Hyperexcitability of female serotonin neurons underlies sex-specific anxiety responses

Hyperexcitability of female serotonin neurons underlies sex-specific anxiety responses | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Hyperexcitability of female serotonin neurons underlies sex-specific anxiety responses View ORCID Profile Suzanne van der Veldt , Fiona Henderson , Lucia Pizzoccaro , Anne-Sophie Simard , Félix Perreault , Justine Fortin-Houde , Guillaume Ducharme , Bénédicte Amilhon doi: https://doi.org/10.1101/2025.08.17.670715 Suzanne van der Veldt 1 CHU Sainte-Justine Research Center , Montréal, Québec, Canada 2 Département de Neurosciences, Université de Montréal , Montréal, Québec, Canada 3 Groningen Institute for Evolutionary Life Sciences, University of Groningen , the Netherlands Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Suzanne van der Veldt Fiona Henderson 1 CHU Sainte-Justine Research Center , Montréal, Québec, Canada 2 Département de Neurosciences, Université de Montréal , Montréal, Québec, Canada Find this author on Google Scholar Find this author on PubMed Search for this author on this site Lucia Pizzoccaro 1 CHU Sainte-Justine Research Center , Montréal, Québec, Canada 2 Département de Neurosciences, Université de Montréal , Montréal, Québec, Canada Find this author on Google Scholar Find this author on PubMed Search for this author on this site Anne-Sophie Simard 1 CHU Sainte-Justine Research Center , Montréal, Québec, Canada 2 Département de Neurosciences, Université de Montréal , Montréal, Québec, Canada Find this author on Google Scholar Find this author on PubMed Search for this author on this site Félix Perreault 1 CHU Sainte-Justine Research Center , Montréal, Québec, Canada 2 Département de Neurosciences, Université de Montréal , Montréal, Québec, Canada Find this author on Google Scholar Find this author on PubMed Search for this author on this site Justine Fortin-Houde 1 CHU Sainte-Justine Research Center , Montréal, Québec, Canada 2 Département de Neurosciences, Université de Montréal , Montréal, Québec, Canada Find this author on Google Scholar Find this author on PubMed Search for this author on this site Guillaume Ducharme 1 CHU Sainte-Justine Research Center , Montréal, Québec, Canada Find this author on Google Scholar Find this author on PubMed Search for this author on this site Bénédicte Amilhon 1 CHU Sainte-Justine Research Center , Montréal, Québec, Canada 2 Département de Neurosciences, Université de Montréal , Montréal, Québec, Canada Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: benedicte.amilhon{at}umontreal.ca Abstract Full Text Info/History Metrics Preview PDF Abstract Mood disorders display robust sex differences in prevalence, symptom profile, and treatment outcomes, with women nearly twice as likely as men to be diagnosed. However, the neural substrates mediating sex-specific regulation of mood and related disorders remain incompletely understood. Here, we identify a neural circuit mechanism involving serotonergic (5-HT) projections from the median raphe region (MRR) to the ventral hippocampus (vHP) responsible for sex-specific regulation of anxiety-like behavior in mice. Using a multimodal approach combining electrophysiology, fiber photometry, and optogenetics, we show that 5-HT neurons targeting the vHP (5-HT vHP neurons) display heightened intrinsic excitability and delayed deactivation in female mice during exposure to aversive environments. Optogenetic activation of this pathway enhanced anxiety-like behavior, and decreased risk-assessment behavior in females, but not in males. Collectively, our findings establish 5-HT vHP neurons as a critical circuit for the regulation of anxiety and provide a mechanistic framework for understanding how serotonergic modulation contributes to sex-specific vulnerability to mood disorders. Introduction Mood disorders disproportionately affect women, who are not only more likely to be diagnosed, but also exhibit different symptom profiles and treatment responses compared to men 1 , 2 . Although this sex bias is well-documented, its neurobiological underpinnings remain poorly understood and largely understudied. Elucidating how biological sex influences mood and vulnerability to mood disorders at the neural circuit level is essential for developing more effective treatment strategies. Anxiety disorders which are among the most prevalent mood-related conditions, show nearly a twofold higher incidence in women relative to men 2 . Drugs increasing serotonin (5-hydroxytryptamine, 5-HT) transmission remain first-line treatment for anxiety and related mood disorders, demonstrating the central role of 5-HT in the neurobiology of anxiety 3 . Yet, paradoxically, acute manipulations of 5-HT signaling, whether via pharmacology or optogenetic stimulation, have shown that increased serotonergic activity can exacerbate anxiety-like behavior in rodents 4 – 8 . These conflicting effects underscore the developmental, electrophysiological and functional heterogeneity of the 5-HT system 9 , 10 , which comprises a diverse population of neurons distributed across multiple raphe nuclei, with widespread, frequently collateralized projections that collectively innervate the entire forebrain 4 , 11 – 15 . Moreover, multiple 5-HT receptors, some with opposing action, can be co-expressed in the same brain regions and neurons 16 – 20 . This complexity has long hampered our understanding of how serotonergic signalling regulates mood states. Among the many targets of serotonergic cells, the ventral hippocampus (vHP), a core component of the limbic system, stands out as a critical node in the regulation of anxiety and behavioral inhibition 21 – 24 . The vHP shares connections with the lateral septum, prefrontal cortex (PFC), lateral hypothalamus and raphe, circuits previously described for their role in anxiety processing 25 – 31 . Lesions of the vHP consistently produce anxiolytic effects 21 , 24 . vHP pyramidal cells and interneurons represent aversive and potentially threatening environments 25 , 27 , 30 , 32 – 34 and manipulation of vHP activity or outputs modulates anxiety-like behaviors in rodents 25 , 26 , 31 , 34 , 35 . In humans, reductions in anterior hippocampal volume are observed in individuals with anxiety disorders 36 , and the anterior hippocampus is engaged during approach-avoidance behaviors 37 , 38 , reinforcing the conserved role of this structure in anxiety regulation across species. The median raphe region (MRR), comprising the median raphe nucleus (MnR) and the paramedian raphe (PMnR), is the main source of serotonergic inputs to the hippocampus in mice 13 . Although comparatively less explored than the dorsal raphe nucleus 9 , 39 – 42 , growing evidence highlights the MRR as a key group of 5-HT neurons with roles in aversion, avoidance and more 43 – 49 . While the impacts of 5-HT release on dorsal hippocampus (dHP) activity and functions have been documented 7 , 20 , 50 – 55 , effects of 5-HT on the vHP have received less attention. Altered 5-HT transmission in the vHP 8 , 56 enhances anxiety-related behaviors, suggesting a critical pathway for 5-HT-mediated regulation of anxiety states 8 , 56 – 58 . The involvement of this pathway, and the extent to which sex-specific differences in vHP-projecting 5-HT neurons (5-HT vHP neurons) from the MRR contribute to anxiety regulation, await clarification. This study aims to advance understanding of how serotonergic circuits contribute to the sex disparity in mood disorders. We postulate that 5-HT vHP neurons are a central component of the neural circuits governing anxiety. To test this hypothesis, we use a combination of circuit-specific fiber photometry, electrophysiology, optogenetics and behavioral analysis. We show that these neurons exhibit heightened intrinsic excitability, spontaneous activity and increased in vivo recruitment in females. These sex differences translate into circuit- and behavior-specific consequences: 5-HT vHP neurons in females exhibit delayed disengagement from aversive contexts, selectively alter hippocampal theta dynamics during novelty exposure, and promote anxiety-like behavior, increased risk-assessment, and grooming when optogenetically activated. Together, our findings delineate a circuit-specific mechanism by which serotonergic signalling in the vHP contributes to sexually divergent anxiety responses, offering insights in how sex differences at the cellular and circuit level may underlie vulnerability to mood disorders. Results MRR 5-HT vHP neurons are more excitable in female mice Serotonergic neurons display strikingly diverse electrophysiological properties, including a wide range of firing frequencies, action potential waveforms and resting membrane potential. Although investigations of sex-differences in these electrophysiological properties are scarce, a handful of studies have reported differences in excitability of 5-HT neurons 59 – 62 . To probe the contribution of raphe-vHP circuits in sex-specific anxiety behaviors we examined whether 5-HT vHP neurons differ in their intrinsic electrophysiological properties. We first mapped the distribution of 5-HT vHP neurons within raphe sub-regions. 5-HT vHP neurons were identified using retrograde expression of eYFP, obtained with injections of a Cre-dependent retrogradely expressed adeno-associated virus in the vHP of SERT-Cre mice ( Fig. 1a ). Transfected neurons were similarly distributed across multiple raphe subregions for males and females, with labeled neurons primarily located in the B9 cell group, the MRR and the interfascicular part of the DR (DRI; Fig. 1b,c ; Extended Data Fig. 1 ). Importantly, the proportion of 5-HT cells retrogradely labeled from the vHP was similar between males and females, representing in % of 5-HT neurons: 17.91 and 18.90 in B9, 14.36 and 19.24 in the MRR, and 15.18 and 20.33 in the DRI, in females and males respectively ( Fig. 2c ; Extended Data Fig. 1b-c ). Download figure Open in new tab Extended Data Fig. 1: Sex-independent distribution of 5-HT vHP neurons across raphe subregions. a, Schematic delineations of raphe regions with 5-HT+ neurons (Tph2+, blue). Distance from bregma shown in mm. Scale bars = 250 μm. b, Total number of 5-HT+ neurons for each raphe subregion, for both sexes (females: n = 4; males: n = 5). c, Total number of eYFP+ neurons per subregion, for both sexes. For the DR, two-sample t-test, P = 0.0394. d, Schematic of experimental strategy to assess in-vitro electrophysiological characteristics of 5-HT vHP cells in raphe slices. e, Representative examples of eYFP-positive (eYFP+, white) cell. f , Same data as shown in Fig. 1f, l and r , grouped on the same graph for direct comparison and statistical testing, showing that female MRR 5-HT vHP neurons have the highest firing frequency among all regions and sexes. Two sample t-tests, MRR: female vs male, P = 0.0089; Female B9 vs MRR, P = 0.0017; Female MRR vs DRI, P = 0.0003; Female MRR vs male DRI, P = 0.0010; Female MRR vs Male B9, P < 0.0001. All data are shown as mean ± s.e.m; ** : P < 0.01, *** : P = < 0.001; For detailed statistical information and for the numbers of female and male mice used, see Extended Data Table 1 . Abbreviations: IP: interpeduncular nucleus, B9: B9 nucleus; MnR: median part of the median raphe region; PMnR: paramedian part of the median raphe region; MRR: median raphe region; DRI: dorsal raphe, interfascicular subregion; DR: dorsal raphe; DRC: caudal part of the dorsal raphe; Tph2: tryptophan hydroxylase type 2; eYFP: enhanced yellow fluorescent protein; For detailed statistical information, see Extended Data Table 1 . Download figure Open in new tab Figure 1: Female MRR 5-HT vHP neurons exhibit increased intrinsic excitability. a, Schematic of experimental configuration: a retrograde AAV containing Cre-dependent construct eYFP was bilaterally infused into the vHP of SERT-Cre mice. b, Representative coronal sections showing eYFP-labeled neurons in the B9, MRR and DRI regions at different anteroposterior levels (distance from bregma in mm indicated in bottom left corner). 