pillry t slow rte of 8 nl min, nl per injetion site (Nnojetor II, Drummond Sientifi; Miro4 ontroller, World Preision Instruments). The glss pillry ws withdrwn 5 2 min fter the esstion of injetion nd ustom-uilt tue of hypodermi grde stinless steel ws lowered to the VMHvl trget oordintes to rete spe tht would ommodte the endosope lens. The tue ws removed fter 2 3 min nd immeditely repled y GRIN endosope lens (Insopix,.5 or.6 mm dimeter, minimum 6 mm length). The lens ws emented to the skull using smll drop of dentl ement (Metond, Prkell), nd susequently uring thin lyers of dentl ement were pplied to over the entire exposed skull. Next, multiple lyers of dentl ryli (Coltene, Whledent) were pplied into whih hed-fixtion r ws llowed to set. One the dentl ryli hd ured, the mie were tken off isoflurne nd llowed to reover in len, utolved ges to minimize hne of infetion. Bupivine nd ketoprofen were dministered s lol nd systemi nlgesis, respetively, during surgery nd mie were provided motril nd iuprofen in their drinking wter fter surgery for week. Mie were mintined in isoltion until reording experiments were performed (typilly more thn 4 weeks; see elow). Virus. Esr + neurons expressing Cre-reominse in the VMHvl were trnsdued with n deno-ssoited virus (AAV) expressing GCMP6s under the humn Synpsin I promoter (AAV.Syn.GCMP6s; Penn Vetor Core). A series of titrtion experiments ws performed to determine the highest dilution t whih right ytoplsmi, non-nuler GCMP6s loliztion ould e oserved in slies of fixed tissue from injeted nimls 32,33 efore performing injetions for in vivo imging pplitions. Mirosope lignment. Mie were initilly heked for epifluoresene signls four weeks fter virus injetion nd endosope lens implnttion. Mie were nesthetized using isoflurne, mounted in stereotxi frme nd the hed ws levelled to the horizontl plne. A hed-mounted miniturized mirosope (nvist, Insopix) ws then lowered over the implnted lens until GCMP6s-expressing fluoresent neurons were in fous uniformly ross the imging plne. If fluoresent neurons were not oserved, the mouse ws returned to the ge nd tested gin on weekly sis. If GCMP6s-expressing fluoresent neurons were deteted, the mirosope ws ligned nd permnent seplte tthed to the hed ording to pulished protools 33. A seplte over (BPC-2, Insopix) ws used to prevent edding dust from the home ge from settling on the lens. After 48 h, the seplte over ws repled y weight-mthed dummy mirosope (DMS-2, Insopix), to limtize the mouse to the weight of the mirosope for t lest three dys efore soil ehviourl testing. Seletion of nimls for imging experiments. Imging dt were olleted from totl of 25 Esr Cre/+ mie (out of 22 injeted/implnted nimls generted in multiple ohorts). Sixteen out of 25 mie were used for experiments reported here. Mie were seleted for omined imging nd ehviour experiments sed on the following riteri: () they hd individully disernle neurons ross t lest hlf the imging field; (2) the imging plne ws ler nd not oluded y lood or deris; nd (3) the imge registrtion ould e pplied suessfully to orret for motion rtefts (ll exept one mouse). The most frequent filure modes were mistrgeting of the virl injetion nd/or GRIN lens implnttion, indequte levels of GCMP6s expression nd filure to otin ler imging plne. Determintion of smple size. For imging experiments, etween three nd seven mie were imged per experimentl prdigm; the numer of mie ws dependent on the numer of usle mie nd the numer of nimls initilly implnted nd injeted. Seletion of mie ws determined y sreening with the minisope efore experimenttion. Seletion ws not sed on ny pre-speified effet. Dt presented in Figs 3 is from five out of seven mie imged tht eventully showed mounting nd ttk ehviour y the third dy of imging. The two nomlous mie tht showed neither onsummtory soil ehviour (mting, fighting), nor seprtion of ensemle representtions, were treted seprtely; dt from one of these mie (mouse 6) is shown in Fig. 4 nd in Extended Dt Fig. 6. The smple size for the numer of ells ws 75 3 ells per mouse, nd ws ffeted y the volume of virl GCMP6s injeted, the effiieny of Cre-medited reomintion nd level of expression. During the post-proessing of the imging dt (fter identifition of individul ells y prinipl omponent nd independent omponent nlyses (PCA ICA)), 5% of the identified omponents were disrded s rtefts nd the remining 95 99% were treted s ells nd used for further nlysis. Smple sizes for ehviourl experiments using wild-type unoperted mie were determined y the urrent stndrd used for mie in ehviourl neurosiene experiments, sed on the miniml numer of mie required to detet signifine with α rte set to.5 in stndrd-powered experiment. Rndomiztion. Animls were ssigned rndomly to experimentl nd ontrol groups. Blinding. Soil ehviours were mnully nnotted lind to the sex of the intruder mouse y trined humn nnottor. Behviorl ssys, imging nd omputtionl nlysis were not performed linded. Resident intruder ssy. Before resident intruder testing, implnted mie were individully housed for t lest four weeks, to llow reovery from surgery nd dequte levels of GCMP6s expression. The lens-implnted Esr Cre/+ mouse ws lwys the home