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Serotonergic modulation of the BNST–CeA pathway reveals sex differences in fear learning

Nature Neuroscience volume 28, pages 1897–1909 (2025)Cite this article

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Abstract

Post-traumatic stress disorder (PTSD) is characterized by intense fear memory formation and is diagnosed more often in women than men. Here we show that increasing serotonin pharmacologically before auditory fear conditioning promoted memory recall in female and male mice, and that females were more sensitive to this effect. Optogenetic stimulation of raphe terminals in the anterior dorsal bed nucleus of the stria terminalis (adBNST) during fear conditioning increased c-Fos expression in the BNST and central nucleus of the amygdala (CeA) and enhanced fear memory recall through activation of 5-HT2C receptors in the adBNST in females only. Likewise, serotonin stimulation during fear learning enhanced adBNST–CeA high gamma (90–140 Hz) synchrony and adBNST-to-CeA communication in high gamma during fear memory recall in females only. These findings suggest that sex differences in the raphe–BNST–CeA pathway may contribute to the higher risk of PTSD in women.

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Fig. 1: Acute systemic administration of the SSRI citalopram before auditory fear conditioning has a stronger effect on tone recall in females than males.
Fig. 2: Acute systemic administration of citalopram before auditory fear conditioning increases c-Fos expression in the adBNST and CeA of females only.
Fig. 3: Optogenetic stimulation of 5-HT terminals in the adBNST during auditory fear conditioning enhances tone recall in females only.
Fig. 4: Optogenetic stimulation of 5-HT terminals in the adBNST during auditory fear conditioning increases c-Fos expression in the adBNST and CeA of females only.
Fig. 5: Females express more Htr2c transcript than males in the adBNST.
Fig. 6: Blocking 5-HT2CRs in the adBNST prevents 5-HT-induced enhancement of tone recall in females.
Fig. 7: Serotonin increases high gamma synchrony in the adBNST–CeA circuit of female mice during retrieval.
Fig. 8: Serotonin-mediated increases in tone recall in females require output from the adBNST to the CeA.

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Data availability

With the exception of electrophysiology recordings, all raw data are available for download. Awake-behaving electrophysiology data and all videos of behavioral testing are available upon request. Source data are provided with this paper.

Code availability

The code related to this paper can be found at https://github.com/ELikhtik/Continuous/tree/HighLowGammaBNSTCeA.

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Acknowledgements

We dedicate this paper to the memory of Dr Carolina Fernandes-Henriques, a talented scientist and wonderful friend. She is greatly missed. We thank members of the animal care staff at Hunter College, particularly B. Wolin and S. Acevedo. We thank S. Hanif for assistance with tissue processing. We would also like to acknowledge G. Schafe, A. Garcia-Garcia and C. A. Denny for their helpful comments and suggestions. This project was supported by NIMH R21MH114182 (N.S.B. and E.L.), NIMH R21MH135430 (N.S.B and E.L.), National Institute on Minority Health and Health Disparities G12MD007599 (N.S.B.), NIMH R01MH118441 (E.L.) and PSC-CUNY Awards (N.S.B. and E.L.).

Author information

Author notes

  1. Deceased: Carolina Fernandes-Henriques.

  2. These authors contributed equally: Jinah Lee, Carolina Fernandes-Henriques.

  3. These authors jointly supervised this work: Ekaterina Likhtik, Nesha S. Burghardt.

Authors and Affiliations

  1. Division of Systems Neuroscience, Research Foundation for Mental Hygiene, Inc., New York State Psychiatric Institute, New York, NY, USA

    Rebecca Ravenelle

  2. Department of Psychiatry, Columbia University Irving Medical Center, New York, NY, USA

    Rebecca Ravenelle

  3. National Institute of Mental Health, Late-Life Neuropsychiatric Research Training Program, Columbia University Irving Medical Center, New York, NY, USA

    Rebecca Ravenelle

  4. Psychology Program, The Graduate Center, City University of New York, New York, NY, USA

    Jinah Lee & Nesha S. Burghardt

  5. Department of Psychology, Hunter College, City University of New York, New York, NY, USA

    Jinah Lee & Nesha S. Burghardt

  6. Biology Program, The Graduate Center, City University of New York, New York, NY, USA

    Carolina Fernandes-Henriques, Allyson K. Friedman & Ekaterina Likhtik

  7. Department of Biological Sciences, Hunter College, City University of New York, New York, NY, USA

    Carolina Fernandes-Henriques, Allyson K. Friedman & Ekaterina Likhtik

  8. Neuroscience Initiative, Advanced Science Research Center, The Graduate Center, City University of New York, New York, NY, USA