5-HT+ cells are detected using Tph2 immunostaining. Scale bars = 200 μm (overviews), 100 μm (magnified). c, Percentage of eYFP+ 5-HT neurons in raphe of females (teal, n = 4 mice) and males (orange, n = 4 mice). d, Examples of membrane potential responses to current steps (−50 pA for lower trace and + 190 pA for upper trace) as part of the electrophysiological characterization of eYFP+ neurons recorded from female (teal) and male (orange) animals for 5-HT vHP , from the B9 nucleus. Number on the left indicates resting membrane potential. e – i, For B9 5-HT vHP neurons, firing frequency responses as a function of current intensity stimulations ( e ), firing frequency at +190 pA ( f ), resting membrane potential ( g ), input resistance ( h ) and adaptation index ( i ) for females (teal, n = 18 cells from 9 mice) and males (orange, n = 15 cells from 10 mice). j, Same as d, for 5-HT vHP neurons recorded from the MRR. k – o, Same as e-i , for eYFP+ neurons recorded from the MRR. l, two sample t-test P = 0.0089, l, two sample t-test, P = 0.0079, and o, two sample t-test, P = 0.0225, for females (teal, n = 17 cells from 6 mice and males (orange, n = 16 cells from 6 mice). p , Same as d and j , for eYFP+ neurons recorded from DRI. q – u, Same as for e - i and k - o , for eYFP+ neurons recorded from DRI. s, two sample t-test, P = 0.0207, for females (teal, n = 18 cells from 9 mice) and males (orange, n = 19 cells from 10 mice). All data are shown as mean ± s.e.m. *: P < 0.05; **: P < 0.01. For detailed statistical information, see Extended Data Table 1 . B9: B9 nucleus; MRR: median raphe region; DRI: dorsal raphe, interfascicular subregion; eYFP: enhanced yellow fluorescent protein; Tph2: tryptophan hydroxylase 2. View this table: View inline View popup Extended Data Table 1: Summary of statistical analyses Download figure Open in new tab Figure 2: 5-HT vHP activation increases anxiety-like behaviors and decreases risk assessment selectively in females. a, Schematic of experimental configuration: a retrograde AAV containing Cre-dependent excitatory opsin ChETA or YFP control is bilaterally infused in the vHP of SERT-Cre mice of both sexes and an optic fiber is implanted above the MRR region. b, Experimental set up: EPM (left) and SA (middle) experiments, with optogenetic stimulation paradigm (right). c - j , Change (stim – pre) in behavioral measures during EPM and SA testing. Data are from two independent experiments and analyzed by three-way ANOVA with Holm’s multiple comparison testing. Significant comparisons are reported as P values. c , Time in closed arms of the EPM: females (ChETA vs control), P = 0.0378; ChETA (female vs male), P = 0.0126. d, Open arm entries of the EPM: females (ChETA vs control), P = 0.0278; ChETA (female vs male). P = 0.0204. e, Anxiety score (see methods) in the SA: ChETA (female vs male), P = 0.0134. f, Number of open zone (zone 2, 3 and 4) entries: females (ChETA vs control), P = 0.0182; ChETA (female vs male), P = 0.0237. g, Number of head dips in the EPM: females (ChETA vs control), P = 0.0052; ChETA (female vs male), P = 0.0068. h, Number of stretchings in the EPM: females (ChETA vs control), P < 0.0001; ChETA (female vs male), P < 0.0001. i, Number of head dips in the SA: ChETA (female vs male), P = 0.0009. j , Number of head dips in the SA: females (ChETA vs control), P = 0.0026; ChETA (female vs male), P = 0.0039. k, Representative labeling of cFos (magenta) and retrograde eYFP (green) expression in coronal sections of the B9, MRR and DRI. Arrows indicate examples of c-fos+/eYFP+ neurons. Scale bars = 50 μm. l, Percentage of eYFP+ neurons co-expressing c-fos in females (teal, eYFP: n = 7; ChETA: n = 7) and males (orange, eYFP: n = 7; ChETA: n = 7). Top: B9; two-way ANOVA with two-sample t-tests: females (ChETA vs control), P < 0.0001; males (ChETA vs control): P < 0.0001. Middle: MRR; two-way ANOVA with two-sample t-tests: females (ChETA vs control): P < 0.0001; males (ChETA vs control): P = 0.0114. Bottom: DRI; two-way ANOVA with two-sample t-tests, females (ChETA vs control), P < 0.0001; males (ChETA vs control: P < 0.0001. m , Stimulation induced change (stim – pre) for females relative to males in the number of c-fos+/eYFP+ neurons for each subregion. One sample t-test, MRR: P < 0.001. Data are shown as mean ± s.e.m. * : P < 0.05, ** : P < 0.01; ***: P < 0.001. For detailed statistical information, see Extended Data Table 1 . For an overview of all behavioral variables analyzed, see Extended Data Table 2 . View this table: View inline View popup Download powerpoint Extended Data Table 2 Comprehensive summary of all statistical testing for behavioral experiments detailed in Fig. 2 , Fig. 3 , Extended Data Fig. 2 , Extended Data Fig. 7 . We performed whole-cell patch-clamp recordings in acute brain slices from retrogradely labeled eYFP-positive cells in the B9, MRR and DRI from male and female mice ( Fig. 1 , Extended Data Fig. 1d-e ). In the B9 region, no sex differences were observed in firing frequency ( Fig. 1d-f ), resting membrane potential (RMP, Fig. 1g ), input resistance ( Fig. 1h ), or spike frequency adaptation during sustained activation, quantified by the adaptation index ( Fig. 1i , see methods). In contrast, 5-HT vHP neurons in the MRR from female mice exhibited consistently higher firing rates across all depolarizing current steps ( Fig. 1j-l ) and a more depolarized RMP compared to males ( Fig. 1m ). Notably, these neurons showed a significantly reduced adaptation index compared to males ( Fig. 1n ). The absence of differences in input resistance ( Fig. 1o ) suggests that sex differences in excitability are not due to changes in passive membrane properties. In turn, 5-HT vHP neurons in the DRI showed comparable firing frequency between sexes ( Fig. 1p-r ) but, similarly to observations in the MRR, their RMP was more depolarized in female mice ( Fig. 1s ). No differences between sexes were found for DRI 5-HT vHP neurons input resistance ( Fig. 1t ) or adaptation index ( Fig. 1u ). Notably, the firing frequencies of female MRR 5-HT vHP neurons were the highest among all regions and sexes ( Extended Data Fig. 1f ). Moreover, under baseline conditions, 29% of female MRR 5-HT vHP neurons (5/17 cells) exhibited spontaneous firing, which was not observed in any neuron recorded from the B9 and DRI regions in either sex, or in male MRR 5-HT vHP neurons. These findings demonstrate that while 5-HT vHP neurons are similarly distributed in male and female mice, female 5-HT vHP neurons display subregion- and sex-specific differences in intrinsic properties. Whereas B9 and DRI neurons were largely similar between the sexes, female MRR 5-HT vHP are uniquely hyperexcitable, exhibiting more depolarized resting potentials, higher firing rates, reduced spike adaptation and spontaneous activity not observed in males or other subregions. Optogenetic activation of 5-HT vHP neurons promotes anxiety-like behavior and decreases risk-assessment in females The MnR is the primary source of 5-HT inputs to the vHP in mice 13 and has previously been implicated in the regulation of anxiety behavior 43 , 63 , 64 . As females show higher anxiety than males across species 1 , 2 , 65 , we hypothesized that increased excitability of MRR 5-HT vHP female neurons might contribute to sex differences in the behavioral response to aversive contexts and potential threat. We used the elevated plus maze (EPM), a classical anxiety assay, and the successive alleys (SA) task, that both reliably elicits approach-avoidance conflict behaviors in male and female mice 66 , 67 , and tested whether stimulating 5-HT vHP neurons would alter anxiety-like and risk assessment behaviors. To assess the functional consequences of selective activation of 5-HT vHP neurons, we expressed an excitatory opsin, ChETA, or a control eYFP construct ( Fig. 2a ). ChETA expression was highly specific to 5-HT neurons, with only 0.21 ± 0.11% of ChETA-expressing neurons lacking Tph2 co-expression. Two cohorts of male and female mice were tested by experimenters of opposite sex and analyzed by three-way ANOVA to control for effect of cohort, sex and stimulation ( Extended Data Table 1 , see methods; Extended Data Fig. 2 ). Light was delivered to the raphe during subsequent exposures to the EPM and the successive alleys (SA) tasks ( Fig. 2b ; Extended Data Fig. 2a ). In the EPM, activation of 5-HT vHP neurons increased time spent in the closed arms in female ChETA mice as compared to both female eYFP controls and male ChETA mice ( Fig. 2c , Extended Data Fig. 2c-d ). Likewise, during activation female ChETA mice made fewer open arm entries than both same-sex eYFP controls and male ChETA mice ( Fig. 2d ), consistent with increased anxiety-like behavior. These sex-specific anxiogenic effects were also evident in the SA task. Activation of 5-HT vHP neurons reduced exploration of aversive zones in female ChETA mice, as reflected by higher occupancy scores ( Fig. 2e , see methods) and fewer entries in the open alleys ( Fig. 2f ), compared to both female eYFP controls and male ChETA mice. Importantly, these behavioral changes occurred largely independent of alterations in gross locomotor activity. In the EPM, optogenetic stimulation did not significantly affect distance traveled ( Extended Data Fig. 2h ). In the SA task, however, stimulation of 5-HT vHP neurons significantly reduced distance traveled in female ChETA animals compared to male ChETA mice ( Extended Data Fig. 2i ), consistent with increased avoidance. Download figure Open in new tab Extended Data Fig. 2: Effects of 5-HT vHP activation on closed-arm time and grooming across cohorts. a, Fiber optic placement in raphe of experimental mice used for optogenetic stimulation during behavioral tests. Numbers above diagrams indicate distance from bregma in mm. Numbers in boxes indicate identification number of experimental subject. b , c, Time spent in the closed arms during the pre-stimulation (pre), stimulation (stim) and post-stimulation (post) epochs for cohort 1 ( b ) and cohort 2 ( c ). d, Change in closed-arm time for both cohorts. e , f, Total time spent grooming during pre, stim and post epochs for cohort 1 ( e ) and cohort 2 ( f ). g, Change in grooming time for both cohorts. h, Difference between the distance travelled during the 4 min of optogenetic stimulation (ON) and the first 4 min of baseline (pre) periods in the elevated-plus maze for each group. No difference was observed across sexes or groups. i, Same as h , but in the successive alleys (SA). Three-way ANOVA with Holms multiple comparison testing, ChETA (female vs male), P = 0.0041. Data are shown as mean ± s.e.m. **: P < 0.01. Data are from two independent experiments and were analyzed by three-way ANOVA. For detailed statistical information and for the numbers of female and male mice used, see Extended Data Table 1 . For an overview of all behavioral variables analyzed, see Extended Data Table 2 . We hypothesized that if reduced exploration of the open arm in the EPM or the open zone in the SA by female ChETA mice reflected heightened anxiety, rather than a general decrease in exploratory drive, it would be accompanied by a reduction in risk assessment behaviors. Head dipping, an exploratory behavior in which the animal lowers its head over the edge of an open arm, was decreased upon stimulation of 5-HT vHP neurons in female ChETA mice in the EPM ( Fig. 2g ). Stretch-attend postures, where mice elongate their bodies in a low stance to cautiously assess their environment, were also strongly reduced in female ChETA animals in the EPM ( Fig. 2h ). This pattern was replicated in the SA, where optogenetic activation similarly reduced both head dipping ( Fig. 2i ) and stretch-attend postures ( Fig. 2j ) in female ChETA mice compared to eYFP controls. Together, these findings indicate that activation of 5-HT vHP neurons enhances anxiety-like states in females, suppressing risk-assessment behaviors characteristic of approach-avoidance conflict, rather than merely reducing overall exploratory drive. Because serotonergic axons form extensive collaterals 14 , we asked whether off-target effects via collateral projections could contribute to the observed behavioral changes. Using eYFP labeling of 5-HT vHP neurons, we mapped their axonal distribution and confirmed collateral innervation across several regions, including the dHP, lateral and medial septum and prefrontal cortex ( Extended Data Fig. 3 ). We observed no sex differences in the pattern or density of these collateral projections, suggesting that the sex-specific behavioral effects are unlikely to arise from differential collateralization between the sexes. Download figure Open in new tab Extended Data Fig. 3: 5-HT vHP collateralization to the forebrain. eYFP-positive collaterals are detected using anti-GFP immunostaining. a, Sparse axons in the prefrontal cortex (PFC). b, High density of axons present in the lateral septum (LS). Overlay of the eYFP fibers with anti-5-HT immunostaining ( b1 - b3 ). c, Collaterals are found in the lateral parts of the medial septum (MS). d, 5-HT axons are found in the preoptic area with the highest density in the lateral part (LPO). e, Axons pass through the medial forebrain bundle (mfb) and a few are observed in the lateral hypothalamic area (LHA). f, Medial amygdaloid nucleus (MeA), basomedial amygdaloid nucleus (BMA) and anterior cortical amygdaloid area (ACo) all contain high density of axons while very few to none are observed in the central amygdala (CeA), lateral amygdala (LaA) and the basolateral amygdala (BLA). g, Few axons are consistently present in the insular cortex (IC). h, The dHP contains 5-HT positive collaterals from neurons targeting its ventral counterpart. i, Example of the injection site in the vHP covering the dentate gyrus (DG), CA1, CA3 and VS (ventral subiculum). High density of eYFP labeled axons are present in CA1 ( i1-i3 ) and CA3 ( i1’-i3’ ). Few axons are observed in the dorsolateral, dorsal intermediate and ventral intermediate part of the entorhinal cortex (DLEnt, DIEnt and VIEnt, respectively) but leakage from the injection site should be considered. j, Qualitative assessment of 5-HT collaterals density in female (n = 4) and male (n = 4) mice. Data are shown as mean. ACo: anterior cortical amygdaloid area; BLA: basolateral amygdala; BMA: basomedial amygdaloid nucleus; CeA: central amygdala; DG: dentate gyrus; DIEnt: dorsal intermediate enthorinal cortex; DLEnt: dorsolateral entorhinal cortex; eYFP: enhanced yellow fluorescent protein; IC: insular cortex; LaA: lateral amygdala; LHA: lateral hypothalamic area; LPO: lateral part of preoptic area; LS: lateral septum; MeA: medial amygdaloid nucleus; mfb: medial forebrain bundle; MS: medial septum; PFC: prefrontal cortex; VIEnt: ventral intermediate part of the entorhinal cortex; VS: ventral subiculum; Scale bar: 100 μm in A-I, 50 μm in b1-b3; f1-f2; h1-h3; i1-i3; i1’-i3’. We next asked whether intrinsic electrophysiological differences translate into heightened neuronal activation during behavior. To address this, we quantified c-fos expression as a proxy for in vivo activity following exposure to the SA paradigm combined with 20 Hz optogenetic stimulation. Mice were perfused 70 minutes post-test to capture peak c-fos induction. Immunohistochemical analysis revealed c-fos expression in eYFP-labeled neurons across the B9, MRR and DRI ( Fig. 2k ). Quantification of the percentage of eYFP+ neurons co-expressing c-fos revealed a significant main effect of optogenetic stimulation across all three subregions in both sexes ( Fig. 2l ). In the B9 and DRI, optogenetic stimulation robustly increased the number of c-fos+ neurons in males and females to a similar extent ( Fig. 2l ). In contrast, in the MRR, a two-way ANOVA revealed both a significant main effect of treatment and a significant sex x treatment interaction, consistent with enhanced excitability of MRR 5-HT neurons in females ( Fig. 2l ). To quantify this differential increase in c-fos, we computed the stimulation-induced change from eYFP-controls in each sex, expressed as [(female ChETA – female eYFP) - (male ChETA – male eYFP)] ( Fig. 2m ). These findings indicate that while optogenetic activation of 5-HT vHP neurons reliably drives neuronal activity across raphe subregions, MRR activation was greater in females, mirroring the observed sex-specific behavioral effects and increased intrinsic excitability of female MRR 5-HT vHP neurons. Collectively, these results suggest that heightened intrinsic excitability of female MRR 5-HT vHP neurons drives their disproportionate recruitment during behavior, as revealed by c-fos mapping. The larger recruitment of hyperexcitable MRR 5-HT vHP neurons in potentially threatening contexts provides a cellular mechanism for sex differences anxiety behaviors. Activation of 5-HT vHP neurons promotes anxiety-related grooming in females Self-grooming is increasingly recognized as a displacement behavior indicative of elevated stress and conflict, as well as a translationally relevant readout of affective states 68 – 70 . Under conditions of heightened arousal, grooming can serve as an adaptive coping strategy 71 . We tested whether optogenetic activation of 5-HT vHP neurons was sufficient to alter grooming behavior across different environments. During EPM and SA exploration, both anxiogenic contexts, optogenetic activation produced a robust increase in time spent grooming selectively in female ChETA mice relative to female eYFP controls and male ChETA mice ( Fig. 3a,b ; Extended Data Fig. 2e-g ), as well as number of grooming episodes and total grooming duration ( Extended Data Table 1 ). We quantified grooming during exploration of an open field with low-threat characteristics: a small open field in low lighting conditions (40 cm length, 70±5 lux), designed to minimize aversive features such as bright illumination and open space. In contrast to EPM and SA, stimulation of 5-HT vHP in the open field did not significantly affect grooming duration in either sex ( Fig. 3c , Extended Data Table 1 ). These findings indicate that vHP-projecting serotonergic neurons contribute to the expression of stress-related grooming in a sex- and context-dependent manner. The absence of effects in the open field suggests that elevated environmental threat is necessary for this circuit to drive displacement-like behaviors. Download figure Open in new tab Figure 3: Activation of 5-HT vHP neurons increases grooming in a context-dependent manner. a, Change in total grooming time (stim – pre) in the EPM. Three-way ANOVA with Holms multiple comparison testing: females (ChETA vs control), P = < 0.0001; ChETA (female vs male), P < 0.0001. b, Same as a , but in the SA. Three-way ANOVA with Holms multiple comparison testing: females (ChETA vs control), P = 0.0316; ChETA (female vs male), P = 0.0098. c, Same as a , but in the open field. For a - c , data are from two independent experiments and were analyzed by three-way ANOVA. d, Schematic of experimental configuration: a retrograde AAV containing the Cre-dependent calcium indicator GCaMP6s was infused bilaterally in the vHP of SERT-Cre mice. 5-HT vHP population activity was recorded using an optic fiber implanted above the MRR. e, Example 5-HT vHP Ca 2+ ΔF/F (green) over time, with locomotion velocity (black) during EPM exploration. f – g, ΔF/F aligned to grooming onset ( f ) and end ( g ) as indicated by vertical dotted line, for females (teal, n = 6) and males (orange, n = 7). h – i, Locomotor velocity for the same animals aligned to start ( h ) and ( i ) end of grooming bouts. j, Quantification of the AUC for 0 - 5 s of panel f after grooming onset. k, Quantification of the AUC for 0 - 5 s of panel g after grooming offset. Data are shown as mean ± s.e.m. * : P < 0.05, ** : P < 0.01; For detailed statistical information, see Extended Data Table 1 . For an overview of all behavioral variables analyzed, see Extended Data Table 2 . Activation of 5-HT vHP neurons increases anxiety levels and promotes stress-induced grooming, presumably through increased recruitment of hyperexcitable MRR 5-HT vHP neurons in female mice. To monitor circuit-level activity of MRR 5-HT vHP neurons we used in vivo fiber photometry in freely moving