ge resident. Dt were olleted from eh resident over three onseutive dys of testing, eginning when the resident ws still nive to dult non-littermte onspeifis (tht is, housed in soil isoltion eginning five dys efore surgery). The results in Figs, 2 re derived from resident nimls on their third dy of testing, y whih time most of the mie expressed norml sexul nd ggressive ehviours. Dt for eh dy were olleted s sequene of 4 individul trils, omprising two seline trils (tht is, trils performed in the sene of n intruder or ojet), two trils with toy mouse (OurPets Ply-N-Squek) nd five trils eh with mle or femle intruder. The sequene of trils with n intruder or ojet ws interleved; the sex of the first intruder ws rndomized ross nimls. Eh tril with n ojet or intruder lsted 2 3 min, to minimize photolehing of GCMP6s, nd ws preeded nd followed y 3 s of imging efore nd fter the intruder ws introdued, respetively. Intruders were mnully introdued into the resident s home ge held y the til, during whih the residents were llowed to nogenitlly investigte the intruders for 5 s. Cre ws tken to remove the intruder or toy from the ren only when the resident ws t distne to prevent rtifiilly trunting ny nturlly ourring soil intertion. The inter-tril intervl ws min. A different intruder mouse ws used for eh tril with mle or femle intruder. One ohort of seven implnted mie were used for imging in these three-dy ssys; of these seven mie, two did not show mting or ggressive ehviour or ensemle seprtion nd were omitted from the nlyses performed in Figs 3 (see min text nd Extended Dt Fig. 6). Another ohort of 2 unoperted wild-type mie ws used for ehviourl nlysis only (Fig. 4j, k). A third ohort of three implnted mie ws used for the restrited-ess ssy (Fig. 4d g); fourth ohort of six implnted mie ws used for imging in the femle priming ssy in Fig. 4l o; fifth ohort of two implnted mie ws used for imging in the mesh ontiner version of the restrited-ess ssy (Extended Dt Fig. ). Restrited-ess ssy: held-intruder. For the ohort of implnted mie (n = 3), the experimentl design ws identil to the resident intruder ssy; however, to prevent free soil intertion, intruder mie were held y the experimenter for the durtion of eh tril. Intertions with five mles, five femles, two toy nd two seline trils were reorded for eh resident eh dy, for three onseutive 27 Mmilln Pulishers Limited, prt of Springer Nture. All rights reserved.

7 RESEARCH Letter dys of testing. The sme implnted mie were susequently given two dditionl onseutive dys of testing in the originl (free ess) resident intruder ssy (see Resident intruder ssy ). One of the three implnted mie eme ill nd hd to e euthnized fter the restrited-ess portion of the experiment. Restrited-ess ssy: mesh rrier. For seond group of implnted mie (n = 2), the experimentl design ws identil to the resident intruder ssy, however intruders were pled inside wire-mesh rrier (n inverted penil up) when introdued into the resident s home ge. Intertions with five mles, five femles, nd two toy nd two seline trils were reorded for eh resident eh dy, for three onseutive dys of testing. The mie were susequently tested for two onseutive dys of soil intertions s in the primry resident intruder ssy (dys 4, 5), followed y n dditionl dy of testing with intruders ehind the mesh rrier (dy 6). Priming ssy (Fig. 4l o). Nive, implnted mie were llowed to freely intert in single session with either femle or mle for 3 min on dy (priming), nd then tested with onspeifi of the opposite sex on dy 2 in min freeintertion tril. Before nd fter the priming on dy, proe trils were performed with (different) proe nimls of eh sex (n = 3 mles, n = 3 femles), to test for ensemle seprtion efore nd fter priming. During these proe trils, mle or femle proe niml ws suspended y the til in the ge for s, during whih time sniffing (ut no ttk or mounting) y the implnted resident ourred. Three implnted mie were primed with femles on dy nd three mie were primed with mles on dy. Of the three mie primed with femles, one mouse filed to mte with the femle during priming, or to show ggression towrds mle on dy 2, nd ws omitted from imging nlysis, euse the purpose of the experiment ws to investigte whether ensemle seprtion orrelted with the pperne of ggression following mting with femles. (This mouse my hve een one of the nomlous mie we desrie, whih exhiit neither mting nor fighting ehviour.) Of the three mie primed with mles on dy, one tht lredy showed sex-speifi ensemle seprtion on the first proe tril (dy ), tht is, efore priming, ws omitted from the nlysis, s the purpose of the experiment ws to investigte whether priming with mle ws suffiient to use sex-speifi ensemle seprtion in mie where suh seprtion hd not yet ourred. An dditionl 2 wild-type unoperted mie tht hd een soilly isolted for two weeks were tested in this priming ssy only to quntify ehviour (Fig. 4j, k). Four mie were presented with femle on dy, nd eight mie only experiened single, 3-min enounter with mle on dy 2. Neuronl nd ehviourl dt quisition. Mie were temporrily hed-fixed efore imging sessions nd the hed-mounted dummy ws repled y the miniturized mirosope. Anesthesi