    Jia Liu

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  3. Carolina Fernandes-Henriques
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  4. Jia Liu
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  6. Ekaterina Likhtik
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Contributions

R.R. performed the SSRI, 5-HT2C antagonist and anterograde chemogenetic experiments. R.R. and J. Lee performed the optogenetic, in vivo electrophysiology, in vivo microdialysis and retrograde chemogenetic experiments. J. Liu performed the RT–qPCR experiments. A.K.F. performed the whole-cell patch-clamp experiments. R.R., C.F.-H., E.L. and N.S.B. analyzed the data. R.R. and N.S.B. designed the experiments. E.L. and N.S.B. supervised the study. R.R., E.L. and N.S.B. wrote the manuscript with help from other authors.

Corresponding authors

Correspondence to Ekaterina Likhtik or Nesha S. Burghardt.

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Nature Neuroscience thanks Rebecca Shansky and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Stage of estrous cycle does not influence fear conditioning.

(a) Schematic of procedures indicating when vaginal swabs were taken each day. (b) Representative images of vaginal cytology of TpH2-ChR2-YFP BAC female mice. Estradiol status on the day of auditory fear conditioning did not affect (c) acquisition (two-way RM ANOVA, group, F(1,25) = 0.14, p = 0.71), (d) tone recall (two-way RM ANOVA, group, F(1,25) = 0.04, p = 0.84) or (e) recall of contextual fear memory (two-tailed unpaired t test, t(24) = 0.44, p = 0.66). n = 15 low estradiol; n = 12 high estradiol. (f) Estradiol status on the day of the tone test did not affect tone recall (two-way RM ANOVA, group, F(1,25) = 0.69, p = 0.41). n = 14 low estradiol; n = 13 high estradiol. (g) Estradiol status on the day animals were placed back in the training context (Context A) did not affect recall of contextual fear memory (two-tailed unpaired t test, t(24) = 1.56, p = 0.13). n = 13 low estradiol; n = 13 high estradiol. Data are represented as mean ± SEM. RM ANOVA = repeated measures ANOVA.

Source Data

Extended Data Fig. 2 Generalization of fear to the testing context is short lasting in saline-treated males.

(a) In males, acute systemic administration of citalopram prior to auditory fear conditioning reduced pre-tone freezing in a different context the next day (one-way ANOVA, F(2,52) = 12.72, p < 0.0001, followed by Tukey’s post-hoc test). The same data are shown as baseline freezing in Fig. 1f. (b) Subtracting pre-tone freezing from tone-evoked freezing during tone-bin 1 reveals elevated freezing in citalopram treated groups compared to the saline group (one-way ANOVA, F(2,52) = 14.31, p < 0.0001, followed by Tukey’s post-hoc test). n = 14 per citalopram dose; n = 28 combined saline. (c) Schematic of behavioral procedures to determine if elevated pre-tone freezing is sustained throughout the recall session. The same two doses of citalopram or saline were injected in a new cohort of male mice before auditory fear conditioning. On the following two days, mice were tested in the testing context (Context B) without exposure to tones and the training context (Context A). (d) Acute citalopram treatment did not affect percentage of time spent freezing to the tone during conditioning (two-way RM ANOVA, group, F(2,22) = 0.90, p = 0.42). (e) The next day, drug-free mice were placed in the testing context (Context B) and freezing was measured when tones are usually presented. Saline-treated mice only froze more than citalopram-treated mice at baseline (b) and when the first two tones are usually presented (Time-Bin 1) (two-way RM ANOVA, time-bin x group, F(10,110) = 6.14, p < 0.0001, followed by Tukey’s post-hoc test). (f) Citalopram treatment did not affect recall of contextual fear memory (one-way ANOVA, F(2,22) = 0.39, p = 0.68). n = 9 saline; n = 7, 10 mg/kg; n = 9, 20 mg/kg. Data are represented as mean ± SEM. RM ANOVA = repeated measures ANOVA. ****p < 0.0001 vs. saline, ***p < 0.001 vs. saline, **p < 0.01 vs. saline, *p < 0.05 vs. saline.

Source Data

Extended Data Fig. 3 Serotonin input to the adBNST is similar in males and females.