SERT-Cre mice. A retrogradely transported AAV encoding the calcium (Ca 2+ ) indicator GCaMP6s was bilaterally infused in the vHP, enabling selective monitoring of Ca 2+ dynamics in 5-HT vHP neurons ( Fig. 3d ). Fiber placement and viral expression were histologically confirmed to target MRR ( Extended Data Fig. 3a,b ). GCaMP6s expression was highly specific to 5-HT neurons, with 99.86% of GCaMP6s-expressing cells in the raphe co-expressing the selective 5-HT neuron marker tryptophan hydroxylase 2 (Tph2, n = 2 mice, 709 neurons, data not shown). To assess 5-HT vHP neuronal dynamics related to grooming under anxiogenic conditions, we tested mice in the EPM and aligned population activity to onset and end of grooming bouts. Activity of 5-HT vHP closely tracked grooming behavior ( Fig. 3e ): grooming episodes coincided with an immediate reduction in calcium signal in both sexes ( Fig. 3f ). Population activity returned upon grooming termination ( Fig. 3g ), consistent with the idea that grooming acts as a strategy to transiently downregulate heightened serotonergic activity. Quantification of event-aligned fluorescence revealed no significant sex differences in activity dynamics, either during the first 5 seconds of grooming initiation ( Fig. 3j ) or in the 5-second periods following termination ( Fig. 3k ). Grooming bouts were predominantly surrounded by low locomotor activity (<5cm/s) in both males and females ( Fig. 3h,i ), indicating that increased 5-HT vHP activity following grooming likely reflects the resumption of exploration. This suggests that grooming may serve as an alternative strategy to downregulate heightened serotonergic activity, consistent with its role as a displacement behavior under conditions of conflict or arousal. 5-HT vHP neurons in the MRR display sex-specific activity dynamics in the elevated plus maze The MRR has been implicated in the response to aversive stimuli 45 , 46 . Notably, the MRR receives direct input from the lateral habenula (LHb), a core structure in aversion-related circuits that signals negative motivational value and modulates serotonergic systems 47 , 72 – 74 . Prior work has shown that EPM exploration globally increases 5-HT levels in the vHP, as measured over slow time scales with microdialysis 56 , 75 . We hypothesized that exploration of a novel and potentially threatening environment such as the EPM would engage 5-HT vHP neurons. We tracked mouse movements and identified locomotor epochs corresponding to transitions from closed arms (“safer” zones) to open arms (aversive zones) of the EPM ( Fig 4a ), or transitions restricted to the closed arms ( Fig. 4c ). Closed-to-open epochs were defined as the mouse spending minimally 10 s in a closed arm before exploring center/open arms for at least 2 s. Closed-to-closed epochs were defined as transitions between closed arms with a maximum of 1 s spent in the center. Changes in fluorescence were averaged and aligned to the timepoint where center-of-body marker crossed the closed arm-center limit. In both sexes and trajectories, 5-HT vHP population activity peaked around transitions ( Fig. 4a,c ). We observed similar transient increases in 5-HT vHP population activity when mice initiated high velocity locomotion bouts in the EPM, independent of location in the maze, or in the open field (OF) ( Extended Data Fig. 4c-j ). Velocity and area under the curve (AUC) of fluorescence signal during 1 s surrounding the locomotion onset did not differ between sexes ( Extended Data Fig. 4d,f,h,j ). Consistent with previous evidence 46 , 76 , 77 , these results indicate that 5-HT vHP neurons are engaged during locomotion. Transitions from closed arms into the aversive open compartments were associated with a ramping increase of 5-HT vHP population activity in both sexes ( Fig. 1d,e ), suggesting progressive recruitment of this population prior to open-arm entry. By contrast, 5-HT vHP activity remained stable before transitions between closed arms ( Fig. 1f,g ). Download figure Open in new tab Extended Data Fig. 4: Locomotion-related activity of 5-HT vHP neurons does not differ between sexes. a, Example fiber placement above the MRR, with magnified view of cell bodies (right). 5-HT neurons (Tph2+) are shown in orange; GCaMP6s-labelled 5-HT vHP neurons shown in cyan. Scale bars = 200 μm. b, Optic fiber placements for all animals used for fiber photometry experiments (n = 6 females, n = 7 males). Numbers in boxes indicate individual animal IDs; brain schematics show distance from bregma in mm. c, Mean normalized GCaMP6s fluorescence signal aligned to the initiation of high velocity locomotion bouts for females (teal, n = 6) and males (orange, n = 7) in the EPM. Dotted vertical line: onset of locomotion. Gray bar indicates the period used for panel d . d, AUC of ΔF/F from –0.5 to +0.5 s around high velocity locomotion onset in the EPM. e, Corresponding instantaneous velocity across all detected high velocity locomotion bouts. Dotted vertical line indicates onset of locomotor bout. f, Quantification of peak velocity for each animal in e. g, Same as c, but for the open field. Gray bar indicates the period used for panel h . h, Same as d, but for the open field. i, Same as e, but for the open field. j, Same as f, but for the open field. k, Mean normalized ΔF/F for closed arm exits towards center/open zones for females (teal, n = 6) and males (orange, n = 7). Dotted vertical line indicates timepoint where center-of-body marker crossed the center-closed arm limit. Gray bar indicates the period used for panel l . l, AUC of ΔF/F obtained over 1 - 2 s after closed arm entry (gray bar in k ). Two-sample t-test, P = 0.0430. m, Velocity around closed arm exits for females (teal, n = 6) and males (orange, n = 7). n, Peak velocity quantification (−0.5 to 0.5 s) for females (teal, n = 6) and males (orange, n = 7). o, Example correlations between AUC of ΔF/F obtained over 1 - 2 s after each closed arm entry and time spent in the EPM for a representative female (left) and male (right). c – n , all data are shown as mean ± s.e.m. For detailed statistical information, see Extended Data Table 1 . Download figure Open in new tab Figure 4: Female 5-HT vHP neurons exhibit delayed disengagement from an aversive context. a, Mean normalized ΔF/F aligned to transitions from a closed arm to an open arm for females (teal, n = 6) and males (orange, n= 7). Dotted vertical line indicates timepoint where center-of-body marker crossed the center-closed arm limit. Gray bar indicates the time window used for panel b . b, Area under the curve (AUC) of ΔF/F obtained over 10 - 9 s preceding closed arm exit (gray bar in a ). c, Same as a , but for closed→closed arm transitions. Dotted vertical line indicates timepoint where center-of-body marker crossed the closed-arm center limit exiting the first closed arm. Gray bar indicates the time window used for panel d . d, AUC of ΔF/F obtained over 10 - 9 s preceding transition timepoint (gray bar in c ). e, Mean ΔF/F for closed arm entries from center/open zones for each female (top, n = 6) and each male (bottom, n = 7). f, Mean normalized ΔF/F for closed arm entries from center/open zones for females (teal, n = 6) and males (orange, n = 7). Dotted vertical line indicates timepoint where center-of-body marker crossed the center-closed arm limit. Gray bar indicates the period used for panel g . g, AUC of ΔF/F obtained over 1 - 2 s after closed arm entry (gray bar in f ). Two-sample t-test, P = 0.0430. h, Velocity around closed arm entries for females (teal, n = 6) and males (orange, n = 7). i, Peak velocity quantification (−0.5 to 0.5 s) for females (teal, n = 6) and males (orange, n = 7). j, Change in mean ΔF/F (last 10 min – first 10 min) after closed arm entry (1 - 2 s) for females (teal, n = 6) and males (orange, n = 7). Dotted vertical line indicates timepoint where center-of-body marker crossed into the closed arm. Gray bar indicates the period used for panel k . k, Corresponding AUC quantification of change in ΔF/F for period indicated by gray bar in j . Two-sample t-test, P = 0.0284. All data are mean ± s.e.m; * : P < 0.05. For detailed statistical information, see Extended Data Table 1 . We also focused more closely on 5-HT vHP activity around transitions in and out of the “safer” closed arms of the EPM. When mice retreated from center/open arms back into the closed areas (≥ 2 s in the center/open followed by ≥ 2 s in closed) activity of 5-HT vHP neurons decreased in both sexes; however, this reduction was significantly attenuated in females ( Fig. 4e-g ), despite comparable velocities ( Fig. 1h,i ). These results suggest that 5-HT vHP population activity remains elevated in females upon return to a less aversive environment, indicating a sex difference in how this circuit disengages from anxiogenic contexts. Conversely, exiting the closed arms did not lead to a subsequent change in 5-HT vHP population activity, and no differences between males and females were observed ( Extended Data Fig. 4k-n ). During EPM exploration, locomotor activity typically declines over time, likely reflecting habituation as animals become familiar with the environment 78 , 79 . We therefore examined how the decrease in activity of 5-HT vHP neurons during closed arms retreats evolved over the duration of the test. Specifically, we correlated AUC of the calcium signal during the 1 to 2 second period following closed arm entry (as in Fig. 4f ) with elapsed time in the EPM for both female and male mice. In males, the decrease in 5-HT vHP neurons population activity remained stable across the session (see example correlation Extended Data Fig. 4p ). In contrast, in females, this suppression correlated with time (see example correlation Extended Data Fig. 4o ): during the early phases of the test, 5-HT vHP neuron activity remained high upon retreat into a closed arm, but a reduction gradually emerged as the session progressed and animals habituated to the test apparatus. Changes in AUC over time were quantified by subtracting the retreat-related response in the last 10 min of the EPM to response in the first 10 min ( Fig. 4j ). While activity of MRR 5-HT vHP neurons remained stable in male mice, post-retreat responses in females evolved with time ( Fig. 4j,k ) showing that during early exploration of the EPM, female 5-HT vHP neurons maintain elevated activity upon retreat into less aversive compartments of the maze. Together, these findings indicate that 5-HT vHP neurons exhibit sex-specific activity dynamics during exploration and habituation in the EPM, with hyperexcitable female 5-HT vHP neurons disengaging more slowly from aversive contexts. Hippocampal response to novelty is modulated by 5-HT selectively in female mice Approach-avoidance conflict behaviors arise from