ws not used t this step, euse it strongly suppressed hypothlmi neuronl tivity. The mie were pled in their home ges situted within the ehviourl ren with hed-mounted mirosopes tthed, nd llowed to hitute 5 3 min efore dt quisition. The ehviourl ren ws ustom-uilt (ll prts from Thorls) with one top-view nd one side-view mer (Fle3, Point Grey) for quiring ehviourl dt, whih were sved s AVI files on Windows omputer. A TTL pulse from the Syn port of the dt quisition ox (DAQ, Insopix) of the mirosope ws used to trigger individul frmes through the GPIO pins on the mer. This llowed : orrespondene etween ehviourl frmes nd neuronl imging frmes, whih ws neessry for synhronizing neuronl tivity to ehviour. Shortly efore dt quisition, the imging prmeters were onfigured nd sved using nvist (Insopix) ontrol softwre. All ut two mie used in this study were imged t 3 Hz with 33.2 ms exposure per frme (the other two were imged t 5 Hz with 66.2 ms exposure per frme, s they exhiited lower levels of GCMP6s fluoresene). Mirosope LED illumintion ws 4% of the mximum llowed, t gin. The field of view ws ropped to the region enompssing the fluoresent neurons to mximize uninterrupted dt quisition t 3 Hz. All imging prmeters were sved nd were the sme for every tril for eh mouse. Dt were sved s stks of frmes in TIFF or RAW formt. Position trking. Instntneous positions nd poses (estimted y fit ellipse) of oth mie ws trked from the top-view video, using ustom softwre written in MATLAB 34. The distne etween nimls ws defined s the distne etween the entroids of the fit ellipses. Dt proessing. The ehviourl videos were loded into MATLAB using ustom-sripted ehviourl nnottor 7,4 nd trined individul lind to the experimentl design nnotted the videos frme-y-frme for ttk, mounting, hed nd/or flnk-direted sniffing (sniffing) nd nogenitl sniffing (AG sniffing). Imging frmes were sptilly downsmpled y ftor of two (tht is,.5 ) in the x nd y dimensions, nd normlized y dividing y sptilly nd-pssed version of the frme (lower ound, μm; upper ound,, μm). Frmes olleted over the ourse of single dy were ontented into single stk nd registered to eh other to orret for motion rtefts using the Mosi softwre pkge (Insopix) 8,33. Imging dt were lulted s reltive hnges in fluoresene, ΔF(t)/F = (F(t) F )/F where F ws the men of ll frmes ross the stk. To extrt single ells nd their C 2+ tivity tres from the fluoresent imging frmes, we pplied n estlished omputtionl lgorithm for ell sorting sed on PCA ICA 35 to generte series of sptil filters tht eh orresponded to single ell. Filters were individully inspeted mnully, nd those tht did not pper to orrespond to single neurons were disrded,33. Thresholded, segmenttion msks for eh sptil filter (tht is, ell) were projeted onto the ΔF(t)/F stk of frmes to extrt time ourse of fluoresene hnges for tht ell. On verge, we identified 228 ± 64 ells per mouse per dy using PCA ICA (totl of 3,396 ells imged in seven mie; 2,379 ells from the five mie tht showed mounting nd ttk). For eh ell, ΔF(t)/F tres were normlized y the stndrd devition of the first seline tril, nd reported in units of stndrd devition (σ). Registrtion ross dys. Individul sptil filters, eh orresponding to ell, for eh dy of imging, were thresholded nd omined into mximum intensity projetion mps (Extended Dt Fig. 7). Intensity-sed imge registrtion ws used to identify pir of ffine trnsformtions for ligning the dy nd dy 3 mps to the dy 2 mp. Overlpping filter triplets from the three dys were mnully sreened for ury. All seleted triplets hd n verge of less thn 3 μm Euliden distne etween entroids on pirs of dys (dy versus dy 2, dy 2 versus dy 3, nd dy versus dy 3; verge entroid seprtion of registered filters =.98 ±.3 μm). Roughly 4% of ll sptil filters (ells) were registered ross three dys of imging (n = 99 ± 37 ells per mouse). Ensemle response vetor. The time-verged ΔF(t)/F tivity of eh ell ws omputed over ll frmes of lose soil intertions (sniffing, mounting or ttk) during eh tril, nd ontented into N vetor (N = ells) lled the popultion tivity (or ensemle response) vetor. The use of responses from periods of soil intertion preluded ny effets of inter-niml distne 5 in our representtions. Nevertheless, nlyses using different epohs during tril (for exmple, only frmes from periods of sniffing, or distne-sed riteri s desried elow) did not fundmentlly hnge our oservtions (Extended Dt Fig. 3). Quntifying the distne modultion of ell responses. Cells exited or inhiited y onspeifis (men ΔF(t)/F > 2σ during periods of mle- or femleintertion) showed vrying degrees of modultion y the distne to the intruder. To quntify the distne modultion of responsive ells, we used the distne (d) etween entroids of the two mie (see Position trking ) to identify frmes in whih the two nimls were not interting (nd were not within ± s of intertion) nd were lose (d < 35% digonl length of ge; Extended Dt Fig. 3) or fr (d > 7%). r lose nd r fr were mesured s the verge ΔF(t)/F of eh ell for eh distne ondition nd used to ompute distne modultion index (m) s m = r lose r fr / ( r lose + r fr ); mesurement of the extent to whih ell tivity vried with inter-niml distne. Exmple ells