(a) Representative image demonstrating overlap of TpH2-ChR2-EYFP neurons (green) with TpH2 immunoreactivity (far red) in the DRN and MRN, scale bar = 500 µm. (b) High magnification images of the DRN showing co-labeled cells, scale bar = 10 µm. (c) Alexa Fluor 647-conjugated cholera toxin subunit B (CTB) was injected into the right adBNST of TpH2-ChR2+ mice and expression was visualized in the raphe nuclei 10 days later (created in BioRender). Representative image from a female mouse showing TpH2-ChR2-EYFP+ neurons (green) and CTB+ neurons (far red) in the DRN and MRN. (d) High magnification of the DRN showing a co-labeled cell, scale bar = 25 µm. (e) Quantification of neurons co-labeled with TpH2-ChR2-EYFP and CTB in DRN (two-tailed unpaired t test, t(11)=0.10, p = 0.92). (f) Percent of EYFP+ cells co-labeled with CTB and EYFP in DRN (two-tailed unpaired t test, t(11)=0.73, p = 0.48). (g) Quantification of neurons co-labeled with TpH2-ChR2-EYFP and CTB in MRN (two-tailed unpaired t test, t(11)=0.51, p = 0.62). (h) Percent of EYFP+ cells co-labeled with CTB and EYFP in MRN (two-tailed unpaired t test, t(11)= 1.53, p = 0.16). n = 7 females; n = 6 males. Data are represented as mean ± SEM. DRN = dorsal raphe nucleus, MRN = median raphe nucleus.

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Extended Data Fig. 4 Generalization of fear to the testing context is short lasting in non-stimulated males.

(a) In males, optogenetic stimulation of 5-HT terminals in the adBNST during auditory fear conditioning decreased pre-tone freezing in the testing context the next day (two-tailed unpaired t test, t(41) = 3.63, p = 0.0008). n = 22 ChR2+ males; n = 21 ChR2- males. The same data are shown as baseline freezing in Fig. 3h. (b) Schematic of behavioral procedures (created in BioRender). A separate cohort of TpH2-ChR2- males received light stimulation during auditory fear conditioning but were not exposed to tones in Context B the next day. The following day they were tested in the training context (Context A). (c) During training, freezing to the tone increased with each tone-shock pairing (two-tailed paired t test, tone 2 vs. tone 5, t(11)=9.59, p < 0.0001). n = 12. (d) The next day, ChR2- males (No Tone Recall, n = 10) exhibited freezing in the testing context that reached levels comparable to ChR2+ males by time the tones are usually presented (two-tailed unpaired t test, t(24)=0.35, p = 0.73). Baseline freezing in the previous cohort of TpH2-ChR2- (white squares) and TpH2-ChR2+ males (blue squares) (Fig. 3h) is included for purposes of comparison. (e). Freezing to the training context (Context A) for mice that were not exposed to tones on the previous day (ChR2- No Tones, n = 10) and the previous cohort that was tone tested (ChR2-Tones, n = 21; ChR2+Tones, n = 22, Fig. 3i) (one-way analysis of variance, F(2,50) = 1.27, p = 0.29). Data are represented as mean ± SEM. ***p < 0.001.

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Extended Data Fig. 5 Optogenetic stimulation of serotonin terminals in the adBNST does not affect cell activity in the raphe nuclei.

(a) (Top) Serotonin terminals in the adBNST were optogenetically stimulated while mice were in their home cage (laser group). A control group was connected to the fiber optic cable without stimulation (no laser). Ninety minutes later, all mice were perfused and cell activity in the raphe nuclei was evaluated with c-Fos (created in BioRender). (Bottom) Representative image showing TpH2-ChR2-EYFP+ neurons (green) and c-Fos+ neurons (red) in the DRN and MRN, scale bar = 500 µm. (b) High magnification images of the DRN showing TpH2-ChR2-EYFP+ neurons (green), c-Fos+ neurons (red) and a co-labeled cell (white arrow), scale bar = 25 µm. (c) Quantification of c-Fos+ cells (two-tailed unpaired t test, t(9) = 1.37, p = 0.20) and (d) cells co-labeled with c-Fos and EYFP (two-tailed unpaired t test, t(9) = 0.38, p = 0.71) in the DRN. (e) Quantification of c-Fos+ cells (two-tailed unpaired t test, t(9) = 0.10, p = 0.93) and (f) cells co-labeled with c-Fos and EYFP (two-tailed unpaired t test, t(9) = 0.77, p = 0.46) in the MRN. n = 2 males/no laser; n = 2 males/laser; n = 3 females/no laser; n = 4 females/laser. Data are represented as mean ± SEM. DRN = dorsal raphe nucleus, MRN = median raphe nucleus.

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Extended Data Fig. 6 Effects of the 5-HT2CR antagonist on tone recall are temporary.