competing drives to explore novelty and avoid aversive features 80 , a pattern readily observed in the EPM. We hypothesized that the sex-specific dynamics of MRR 5-HT vHP neuron activity might partly reflect a delayed familiarization to the novel environment in females, and next interrogated the roles of 5-HT vHP neurons in modulating the response to novelty. To measure the hippocampal response to novelty and its modulation by 5-HT we turned to local field potentials (LFP) and optogenetics. Theta oscillations (6-10Hz) dominate hippocampus LFP when mice are exploring an environment 81 – 83 . Theta exhibits a well-established linear relationship with locomotion, with increased speed being accompanied by higher theta frequency 84 – 87 . Prior work shows that exposure to a novel environment robustly modulates two properties of the speed-theta frequency correlation: the slope of the linear frequency-speed relationship (“slope”) and its y-axis intercept (“intercept”) 88 .The slope is a metric known to be modulated by novelty on a timescale of minutes in the dHP, with flatter slopes observed in novel environments; as animals habituate, the slope progressively steepens 86 , 88 , 89 . In contrast, the intercept is sensitive to anxiolytic drugs, which consistently decrease intercept values independent of their mechanism of action 90 . We therefore examined whether activation of 5-HT vHP neurons modulates these novelty- and anxiety-related theta dynamics in males and females. Using an optic fiber placed above the labeled population, 5-HT vHP neurons were activated while recording LFP simultaneously from the vHP and dHP ( Fig. 5a-c ; Extended Data Fig. 5 ). Recordings were conducted during free exploration of a novel environment as in Fig 3 . LFP were recorded across baseline and stimulation (20 Hz) epochs ( Fig. 5b ). As expected, dHP theta frequency linearly scaled with running speed. In eYFP controls the slope of this relationship increased from the first 4 minutes of exploration (baseline, grey lines), to the subsequent 4 minutes (stimulation epoch, black lines) in both females and males ( Fig. 5d,e ). A similar pattern was observed in the vHP ( Fig. 5h,i ). Photoactivation of 5-HT vHP neurons had sex-specific effects on the modulation of hippocampal novelty response. In male ChETA mice, the slope increase during stimulation was similar to eYFP controls ( Fig. 5g,k ,l,n). In contrast, female ChETA mice failed to show the slope increase normally observed as the novelty of the environment decreases, for both dHP ( Fig. 5f,l ) and vHP recorded theta ( Fig. 5j,n ). Thus, 5-HT vHP neurons influence the habituation-related change in slope of hippocampal theta frequency-velocity relationship in a sex-specific manner. Download figure Open in new tab Extended Data Fig. 5: Electrode and optic fiber placements for LFP recordings and optogenetic stimulation. a, Approximate electrode placement in the dHP, with representative electrode tract ( a1 , DAPI in blue; as indicated by white arrow). Scale bar = 1000 μm. Numbers in boxes indicate ID of the animals. Numbers above diagrams indicate distance from bregma in mm. b, Same as a , for the vHP ( b1 : representative tract as indicated by white arrow). Scale bar = 1000 μm. c, Fiber optic placement in the raphe. Download figure Open in new tab Figure 5: Activation of 5-HT vHP neurons disrupts theta-velocity relationship selectively in females. a, Schematic of simultaneous multi-site local field potential recording with pathway specific optogenetic stimulation. b, Stimulation paradigm and example trajectories for a female (top) and male (bottom) animal for baseline (pre) and stimulation (stim) period. c, Example raw LFP traces (gray) recorded from dHP (top) and vHP (middle) and corresponding velocity (bottom), for animals exploring the open field, 2.5 s before and after stimulation onset. Blue: theta filtered traces. Red: instantaneous animal velocity. d – g, Instantaneous theta frequency in the dHP as a function of running speed for eYFP control mice ( d , females n = 5 and e , males n = 5) and ChETA mice ( f , females n = 8 and g , males n = 7). Speed frequency relationship was computed during pre (0-4 min, gray) and stim (4-8 min, black) epochs. Thin lines: individual animals; thick lines: group mean. Shaded areas represent s.e.m. h – k, Same as d-g in the vHP. l, For the dHP, difference between slopes during optogenetic stimulation and baseline epochs. Two sample t-test: females (ChETA vs control), P = 0.0016. m, For the dHP, difference between intercepts during optogenetic stimulation and baseline epochs. Two-sample t-test: females (ChETA vs control), P = 0.0278. n, Same as l , for vHP. Two sample t-test: females (ChETA vs control), P = 0.0251. o, Same as m , for vHP. Two sample t-test: females (ChETA vs control). P = 0.0228. All data are shown as mean ± s.e.m; * : P < 0.05, ** : P < 0.01; dHP: dorsal hippocampus; vHP: ventral hippocampus. For detailed statistical information, see Extended Data Table 1 . Moreover, theta frequency at intercept, a metric modulated by anxiolysis, was also affected by 5-HT vHP neuron activation in a sex-specific manner. In males, the change in intercept over time was similar between eYFP controls and ChETA groups in both dHP ( Fig. 5m ) and vHP ( Fig. 5o ). By contrast, photoactivation of 5-HT vHP neurons prevented the decrease in intercept observed in female eYFP mice, in both the dHP ( Fig. 5m ) and the vHP ( Fig. 5o ), suggesting a sex specific effect of increasing 5-HT vHP neuron activity on anxiety levels. Taken together, activation of this pathway in females attenuates the normal vHP and dHP activity changes associated with familiarization during exploration of a new environment. These findings show that 5-HT vHP neurons selectively influence theta oscillations properties underlying novelty processing and anxiety-related behavior in a sex-dependent fashion. Using double retrograde tracing we confirmed that, under our experimental conditions, a subset of 5-HT vHP neurons also innervate the dHP, likely contributing to the similar modulation of theta oscillations observed in both dHP and vHP during 5-HT vHP activation ( Extended Data Fig. 6 ). To control for the absence of altered locomotor behavior, we quantified movement across stimulation conditions in mice implanted with only optic fibers ( Extended Data Fig. 7a ). Importantly, optogenetically evoked changes in hippocampal theta occur independently of gross locomotor changes in females ( Extended Data Fig. 7b-h ), consistent with prior reports that DR, but not MnR, 5-HT neurons modulate rodent locomotion 7 , 63 , 91 , 92 . Altogether, these findings demonstrate that, in a novel and low-threat environment, 5-HT vHP neurons shape circuit-level signatures of novelty in a sex-dependent fashion, without prominent influence on locomotion. In addition, sex-specific changes in the intercept of the speed-theta frequency relationship suggest that increased 5-HT vHP neurons activity modulates anxiety levels predominantly in females. Download figure Open in new tab Extended Data Fig. 6: Subpopulation of 5-HT vHP neurons collateralizes to dHP. a, Experimental strategy for dual retrograde labeling of 5-HT vHP neurons projecting to both the dHP and the vHP. b, Representative image of CTB-A647 (magenta) in the dHP. c, Representative image of triple-labeled 5-HT neurons (Tph2+, red), expressing eYFP (eYFP+, green) and CTB-A647 (CTB+, magenta) in the MRR. Scale bar = 100 μm. d, Proportion of double-labeled 5-HT vHP neurons across raphe subregions for females (teal, n = 4) and males (orange, n = 3): 13.39% in B9, 32.83% in the MRR and ≥ 35.71% in the DR. Kruskall-Wallis ANOVA, with Dunn’s post-hoc testing. B9 vs MRR: P = 0.0258, B9 vs DRI: P = 0.0017. Data are shown as mean ± s.e.m. * : P < 0.05, ** : P < 0.01. CTB: Cholera Toxin Subunit B; dHP: dorsal hippocampus; DRI: dorsal raphe, interfascicular subregion; eYFP: enhanced yellow fluorescent protein; MRR: median raphe region; For detailed statistical information, see Extended Data Table 1 . Download figure Open in new tab Extended Data Fig. 7: Optogenetic activation of 5-HT vHP neurons does not consistently alter locomotion. a, Difference between the distance travelled during the 4 min of optogenetic stimulation (ON) and the first 4 min of baseline (pre) periods in the open field for each group. No difference was observed for females while ChETA-expressing males showed a modest reduction in distance traveled. Three-way ANOVA with Holms multiple comparison testing: males (ChETA vs control), P = 0.0452). b, Same as a , but for mean velocity, no difference was observed for either sex. c, Same as a , but for time spent immobile. Here again, no difference was observed for either sex. d, Instantaneous velocity over time, around light stimulation onset (time = 0) for female eYFP (gray) and ChETA (teal) mice in the open field. e, Velocity averaged by 10 s bouts, around light stimulation onset (time = 0) for female eYFP (gray) and ChETA (teal) mice in the open field. Time-binned analyses of average velocity confirmed the absence of prominent stimulation effects, aside from a transient (10 s) decrease in average velocity. Two-sample t-test, eYFP vs control, 0 - 10 s, P = 0.0461. f, Same as d , but for male eYFP (gray) and ChETA (orange) mice. g, Same as e , but for male eYFP (gray) and ChETA (orange) mice. No change in velocity was observed in males. For a-c data are from two independent experiments and were analyzed by three-way ANOVA. For detailed statistical information and for the numbers of female and male mice used, see Extended Data Table 1 . For an overview of all behavioral variables analyzed, see Extended Data Table 2 . Discussion Mood and anxiety disorders disproportionately affect women, yet the neural substrates driving this sex bias remain poorly defined. Here, we identify serotonergic projections from the MRR to the vHP as a key neural substrate underlying sexually divergent anxiety responses. By integrating viral circuit tracing, retrograde fiber photometry, in vivo local field potential recordings, pathway specific optogenetics and patch-clamp electrophysiology, we demonstrate that this circuit displays robust sex differences that converge across multiple levels of analysis. Female MRR 5-HT vHP neurons display increased intrinsic excitability, greater functional recruitment and a causal role in promoting anxiety-like responses. These findings offer a mechanistic framework for understanding how serotonergic circuits can differentially shape anxiety-related behaviors in females and contribute to sex-specific risk for mood disorders. Consistent with previous work 13 , 73 , 93 , 94 , we find that 5-HT vHP neurons are distributed over multiple raphe subregions, notably the B9, MRR and