with low vlue of m ( < m <.2) showed little modultion of tivity depending on distne etween the nimls (Extended Dt Fig. 3e2). PCC of mle femle similrity. Beuse on ny given tril, the imged resident enountered onspeifi of one sex, the PCC etween mle nd femle representtions ws omputed etween given tril s popultion tivity vetor nd the verge popultion tivity vetor during the two nerest neighouring trils of the other intruder sex. For the first nd lst trils, only the one temporlly losest other-sex tril ws used. Frtion of vrine ptured y intruder sex. The differene of ovrines method 36 ws used to identify the two lrgest mle- nd femle-speifi soures of vrine in the ensemle response. For resident mouse on given dy, the ovrine mtrix etween ells on ll mle trils (C M ) nd on ll femle trils (C F ) ws generted, nd the differene etween them (C = C M C F ) ws lulted. By onstrution, the eigenvetors of C with the lrgest positive nd negtive eigenvlues orrespond to the lrgest mle- nd femle-speifi soures of vrine, respetively. To mesure the frtion of vrine ptured y this pir of eigenvetors, the N T (neurons y time) mtrix of ensemle tivity on given dy of imging ws reonstruted s weighted sum of these two vetors using liner regression. The R 2 vlue of the reonstrution ws reported s the frtion of vrine ptured. Dimensionlity redution. Low-dimensionl representtions for visulizing hnging ensemle dynmis over time were onstruted using PCA 37 or prtil lest squres (PLS) regression 38 (using the plsregress funtion in MATLAB). For PLS, ll tres from dy of imging were ontented nd regressed ginst T vetor with entries vlued t (if mle intruder ws present), (if there ws femle intruder) or (otherwise). Deoding intruder sex (Fig. 3f). To determine whether VMHvl tivity ontined informtion out intruder sex, liner deoder ws used to deode the sex of the intruder from ensemle tivity during periods of soil intertion. Trining dt ws onstruted from the set of N (N = neurons) popultion 27 Mmilln Pulishers Limited, prt of Springer Nture. All rights reserved.

8 Letter RESEARCH tivity vetors from ll frmes ourring during soil intertion. To void effets of dtset size ross imged mie, rndom suset of 5 ells ws drwn from the set of ll ells tht were imged tht dy. This trining dt, long with intruder sex lels, ws then used to trin liner SVM deoder 39. Deoder performne ws evluted using ross-vlidtion, y exluding ll dt from one intruder during trining nd using tht dt to test deoder performne; this ws repeted 5 times for eh intruder, eh time with different rndom suset of ells, nd performne omputed s the verge ury ross test sets. Anlysis ws repeted for eh dy of imging, with seprte deoder trined for eh dy. Deoding intruder sex following intruder introdution (Fig. l). We wnted to identify how quikly, from the moment the intruder entered, liner deoder ould predit the sex of the intruder from the ensemle tivity. The deoder performne ws tested in hlf-seond windows, strting 5 s efore to s fter the moment tht the intruder fully entered the resident s home ge. The trining set ws onstruted y verging the ΔF(t)/F for eh ell within.5 s time in entred t time t, for eh mle nd femle tril on one dy (typilly trils totl). Overfitting to the smll (n = 9) numer of trining exmples ws redued y projeting trining dt onto the first prinipl omponents of ensemle tivity from tht dy; we found tht this improved deoder performne. The projeted dt nd intruder sex lels were then used to trin liner SVM deoder, nd deoder performne ws evluted using leve-one-out ross-vlidtion with eh tril held out one. To llow dynmi hnges in intruder-evoked responses, new deoder ws trined t eh time point. Deoding ehviour (Fig. 2). To determine whether ensemle tivity ontined informtion out speifi soil ehviours, seprte inry liner deoder ws trined for eh ehviour, to predit whether the ehviour ws ourring or not. To void onfounding representtions of ehviour with representtions of intruder sex, seprte deoders were trined for mle- nd femle-direted ehviours. Trining dt ws onstruted from the set of N ensemle tivity vetors from ll frmes of ll dy 3 trils with given intruder sex, lelled positive or negtive for trget ehviour. Negtive trining dt (tht is, dt from periods when the ehviour of interest ws not ourring) ws under-smpled to produe n equl numer of positive nd negtive trining exmples. These inry lels nd their orresponding ensemle tivity vetors were then used to trin liner SVM deoder. Deoder performne ws tested y ross-vlidtion y exluding ll dt from one intruder during trining, nd omputing the verge deoder performne on the held-out dt using the F sore (see elow). Testing ws repeted for eh intruder, nd for 5 itertions of eh trining set. Mesuring performne of ehviour deoders (Fig. 2). Beuse most ehviours ourred on smll frtion of tested frmes, reporting the ury (true positives + true negtives) / (totl smples) of trined ehviour deoder ould e misleding. Insted, we reported the F sore, F = 2 ( preision rell)/ (preision + rell), where preision (lso lled speifiity) is the frtion of predited positives tht re tul (ground-truth) positives (preision = (true positives)/ (true positives + flse positives)) nd rell (lso lled sensitivity) is the frtion of tul (ground-truth) positives tht re orretly predited positive (rell = (true positives)/(true positives + flse negtives)). F sores of trined SVMs were omputed y shuffling oth the ells nd the ehviour nnottions. To permute ehviour nnottions while preserving their temporl struture, ll ehviour outs nd ll inter-out intervls were identified for given dy/intruder sex/ehviour, nd syntheti ehviour rsters were onstruted y rndomly drwing without replement from the set of outs nd the set of inter-out intervls. The trined SVM weights were pplied to the shuffled ells, nd the F sore ws omputed reltive to the shuffled ehviour. Eh form of shuffling ws repeted, giving, shuffled oservtions; we onsidered oserved F sores s signifint if they fell ove the 97.5 perentile of the distriution of hne F sores. Mhlnois distne (Fig. 3h, i). For eh imged mouse, the Mhlnois distne ws used s n dditionl mesurement of mle nd femle representtion seprtion. Two distriutions of N-dimensionl (N = neurons) popultion tivity vetors were generted from ll imging frmes during whih soil intertion ourred: one distriution for mle trils nd the other for femle trils. The Mhlnois distne (d) etween eh vetor (the test vetor) nd these two distriutions were omputed, to give d sme (distne to points of the sme sex s the test vetor) nd d opp (distne to points of the opposite sex), whih were then verged ross ll test vetors. Points within the distriutions tht me from the sme tril s the test vetor were omitted during distne lultion. Choie proility (Fig. 2d-h). Choie proility (CP) nlysis ws used to mesure ell s tuning, defined here s how well two onditions ould e preditively disriminted from single ell s tivity 7. The CP of given ell, for pir of (ehviourl) onditions, ws omputed y onstruting histogrm of tht ell s ΔF(t)/F vlues under eh of the two onditions. These two histogrms were plotted ginst eh other to generte n ROC (reeiver-operting hrteristi) urve. The integrl of the re under this ROC urve generted the CP vlue for eh ell, with respet to eh of the two ehviourl onditions (see Extended Dt Fig. 4). This CP vlue is ounded from to, where CP of.5 orresponded to no differene etween onditions. All frmes from ll ehviour outs lsting two or more seonds were used to ompute the CP, nd dt from the seond nd third dy of imging were omined. Cells from mie tht spent less thn 2% of time engging in ny of the four exmined ehviours (ttk, mounting, mle-direted sniffing nd femle-direted sniffing) were omitted from this nlysis, owing to noisiness of their CP estimtes. The signifine of ell s CP, reltive to hne, ws omputed using shuffled out timings for the two ompred onditions (s ws done for estimting hne deoder performne). Shuffling ws repeted, from whih the men nd s.d. of shuffled CPs ws estimted. Any oserved CP tht ws 2 s.d. ove the shuffled men ws onsidered signifint. Non-negtive LASSO regression of ehviour ginst ensemle seprtion (Fig. 4 ). To identify ehviourl orreltes of ensemle seprtion, the PCC nd the umultive time spent engging in eh ehviour ws omputed for eh niml nd tril (round 3 trils per mouse; n = 6 mie for whih imging dt ws ville for ll three dys; this inludes one of the two mie tht did not show mounting or ttk ehviour, nd whose sex representtions did not segregte). The weighted sum of umultive ehviours most orrelted with the PCC ws found using non-negtive lest-ngle regression (LARS) implementtion of lest solute shrinkge nd seletion opertor (LASSO) 2, to impose sprseness nd non-negtivity onstrints on the fit weights. The sprseness prmeter of LARS ws seleted to minimize the ross-vlidted men squred error of the fit. For ll ehviours, squre-root trnsform of umultive ehviour times, efore LASSO regression, inresed their orreltion with the PCC. Code vilility. Custom ode written for the purpose of this study is essile t Dt vilility. Imging nd ehviourl dt will e mde ville y the orresponding uthor upon resonle request. 3. Ogw, S. et l. Aolition of mle sexul ehviors in mie lking estrogen reeptors α nd β (αβerko). Pro. Ntl Ad. Si. USA 97, (2). 32. Dn, H. et l. Thy GCMP6 trnsgeni mie for neuronl popultion imging in vivo. PLoS ONE 9, e8697 (24). 33. Resendez, S. L. et l. Visuliztion of ortil, suortil nd deep rin neurl iruit dynmis during nturlisti mmmlin ehvior with hed-mounted mirosopes nd hronilly implnted lenses. Nt. Protools, (26). 34. Dollár, P., Welinder, P. & Peron, P. Csded pose regression. In IEEE Conferene on Computer Vision nd Pttern Reognition (CVPR, 2). 35. Mukmel, E. A., Nimmerjhn, A. & Shnitzer, M. J. Automted nlysis of ellulr signls from lrge-sle lium imging dt. Neuron 63, (29). 36. Mhens, C. K., Romo, R. & Brody, C. D. Funtionl, ut not ntomil, seprtion of wht nd when in prefrontl ortex. J. Neurosi. 3, (2). 37. Jolliffe, I. Prinipl Component Anlysis (Wiley Online Lirry, 22). 38. De Jong, S. SIMPLS: n lterntive pproh to prtil lest squres regression. Chemom. Intell. L. Syst. 8, (993). 39. Cristinini, N & Shwe-Tylor, J. An Introdution to Support Vetor Mhines nd Other Kernel-sed Lerning Methods (Cmridge Univ. Press, 2). 27 Mmilln Pulishers Limited, prt of Springer Nture. All rights reserved.