DRI, with no differences in total number of 5-HT or 5-HT vHP neurons between sexes. Our in vitro electrophysiological recordings revealed that MRR 5-HT vHP neurons are intrinsically more excitable in females, exhibiting increased spontaneous activity, more depolarized resting membrane potentials and higher firing rates compared to males. DRI 5-HT vHP neurons in females also show depolarized membrane potentials, suggesting that intrinsic excitability may be an emergent property of subsets of serotonergic populations with shared developmental origins, connectivity and functions 11 , 95 , 96 . A few studies also report sex differences in the excitability of DR 5-HT neurons, but without specifying localization within DRI or projection targets 59 , 60 . In contrast, 5-HT vHP neurons from the B9 group showed no sex differences in intrinsic properties. Together, these findings highlight striking electrophysiological heterogeneity in raphe-vHP circuits arising from B9, MRR and DRI. These raphe-vHP circuits might also show neurochemical diversity. The MRR and B9 contain 5-HT neuron populations of different embryonic origins (derived from rhombomeres 1, 2 and 3) 97 , 98 , that diverge into neurochemically distinct subtypes. Notably, the MnR contains serotonergic neurons co-expressing the vesicular glutamate transporter VGLUT3, which project to the vHP 98 – 101 , raising the possibility that a subset of the virus-transfected neurons in our study co-release glutamate and serotonin 52 , 102 , 103 . These diverse properties of raphe-vHP populations suggest that vHP functions are modulated by multiple specialized raphe circuits. Our data link the intrinsic excitability of female MRR serotonergic neurons to heightened functional activation in vivo . Consistent with increased firing rates in MRR 5-HT vHP neurons observed in slice electrophysiology, c-fos mapping revealed greater stimulation-induced recruitment of MRR 5-HT vHP neurons in female mice. In addition, in vivo calcium imaging revealed that MRR 5-HT vHP neurons are dynamically, and sex-specifically, engaged during transitions into and out of the more aversive compartments of the EPM. While 5-HT vHP activity declined upon retreat into closed arms in both sexes, this decline was attenuated in females and emerged only gradually over the session. This delayed disengagement suggests a sex-specific persistence of neuronal activation, potentially reflecting slower updating of internal safety predictions under environmental novelty or threat. Strikingly, 5-HT vHP neurons also displayed ramping activity in the seconds preceding exploration of the more aversive parts of the EPM, reminiscent of DR neuron ramping activity patterns observed during anticipation of reward or punishment 40 , 104 – 106 . Such progressive increases in DR 5-HT neuron activity over long timescales have recently been proposed to reflect a prospective coding for value 41 , a framework that possibly extends to MRR 5-HT neurons. Notably, MRR and DR 5-HT neurons respond in opposite directions to rewarding stimuli 46 , and also differ during locomotion: whereas DR neurons decrease their activity during locomotion bouts in the open field or avoidance tests 92 , our findings confirm that in the same settings, MRR activity increases. At the network level, targeted optogenetic activation of 5-HT vHP neurons selectively disrupted dHP and vHP theta dynamics in female mice. Stimulation flattened the theta frequency-running speed relationship, mirroring the pattern typically seen during novelty exposure and consistent with a failure to habituate to the environment 86 , 88 , 90 . In contrast, control animals and male ChETA mice exhibited the expected progressive slope increase over time, reflecting familiarization. These findings extend previous results showing serotonergic modulation of hippocampal theta 107 – 110 by demonstrating a sex-dependent effect. Given that, during anxiety states, theta synchrony facilitates long-range communication between the hippocampus and other regions, such as the PFC 28 , 31 , 111 , serotonergic modulation of vHP theta could maintain persistent vigilance. This interpretation is supported by our anatomical mapping which revealed collateral projections from 5-HT vHP neurons to the PFC, raising the possibility that their activation coordinates distributed circuits implicated in anxiety regulation. Behaviorally, optogenetic activation of 5-HT vHP neurons increased anxiety-like behavior selectively in females. In ethologically relevant tests, including the EPM and SA, female ChETA mice exhibited increased avoidance of anxiogenic zones, reduced risk-assessment behaviors such as head-dipping and stretch-attend postures, and increased stress-related grooming. Notably, grooming was selectively elevated in anxiogenic environments but unaffected in the open field, highlighting the context dependence of this circuit’s behavioral impact, in line with past work 45 . The observation that grooming episodes were associated with rapid decreases in calcium signal in both sexes supports the view of grooming as an adaptive displacement behavior 70 , 112 , 113 , which transiently suppresses serotonergic tone. Importantly, the behavioral effects of 5-HT vHP neuron activation occurred in the near absence of locomotor changes, supporting a specific role in anxiety modulation behavior rather than general arousal. While broad activation of the MnR has previously been shown to increase anxiety-like behavior 8 , 43 , 63 , our data refine this understanding by demonstrating that selective stimulation of the vHP projection pathway is sufficient to amplify anxiety-related behaviors in females. Although our c-fos data show robust activation of 5-HT neurons in both sexes, stimulation did not alter anxiety-related behaviors in males. This dissociation suggests that, in males, the vHP-projecting serotonergic pathway may primarily contribute to behavioral domains not captured by the behavioral paradigms used here. The MnR provides dense serotonergic innervation to a wide array of forebrain regions 73 , and individual 5-HT neurons collateralize extensively 93 , 94 . Although 5-HT vHP neurons also collateralize to other forebrain regions, including the dHP and lateral septum, which could contribute to their behavioral effects, we found no sex differences in collateral density or distribution, suggesting that differential downstream targeting is unlikely to account for the sex-specific responses. The postsynaptic mechanisms mediating serotonergic modulation of vHP outputs remain unknown. Given that virtually all 5-HT receptor subtypes are expressed in the vHP 114 , serotonergic input to this region could act on multiple parallel output pathways, including to anxiety-related regions such as prefrontal cortex, lateral hypothalamic area or lateral septum 25 , 115 – 118 . The details of 5-HT modulation of vHP long-range principal cells or interneurons, and their impact on sex specific anxiety responses await clarification. Overall, our findings advance an emerging body of work delineating the roles of topographically organized serotonergic circuits in the modulation of emotion-related behaviors 18 , 46 , 119 , 120 . Our study directly links hyperexcitability of the female MRR 5-HT vHP pathway to enhanced anxiety-like responses. These findings align with the higher prevalence of anxiety and mood disorders in women 121 – 123 , and suggests that sex-specific serotonergic circuit function may constitute a core vulnerability factor across affective disorders. By delineating a projection-specific serotonergic pathway that differentially modulates anxiety-related behaviors in females, our work highlights the potential for sex-informed, circuit-targeted interventions in mood and anxiety disorders. Funding S.v.d.V. was supported by a CIHR postdoctoral fellowship and a Fonds de Recherche du Quebec - Santé (FRQS) postdoctoral fellowship. J.F.H. was supported by a Canada doctoral graduate scholarship from the Natural Sciences and Engineering Research Council of Canada (NSERC) and by a complement scholarship from the Fonds de recherche du Québec (FRQ). A.-S.S. was supported by a recruitment grant from the Department of Neuroscience at the Université de Montréal. This work was funded by the Canadian Institutes for Health Research (CIHR) #173244, NARSAD Young Investigator Grant #25287, the Réseau Québécois sur le Suicide et les troubles de l’Humeur Associés, the Fonds de Recherche du Québec – Santé and the Canadian Foundation for Innovation to B.A. This project has been made possible by the Canada Brain Research Fund (CBRF), an innovative arrangement between the Government of Canada (through Health Canada) and Brain Canada Foundation, and the Azrieli Foundation. The funders had no role in study design, data collection or analysis. Contributions S.v.d.V. performed LFP experiments. F.H. performed fiber photometry and double-projection experiments. L.P. performed patch-clamp electrophysiology experiments. A.-S.S. and F.P. performed optogenetic experiments. J.F.H. and A.-S.S. collected collateral dataset. A.-S.S., F.P. and F.H. performed c-fos experiments. S.v.d.V. and G.D. developed analysis pipelines and code. G.D. provided technical support for all experiments and statistical analysis. All authors analyzed data, performed statistical analysis and provided material for the manuscript. S.v.d.V. and B.A. prepared figures and wrote the manuscript with contribution and input from all authors. B.A. conceived and supervised all aspects of the project, and secured funding. L.P. and F.H. contributed equally to this work. Ethics declarations Competing interests: The authors declare no competing interests. Materials and methods Animals Male or female SERT-Cre (B6.FVB(Cg)-Tg(Slc6a4-cre)ET33Gsat/Mmucd line, MMR,#031028-UCD;) transgenic mice were bred with female or male C57BL/6J mice (Jackson Laboratory, #000664) to generate experimental animals. All mice used in this study were between 60-90 days old at the time of experiments. Animals were group-housed on a 12-hour light/dark cycle at 24°C and 40% humidity with ad libitum access to food and water. All procedures involving animals were approved by the Comité Institutionnel des Bonnes Pratiques Animales en Recherche (CIBPAR) of the Centre de Recherche Azrieli du CHU Sainte-Justine (protocol 2023-5039), in accordance with the standards of the Canadian Council on Animal Care. Stereotaxic surgeries Animals were anesthetized with isoflurane (5% induction, 0.5-1.5% maintenance) and placed in a stereotaxic frame (David Kopf instruments). Carprofen (2 mg/kg) was administered subcutaneously before surgery. Body temperature was maintained at 37°C with a heating pad (Stoelting) and eyes were hydrated with ocular gel (Optixcare, Aventix). Craniotomies were performed using a mountable drill (Stoelting). Viral injections were performed using a glass pipette backfilled with mineral oil connected to a Nanoject III (Drummond Scientific) at a flow rate of 1 nL/second. To target 5-HT vHP neurons, a retrogradely transported adeno-associated virus (AAV) of serotype 2/9 was bilaterally infused in the vHP. Each hippocampus received 400 nL of viral solution injected at two sites (stereotaxic coordinates: [AP] −3.52, [ML] ±3.25, [DV] −4.17 and −3.47). All stereotaxic coordinates are taken from bregma (in mm). For electrode and optic fiber implantation surgeries, the skull was exposed and carefully cleaned with ethanol before craniotomies were drilled. Electrodes and/or optic fiber were then slowly lowered into position and maintained in place using a combination of C&B-Metabond and dental acrylic (Parkell) and 2-3 stainless-steel anchor screws (Antrin Miniature Specialties Inc). Electrodes and optic fiber stereotaxic coordinates are described in the corresponding sections below. Following surgery, mice were monitored for at least 3 days and allowed to recover for at least one week before subsequent procedures or behavioral testing. Fiber photometry recordings SERT-Cre mice received bilateral injections of AAV2/9-Syn-Flex-GCaMP6s-WPRE-SV40 (CS1028, University of Pennsylvania Vector Core) targeting the vHP using the methodology described above, resulting in retrograde expression of the calcium indicator GCaMP6s in 5-HT vHP neurons. Four weeks later, animals were implanted with a 200 µm low-autofluorescence optic fiber cannula (Neurophotometrics) targeting GCaMP6s transfected neurons in the MRR ([AP] – 4.20 to – 5.30 with a 10° rostro-caudal angle, [ML] 0.00, [DV] – 3.5 to – 4.45). Following implantation and recovery, mice were habituated for at least one week. They were handled daily, connected to sham patchcords in individual home cages containing familiar bedding and nesting material, and then returned to their littermates before the dark cycle. Behavioral testing was conducted on separate days in either a in a 40 x 40 cm light grey square open field (40 x 40 cm light grey square) or an EPM consisting of two closed arms (30 x 5 x 19.8 cm) and two open arms (30 x 5 x 0.9 cm), and a 5 x 5 cm center zone. On each testing day, mice were first habituated for 1 hour in individual cages in the experimental room. After habituation, mice were connected to the patchcord and returned to the cage for 20 minutes during which photometry recording was initiated. Animals were then placed in the behavioral apparatus for 20 minutes of free exploration. Excitation light (470 nm and 415 nm) was alternately pulsed at 10 Hz at ∼80 µW (measured at the fiber tip) using an FP3002 photometry console (Neurophotometrics). Arenas were cleaned with water and 70% ethanol between animals. Room illumination was maintained at 70 ± 10 lux. Fluorescence was recorded sequentially for both the 470 nm calcium-dependent signal and the 415 nm isosbestic control. Injection sites, viral expression, and fiber placements were verified post hoc by histology, as described below. Bleaching and movement artifacts were corrected using the method previously described 124 . Briefly, we used the coefficient obtained from a linear fit (obtained from iteratively reweighted least squares regression) of the 415 nm signal to the 470 nm signal, to scale the 415 nm signal. The scaled 415 nm signal was subtracted from the 470 nm signal to obtain ΔF, and ΔF was divided by the scaled 415 nm signal to yield ΔF/F. Signals were z-scored across the recording session and low-pass filtered at 3 Hz prior to further analysis. Peri-event plots were generated for each mouse by averaging z-scored ΔF/F around behavioral events and subtracting the baseline (average signal 1-2 s prior to the event). Within-mouse averages were then averaged across animals to obtain group means. Mouse body parts were tracked using SLEAP 125 , and the center of the body was used for all subsequent analyses. Animal positions were filtered to reduce jitter: the animal position at a specific frame was updated only if the raw animal position for that frame was located further than ½ of the mouse length away from the previous position. Movement initiation was defined as the first frame where velocity exceeded 1 cm/s, provided the mouse moved 10 cm in the subsequent second. For the transitions between closed and open arms in the EPM ( Fig. 1d-g ), closed→open transitions were defined as the mouse spending at least 10 s in a closed arm before entering center/open arms for ≥ 2 s. Closed→closed transitions were defined as transitions between closed arms with ≤ 1 s spent in the center. Changes in fluorescence were aligned to the timepoint at which center-of-body crossed the closed arm-center boundary. For time-locked analysis of closed-arm entries and exits ( Fig. 1h-s ), arm entries were included in the analysis if the mouse spent ≥ 2 s in the center/open arm before entering the closed arm, and ≥ 2 s in the closed arm after entry. Exit transitions were defined similarly, requiring at least 2 s in the closed arm before transition and at least 2 s in the center or open arm after exit. Grooming onset and offset were manually scored frame-by-frame using custom Matlab code. Local field potential recordings SERT-Cre mice received bilateral vHP injection of either AAV2/9-EF1a-DIO-hChR2(E123T/T159C)-eYFP (#35509-AAV9, Addgene), to cause CRE-dependent expression of the excitatory opsin ChETA in vHP projecting 5-HT neurons, or AA2/9-EF1a-DIO-eYFP (#27056-AAV9, Addgene) for control mice, using the methodology described above. Three weeks after virus injection, animals were implanted with a 200 μm optic fiber (#FG200UEA, Thorlabs) above the MRR ([AP] −4.35, [ML] −0.55 at 7° angle, [DV] −3.23), and custom recording electrodes in the hippocampus. For local field potential (LFP) recordings, custom bipolar electrodes were constructed using 50 µm PFA coated tungsten wire (#585088, A-M Systems) and targeted to the ventral ([AP] −3.65, [ML] 3.50, [DV] −3.00) and dorsal hippocampus ([AP]: −1.58, [ML] 1.40, [DV] −1.50). A ground screw was placed over the cerebellum and an EMG electrode was implanted in the trapezius muscle. All electrode and fiber placements were confirmed by histology, as described below. LFP were recorded and digitized at 20 KHz using an Open Ephys system 126 , while mice explored a novel, circular open field (39 cm diameter). Videos were processed with DeepLabCut 127 , 128 (version 2.3rc3) to extract body positions, using the body center as the animal’s position. LFP recordings were down sampled to 1 kHz and aligned with the behavioral tracking data. LFPs were band-pass filtered (5-10 Hz) and subjected to a Hilbert transform to obtain the analytic signal. The power of the signal was taken as the magnitude of the analytic signal squared. The instantaneous phase of the analytic signal was extracted and unwrapped to provide an estimation of instantaneous theta frequency. Periods with power >6 SD were considered movement artefacts and were removed from analysis along with the flacking 250ms of data on each side. Animal positions were filtered to reduce jitter as described in the fiberphotometry section. Running speed was computed from the filtered positions by dividing Δd/Δt where d is the distance and t is time. Finally, to characterize the relationship between theta frequency and running speed, the median of the instantaneous frequency was taken over a time window of approximately 33.3ms to yield a frequency estimate corresponding to each frame of the video (sampled at 30 Hz). A first order polynomial was fitted to theta frequency and animal speed between 1 and 30 cm/s to extract the slope and intercept of the speed – theta frequency relationship. Optogenetic manipulations and behavior 5-HT vHP neurons of SERT-Cre mice were transfected with either the excitatory opsin ChETA or eYFP, and an optic fiber was implanted as described above. Mice were allowed a 2-week recovery period post-implantation, followed by 5 days of handling (∼10min/day) to minimize stress related to the experimenter, the behavior room or the fiber optic connection on the day of the experiment. On testing days, mice were transferred to the experimental room ≥ 45 min before behavioral assays and placed in individual cages containing familiar bedding and nesting material. All experiments were conducted between 9 AM and 2 PM to reduce circadian variability. Behavioral testing occurred in the following order with 48 h intervals between tests: open field, EPM, and SA. Arenas were cleaned with 70% ethanol between mice to reduce olfactory cues, and light sensitivity was maintained at 70 ± 5 lux at the center of each apparatus. Mice were allowed to freely explore the maze for 12 min, with optogenetic stimulation delivered during minutes 4-8. Stimulation consisted of blue light (450 nm) pulses at 20 Hz (10 ms pulse width) at ∼15 mW measured at the fiber tip. Viral expression and fiber placement were confirmed post hoc (see Histology). Mouse body parts were extracted from each video using SLEAP 125 , using the center of the body as the animal’s position. From this raw animal position, the time spent and the number of entries in each zone of the different mazes could be computed. Entries into a zone were recorded if the raw animal position was within the zone for at least 1s. To compute the distance traveled and animal velocity, we filtered the raw animal position as described in the previous section. From this filtered position, the distance traveled was computed as the sum of the frame-to-frame displacement, and velocity was obtained by multiplying frame-by-frame displacements by the video sampling frequency. The time immobile was taken as the total time where the animal was not locomoting and the mean velocity while moving was calculated from all periods of locomotion. For the successive alleys an anxiety index was calculated as: Where T z1 - T z4 are time spent in zones 1-4 and T total is total test duration. Values near 1 indicate that mice spent most of their time in less aversive zones (zone 1). Values near 0.5 reflect uniform exploration across zones. These scores can also be interpreted as the normalized mean position of the mouse along the alleys from zone 1 to zone 4. Grooming, rearing, stretching and head-dipping behaviors were manually scored by the same experimenter blind to treatment (eYFP or ChETA). Histology For all immunohistochemistry experiments, mice were deeply anesthetized with an intraperitoneal injection of ketamine/xylazine/acepromazine (80/12.5/2.5 mg/kg, respectively). Animals were transcardially perfused with 45 mL of 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS), and brains were extracted and