9 RESEARCH Letter normlized ΔF/F perent of units (%) d N N2 N3 N4 N5 g 9 4 in 2σ exited units +2σ sniff AG sniff inhiited units seline toy trils trils ΔF/F (σ reltive to seline) 3 se out e h 7 5 % exited units (>2σ) % inhiited units (< 2σ) 5 utoorreltion N N2 N3 N4 N5 offset (se) 45 f sorted units (n = 435) *** N N2 ** * * T % BL % T BL stimulus identity N3 N4 N5 utoorreltion 3 hlf-width (se) oserved expeted y hne perent of units * oth oth oth ex oth inh ex, inh ex, inh Extended Dt Figure See next pge for ption. 27 Mmilln Pulishers Limited, prt of Springer Nture. All rights reserved.

10 Letter RESEARCH Extended Dt Figure Properties of Esr + neuron responses in VMHvl during free soil intertions. Dt presented re from the third dy of imging., Histogrm of time-verged ΔF(t)/F vlues oserved in the 3 s following stimulus introdution (or the first 3 s of imging, on seline trils). Dshed lines indite thresholds for identifying exited ( 2 s.d. (2σ) of seline ove the men pre-intruder seline) nd inhiited ( 2σ elow pre-intruder seline) ells. (n = 5 imged mie, 5 mle trils, 5 femle trils, 2 toy trils, 2 seline trils per mouse.),, Averge perentge of time ells were exited () or inhiited () in first 3 s of imging. d, Exmple tres from single Esr + ells, showing exmple response profiles during single tril intertion with femle onspeifi. While some ells showed trnsient lium responses (N, N2), others showed slower dynmis (N3 N5), inluding persistent elevtion or suppression of tivity for the durtion of the enounter with the femle. We elieve the ltter responses reflet the ongoing tivity of some VMHvl ells, s filtered y the slow dynmis of GCMP6s. e, Autoorreltion funtions omputed ross ll dy 3 trils, for eh exmple ell in d; red line indites hlf-width. f, Autoorreltion hlfwidth omputed ross ll dy 3 trils, for ll ells from ll mie (n =,435 ells in five mie), sorted from smllest to lrgest, reveling ontinuous rnge of vlues. Autoorreltion hlf-width of the five ells in d re indited. g, Exmple sptil mps of exited (left; men ΔF(t)/F 2σ during periods of soil intertion) nd inhiited (right; men ΔF(t)/F 2σ during intertion ross ll mle nd femle trils on the third dy of imging) ells in the presene of mle nd femle onspeifis. h, The perentge of ells ross ll mie (n = 5) tht were exited nd/ or inhiited y oth mles nd femles, ompred to the perentge expeted y hne ssuming sttistil independene. (oth inhiited, P = ; exited y femle + inhiited y mle, P =.299). 27 Mmilln Pulishers Limited, prt of Springer Nture. All rights reserved.

11 RESEARCH Letter Extended Dt Figure 2 Representtions of mles nd femles in ll individul mie., Dy 3 ensemle representtions of intruder sex for the five mie tht mounted nd fought with onspeifis, projeted onto the first two xes of PLS regression ginst intruder sex. Tres re oloured y intruder sex identity. Perentge of vrine explined y the first two PLS omponents is noted for eh mouse., Projetion of single tril onto the first two prinipl xes of one exmple mouse (mouse 2), olouroded to highlight temporl trjetory of the popultion tivity vetor during the tril. Prinipl xes were identified using PCA (sme xes s in Fig. k)., All trils from mouse 2, oloured y tril numer. 27 Mmilln Pulishers Limited, prt of Springer Nture. All rights reserved.