postfixed in PFA at 4°C for 24-48 h. For cryosectioning, brains were cryoprotected using 15% sucrose in PBS for 24 h, embedded in Tissue-Plus™ O.C.T. Compound (#23-730-571, Fisher Scientific), and frozen in isopentane. Standard immunohistochemistry was performed using antibody solutions prepared in PBS with 0.45% gelatin and 0.25% triton. Image processing and analysis were performed using FIJI software (ImageJ, version 2.14.0). When necessary, Tph2 immunolabelling was used to delineate the raphe nuclei. Histological validation of viral expression, electrode and fiber placement For anatomical validation of viral expression and optical fiber implantation sites, frozen coronal sections (25 μm) were prepared and cut on a cryostat (Leica Biosystems CM3050 S), and stained for eYFP or GCaMP6s and Tph2 as described above, using the following primary and secondary antibodies: anti-Tph2 in guinea pig (1:1000; #TPH2-GP-Af900, Frontier Institute), anti-GFP in chicken (1:2000; #A10262, Invitrogen), anti-chicken A488 in donkey (1:2000; #703-545-155, Jackson ImmunoResearch) and anti-guinea pig A647 in goat (1:2000; #A21450, ThermoFisher). Sections were mounted on slides using Fluoromount-G containing DAPI (ThermoFisher). Images were acquired using a Leica DMi8 wide-field inverted microscope with 10X and 20X objectives, equipped with a Leica DFC9000 camera, and LAS X software (Leica). For electrophysiology experiments, fixed (non-frozen) brains were sectioned at 40 μm using a vibratome (Leica VT1200) and collected sequentially. One-quarter of the sections were stained for eYFP and Tph2, as described above, to assess viral expression and optical fiber placement, while the remaining three quarters were directly mounted to assess HP electrode placement. These sections were imaged using the Zeiss AxioScan.Z1 with 10X objective and ZEN Microscopy Software (Zeiss). Collaterals To assess the proportion of 5-HT neurons projecting to both the dHP and vHP ( Extended Data Fig. 4 ), adult SERT-Cre males (n = 3) and females (n = 4) were injected as described above with AAV2/9-EF1a-DIO-hChR2(E123T/T159C)-eYFP (#35509-AAV9, Addgene) in the left vHP (2 injections of 400 nL of virus). Five weeks later, neurons projecting to the left dHP were retrogradely labeled with CTB-A647, using 100 µg of CTB-A647 previously suspended in 20 µl of PBS1x at 4 °C and short-term stored at 4 °C. dHP injections were performed at: [AP] −1.90 and −2.40, [ML] −1.35 and −2.00, [DV] −1.55 and −1.65. Mice were sacrificed 10 days post CTB injection. Coronal sections were cut (35 µm) on a vibratome (Leica VT1200) and labeled for eYFP and Tph2 using the following primary and secondary antibodies: anti-GFP in goat (1:5000; #NB100-1770, Novus Biologicals), anti-Tph2 in rabbit (1:2000; #NB100-74555, Novus Biological), anti-goat-A488 in donkey (1:2000; #A11055, Life Technologies) and anti-rabbit-A555 in donkey (1:2000; #A31572, Life Technologies). The distribution of HP-projecting 5-HT neurons was quantified on sections collected at 105 µm intervals along the rostro-caudal axis of the raphe nuclei (B5-B9). For qualitative assessment of 5-HT vHP collaterals ( Extended Data Fig. 7 ), a separate group of SERT-Cre males (n = 4) and females (n = 4) was used. 5-HT vHP neurons were transfected using bilateral injections as described above. Free-floating coronal and horizontal slices (25 μm) were cut on a cryostat (Leica Biosystems CM3050 S) and labeled for eYFP and 5-HT using the following primary and secondary antibodies: anti-GFP in chicken (1:2000; #A10262, Invitrogen), anti-5-HT in goat (1:2000; #ab66047, Abcam), anti-chicken-A488 in donkey (1:2000; #703-545-155, Jackson ImmunoResearch) and anti-goat-A555 in donkey (1:2000; #A21432, Thermo Fisher Scientific). All regions with collaterals were identified using anatomical landmarks and a mouse brain atlas 129 as reference. All images were acquired using a Leica DMi8 wide-field inverted microscope as described above. C-fos assessment of neural activity C-fos expression was assessed in mice expressing ChETA or eYFP in 5-HT vHP neurons. Mice were sacrificed 70 minutes after completing 12 minutes in the SA paradigm with optogenetic stimulation performed as described above. Fixed, non-frozen brains were cut into 40 µm sections using a vibratome (Leica VT1200) and triple-labeled for c-fos, eYFP and Tph2 using the same protocol, with the addition of anti-c-fos in rabbit (1:2000; #2250, Cell Signalling) and anti-rabbit –A555 in donkey (1:2000; #A31572, Life Technologies). The distribution of 5-HT+/c-fos+/eYFP+ neurons was quantified on sections collected at 120 µm intervals along the rostro-caudal axis of the raphe nuclei. Patch-clamp Mice were deeply anesthetized with isoflurane and brains were prepared for sectioning on a vibratome using a modified protocol from Ting and colleagues 130 . Each mouse was transcardially perfused with an NMDG-based solution (in mM: 93 NMDG, 93 HCl, 2.5 KCl, 1.2 NaH2PO4, 30 NaHCO3, 20 HEPES, 25 glucose, 5 sodium ascorbate, 2 thiourea, 3 sodium pyruvate, 10 MgSO4, and 0.5 CaCl2 at pH 7.4, oxygenated with carbogen). Coronal sections (300 μm) were cut using a vibratome (Leica VT1200) and incubated at 32°C for 10 to 12 minutes in the same NMDG solution. Slices were then transferred to artificial cerebrospinal fluid (aCSF) at room temperature (in mM: 124 NaCl, 24 NaHCO3, 2.5 KCl, 1.2 NaH2PO4, 5 HEPES, 12.5 glucose, 2 MgSO4, and 2 CaCl2, pH 7.4, oxygenated with carbogen). During recordings, slices were continuously perfused with aCSF at 28°C at 3 mL/min. 5-HT vHP neurons were visually identified via eYFP fluorescence. Whole-seal recordings were obtained using an intrapipette solution (in mM: 126 K-gluconate, 4 KCl, 10 HEPES, 4 Mg2ATP, 0.3 Na2GTP, and 10 PO-Creatine, adjusted to pH 7.25 with KOH; 272 mOsm). Pipette had a resistance between 4 to 8 MΩ. Recordings were performed using an Axon Multiclamp 700B amplifier (Molecular Devices). After achieving whole-cell configuration, resting membrane potential was recorded for 4 min prior to additional protocols. In current-clamp, 500 ms hyperpolarizing steps were applied to assess hyperpolarization-activated current, passive membrane conductance and input resistance. 500 ms depolarizing current steps were applied to assess firing frequency, rheobase and action potential features. The adaptation index was calculated using the last depolarizing current step as: where ISI i and ISI l are the initial and last interspike interval (ISI), respectively. All membrane potentials were corrected offline for a liquid junction potential (LJP) of −15.8mV. Statistics All data are presented as mean ± standard error of the mean (s.e.m.), and the corresponding statistical tests are described in the figure legends and in Extended Data Table 1 , and Extended Data Table 2 . Origin 2024 (OriginLab) was used for all basic statistical analysis, except for behavioral experiments described in Figure 4 and Figure 5 , which were analyzed with R (version 4.4.1 in RStudio 2025.05.0). Behavioral experiments were conducted on two independent cohorts, and therefore analyzed with three-way ANOVA with sex, treatment and cohort as factors. The ANOVA model included all main effects (sex, treatment and cohort) and interactions (sex * treatment, sex * cohort, treatment * cohort and sex * treatment * cohort). Post hoc tests were performed for planned comparisons (female-eYFP vs. male-eYFP, female-eYFP vs. female-ChR2, male-eYFP vs. male-ChR2 and female-ChR2 vs. male-ChR2) using Holm’s correction for multiple testing. Statistical tests were performed in R using the aov function (stats package) for the three-way ANOVA and the emmeans function ( emmeans package) for post hoc comparisons. Homogeneity of variance was tested using Levene’s test (leveneTest function, car package) using median 131 . For clarity, only the plots of the marginal means are shown in the main text, while the full group breakdown is shown in Extended Data Table 1 . A threshold of P < 0.05 was considered statistically significant. Figures use the following significance notation: *, P < 0.05; **, P < 0.01; ***, P < 0.001. Acknowledgements We thank Dr. Elke Küster-Schöck from the Centre Hospitalier Universitaire Sainte-Justine Imaging center for microscopy guidance, and Denise Carrier, Véronique Pelletier and the animal facility team for help with colony maintenance. We thank all members of the Amilhon lab for their valuable comments on the manuscript, and undergraduate students Clémence Brin, Camille Pons, Charlène Manti, Morgane Roger for their help with various aspects of the study. Funder Information Declared Canadian Institutes of Health Research, https://ror.org/01gavpb45 , #173244 NARSAD Young Investigator Grant , #25287 Réseau Québécois sur le Suicide et les troubles de l’Humeur Associés Brain Canada Future Leaders program Fonds de Recherche du Québec - Santé Canadian Foundation for Innovation Footnotes Reorganization of the results section and corresponding figures. References 1. ↵ Bangasser , D. A. & Cuarenta , A . Sex differences in anxiety and depression: circuits and mechanisms . Nat Rev Neurosci 22 , 674 – 684 ( 2021 ). OpenUrl CrossRef PubMed 2. ↵ McLean , C. P. , Asnaani , A. , Litz , B. T. & Hofmann , S. G . Gender differences in anxiety disorders: Prevalence, course of illness, comorbidity and burden of illness . 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Share Hyperexcitability of female serotonin neurons underlies sex-specific anxiety responses Suzanne van der Veldt , Fiona Henderson , Lucia Pizzoccaro , Anne-Sophie Simard , Félix Perreault , Justine Fortin-Houde , Guillaume Ducharme , Bénédicte Amilhon bioRxiv 2025.08.17.670715; doi: https://doi.org/10.1101/2025.08.17.670715 Share This Article: Copy Citation Tools Hyperexcitability of female serotonin neurons underlies sex-specific anxiety responses Suzanne van der Veldt , Fiona Henderson , Lucia Pizzoccaro , Anne-Sophie Simard , Félix Perreault , Justine Fortin-Houde , Guillaume Ducharme , Bénédicte Amilhon bioRxiv 2025.08.17.670715; doi: https://doi.org/10.1101/2025.08.17.670715 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Neuroscience Subject Areas All Articles Animal Behavior and Cognition (7629) Biochemistry (17660) Bioengineering (13881) Bioinformatics (41913) Biophysics (21436) Cancer Biology (18578) Cell Biology (25482) Clinical Trials (138) Developmental Biology (13372) Ecology (19889) Epidemiology (2067) Evolutionary Biology (24302) Genetics (15599) Genomics (22483) Immunology (17728) Microbiology (40365) Molecular Biology (17163) Neuroscience (88540) Paleontology (666) Pathology (2830) Pharmacology and Toxicology (4821) Physiology (7637) Plant Biology (15136) Scientific Communication and Education (2045) Synthetic Biology (4290) Systems Biology (9818) Zoology (2269)