12 Letter RESEARCH lose intermedite fr 9% 4% 3% lose + noninterting intermedite fr interting interted within +/-se 2% 53% d m= frmes used for nlysis f rlose rfr rlose + rfr m =.89 σ lose 2 fr mle-exited femle-exited distne modultion (m), exited units mle-inhiited femle-inhiited 2 σ lose 2 fr lose + noninterting intermedite fr interting 4 min m =.8 4 perent of units (%) e e2 5 frmes used for nlysis.25 e perent of units (%) orreltion oeffiient ttk + mount + sniff sniff only lose + noninterting intermedite fr within-sex vs intruder sex deoder ury (%) distne modultion (m), inhiited units Extended Dt Figure 3 See next pge for ption. 27 Mmilln Pulishers Limited, prt of Springer Nture. All rights reserved.

13 RESEARCH Letter Extended Dt Figure 3 Distne dependene of intruder sex representtions., Exmple video frme showing estimtes of resident nd intruder poses nd entroids (red ounding oxes nd points, respetively) produed y n utomted trker 34. Green, grey, nd purple rs mrk the inter-mouse distnes (reltive to the resident) tegorized s lose, intermedite nd fr., Perentge of time nimls spent either interting with onspeifis or present within the three zones. Time points in whih nimls hve reently interted (tht is, intertion hs oured within ± s) re shown s their own tegory s ontrol for the effet of slow GCMP dynmis., PCC etween trils of the sme intruder sex, or etween mle nd femle trils, where representtions of intruders re omputed y verging eh ell s ΔF(t)/F ross the indited susets of imging time points. d, Aury of liner SVM deoder for intruder sex, using representtions omputed s in. Deoder ury ws not signifintly ffeted y distne. e, Top, the distne modultion index (m), mesurement of the extent to whih ell tivity is modulted y inter-niml distne, is omputed from r lose (the verge response of ell when nimls re lose ut hve not reently interted (within ± s)), nd r fr (the verge response of tht ell when nimls re fr prt nd hve not reently interted). e, Bottom, exmple ΔF/F tres (lk) from two ells tht hve high (e, m =.89) nd low (e2, m =.8) distne modultion index, nd orresponding inter-niml distnes (rown). f, Histogrms of vlues of m oserved in ll ells tht re signifintly exited (ove) or inhiited (elow) during intertion with mles or femles. Note tht inhiited ells re less sensitive to interniml distne thn re exited ells, nd tht the distriution of m is similr for ells tht re responsive to mle nd femle intruders. 27 Mmilln Pulishers Limited, prt of Springer Nture. All rights reserved.

14 Letter RESEARCH normlized ΔF/F (σ) min ttk sniff 4 frmes (ount) pdf plots -3 8 normlized ΔF/F (σ) df plots ttk sniff -3 8 normlized ΔF/F (σ) df of sniff frmes hoie pro. = re under urve =.69 df of ttk frmes ttk vs sniff 4 units (ount) hoie proility Exmple units tuned for: intruder intruder 2 intruder 3 Attk 5 σ Sniff min intruder intruder 2 intruder 3 ttk mount sniff AG sniff 5 σ min intruder intruder 2 intruder 3 Mount σ min intruder intruder 2 intruder 3 Sniff 5 σ onsummtory ehviors ttk mount min overlps in unit ehvior tuning mount-tuned mount- nd ttk-tuned ttk-tuned ttk- nd sniff -tuned sniff -tuned sniff - nd sniff -tuned sniff -tuned d preision - direted preision - direted ppetitive ehviors sniff sniff Extended Dt Figure 4 See next pge for ption. ttk mount sniff AG sniff rell rell 27 Mmilln Pulishers Limited, prt of Springer Nture. All rights reserved.

15 RESEARCH Letter Extended Dt Figure 4 Choie proility histogrms nd exmple ells., Steps for omputing CP, mesurement of the disriminility of two onditions, given the ΔF(t)/F of single ell. Given pir of ehviours (here, mle-direted ttk nd sniffing), we omputed the distriution of ΔF(t)/F vlues for this ell for eh ehviour, nd then integrted eh distriution to produe pir of umultive distriution funtions (dfs). The re under the ROC urve formed from these two distriutions is defined s the ell s CP; CP of this exmple ell ws.69. Right-most pnel shows CP for ll ells (rrow indites CP of the exmple ell); outlined rs denote ells for whih CP ws signifintly higher thn hne., F/F tres nd orresponding ehviour rsters from exmple ells tht showed signifint CPs for the speified ehviour, illustrting the reltive hnges in F/F nd ehviour. Three representtive trils re shown (sme exmples s in Fig. 2 d g)., Proportion of ells signifintly tuned for eh exmined ehviour. Overlp etween loks reflets the proportion of ells tht were signifintly tuned for oth ehviours. Note tht y this metri, ells nnot e tuned for oth ttk nd mledireted sniffing, nor for oth mounting nd femle-direted sniffing, euse the CP of ell is defined y ompring ΔF(t)/F vlues under these pired onditions. All ells signifintly tuned for ttk, mount or sniff lso showed signifint CP for periods of lose soil intertions (inluding sniffing nd/or mount or ttk) versus periods of nonsoil intertions, y onstrution. However, some ells tht showed signifint CP for lose soil intertions versus non-soil periods did not show signifint CP for speifi ehviour (see Fig. 2h). d, Seprte preision (true positives / (true positives + flse positives)) nd rell (true positives / (true positives + flse negtives)) sores for the ehviour deoders presented in Fig. 2,. The F sore (presented in Fig. 2) is defined s 2(preision rell) / (preision + rell). 27 Mmilln Pulishers Limited, prt of Springer Nture. All rights reserved.

16 Letter RESEARCH 5% ttk mle 5% ttk femle % time spent engging in ehvior (<.2% on ll trils/dys) dy dy 2 dy 3 dy dy 2 dy 3 3% mount mle 3% mount femle dy dy 2 dy 3 dy dy 2 dy 3 3% nogenitl sniff mle 3% nogenitl sniff femle dy dy 2 dy 3 dy dy 2 dy 3 75% sniff mle 75% sniff femle dy dy 2 dy 3 dy dy 2 dy 3 umultive ehvior (min) 2 unoperted ontrol mie mount ttk tril numer Extended Dt Figure 5 Expression of soil ehviours ross trils nd dys., Perentge of time mie spent engging in mle- nd femledireted ehviours on eh of five trils on three dys of imging. n = 5 mie, men ± s.e.m., Cumultive time spent mounting nd ttking y ohort of unoperted, soilly isolted mie tht underwent two dys of the stndrd soil experiene prdigm depited in Fig. d (n = 8 mie). 2 umultive ehvior (min) 3 2 operted mie (reprodued from Fig 4A) tril numer 2 The unoperted mie exhiited grdul pperne of mounting, nd showed mounting ehviours efore ttk, inditing tht hnges in ehviour were not due to the effets of surgery or the presene of the sope., Dt from implnted mie (n = 5), reprodued from Fig. 3g nd restrited to the first 2 trils for diret omprison. 27 Mmilln Pulishers Limited, prt of Springer Nture. All rights reserved.

17 RESEARCH Letter : Femle 2: Mle 3: Femle 4: Mle 5: Femle 6: Mle 7: Femle 8: Mle 9: Toy : Femle : Mle Dy Dy 2 ttk mount sniff AG sniff nonsoil : Femle 2: Mle 3: Toy 4: Femle 5: Mle 6: Femle 7: Mle 8: Femle 9: Mle : Femle : Mle 2: Toy 3: Femle -4 PLS omp intruder in time reltive to intruder introdution (se) d Mouse 6 : Mle 2: Femle 3: Mle 4: Femle 5: Mle 6: Femle 7: Mle 8: Femle 9: Toy : Mle : Femle - : Femle 2: Mle 3: Toy 4: Femle 5: Mle 6: Femle 7: Mle 8: Femle 9: Mle : Femle : Mle : Mle 2: Femle 3: Toy 4: Mle 5: Femle 6: Mle 7: Femle 8: Mle 9: Femle : Mle : Femle 3 seline femle mle toy overlp : Mle 2: Femle 3: Toy 4: Mle 5: Femle 6: Mle 7: Femle 8: Mle 9: Femle : Toy : Mle 2: Femle Dy 3 Mouse 2 PLS omp 2 intruder in 5 PLS omp 2 Dy 3 Dy 2 Dy - PLS omp time reltive to intruder introdution (se) Extended Dt Figure 6 Comprison of the soil ehviours nd neurl representtions in exmple mie tht showed nd did not show ggression. d, Behviour rsters (, ) nd the orresponding first two PLS omponents (, d) for exmple mie tht showed (, ) nd did not (, d) show ggression to onspeifi mles over the ourse of the experiment. 27 Mmilln Pulishers Limited, prt of Springer Nture. All rights reserved.

18 Letter RESEARCH overlid filters from dys -3 dy 5 μm dy 2 5 μm 5 μm dy 3 5 μm dy filters dy 2 filters dy 3 filters registered triplets other triplets unit triplets (ount) 6 mouse men distne etween triplet entroids (μm) mouse 4 5 mouse mouse mouse mouse Extended Dt Figure 7 Registrtion of ells ross three dys of imging., Left, mximum projetion mps of sptil filters of ll ells from eh of three dys of imging for n exmple mouse. Right, RGB-omposite imge of the three left imges (dy = red hnnel, dy 2 = green hnnel, dy 3 = lue hnnel), showing overlp of registered filters (overlp in ll three hnnels ppers in white). Outlined re 2 exmple ells (out of 35) tht ould e identified in ll three dys of imging: red/green/lue lines indite filter outlines on first/seond/ third dy, respetively; lk points mrk filter entroids., Histogrms of verge distne etween filter entroids from dys 3, in ells tht ould e trked ross dys (red) s ompred to rndom triplets of ells (lk). Dy 3 entroids from trked ells were seprted y n verge of 2.5 ±.6 μm (men ± s.e.m., n = 593 ells trked ross dys in six mie, during the stndrd resident intruder ssy). 27 Mmilln Pulishers Limited, prt of Springer Nture. All rights reserved.