(a) Schematic of procedures (created in BioRender). The 5-HT2CR antagonist RS102221 was bilaterally infused into the adBNST of female TpH2-ChR2+ mice 10-15 minutes before fear conditioning, with blue laser stimulation during each tone presentation (Train 1). On the following two days, mice were tested to the tone (Tone Test 1) and training context (Context Test). Forty-eight hours later, a subset of these females received an infusion of aCSF into the adBNST before a second fear conditioning session with laser stimulation (Train 2), followed by a second tone test (Tone Test 2). Reconditioning in the absence of the antagonist increased freezing during (b) training (two-way RM ANOVA, group x tone, F(4,36) = 7.61, p = 0.0002, followed by Bonferroni’s post-hoc test) and (c) the tone test (two-way RM ANOVA, group, F(1,9) = 41.85, p = 0.0001). (b-c) Group differences in baseline freezing during training (two-tailed unpaired t test, t(9) = 2.67, p = 0.03 and testing (two-tailed unpaired t test, t(9) = 2.61, p = 0.03) are also shown. n = 7 RS102221 females; n = 4 aCSF females. RM = repeated measures, ANOVA = analysis of variance, aCSF = artificial cerebrospinal fluid. Data are represented as mean ± SEM. *p < 0.05, **p < 0.001, ***p < 0.0001.

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Extended Data Fig. 7 There are no sex differences in the effects of auditory fear conditioning on tone recall or serotonin in the adBNST.

(a) Schematic of procedures. Mice were habituated to the training and testing contexts before being given an injection of saline (i.p.) one hour prior to auditory fear conditioning (5 CS-US pairings). Mice were tested to tones and the training context on the following two days. Males and females exhibited similar tone-evoked freezing during (b) conditioning (two-way RM ANOVA, sex, F(1,53) = 0.03, p = 0.87) and (c) the tone test (two-way RM ANOVA, sex, F(1,53) = 1.77, p = 0.19). Baseline (BL) freezing on the testing day was higher in males than females (two-tailed unpaired t test, t(53) = 6.715, p < 0.0001). (d) Recall of contextual fear memory was stronger in males than females (two-tailed unpaired t test, t(53) = 2.24, p = 0.03). n = 28 females; n = 27 males. (e) To label adBNST projecting raphe neurons, Alexa Fluor 647-conjugated cholera toxin subunit B (CTB) was bilaterally injected into the adBNST of TpH2-ChR2+ mice 10 days prior to fear conditioning (created in BioRender). Mice of both sexes were perfused from the home cage (naïve) or 90 min following conditioning (trained). (f) CTB-treated males and females exhibited similar tone-evoked freezing during conditioning (two-way RM ANOVA, sex, F(1,8) = 0.11, p = 0.75). (g) Images of tissue stained for TpH2-ChR2-EYFP (green), c-Fos (red), and CTB (far red). Arrow indicates a triple-labeled cell. (h) In the DRN, fear conditioning increased the number of c-Fos+ cells (two-way ANOVA, training, F(1,18) = 26.43, p < 0.0001, (i) c-Fos & EYFP co-labeled cells (two-way ANOVA, training, F(1,18) = 32.08, p < 0.0001), and (j) c-Fos & EYFP & CTB triple-labeled cells (two-way ANOVA, training, F(1,18) = 9.62, p = 0.006) similarly in both sexes. (k) In the MRN, fear conditioning increased the number of c-Fos+ cells (two-way ANOVA, training, F(1,18) = 43.49, p < 0.0001), (l) c-Fos & EYFP co-labeled cells (two-way ANOVA, training, F(1,18) = 30.59, p < 0.0001), and (m) c-Fos & EYFP & CTB triple-labeled cells (two-way ANOVA, training, F(1,18) = 8.52, p = 0.009) similarly in both sexes. n = 5 naïve females; n = 7 naïve males; n = 5 trained females; n = 5 trained males. (n) (Bottom) In vivo microdialysis procedures (created in BioRender). Following implantation of cannula in the right adBNST, baseline dialysate was collected from TpH2-ChR2-EYFP- mice for two hours prior to fear conditioning (1 µL/min) and for two hours immediately following conditioning. (Top) Histological verification of canula placements. (o) Cannulated males and females exhibited similar tone-evoked freezing during conditioning (two-way RM ANOVA, sex, F(1,10) = 0.17, p = 0.69). (p) Extracellular concentrations of 5-HT in the adBNST at baseline (two-tailed unpaired t test, t(11) = 0.82, p = 0.43). (q) Extracellular concentrations of 5-HT in the adBNST post-conditioning (two-way RM ANOVA, sex, F(1,11) = 0.24, p = 0.64). n = 6 microdialysis females; n = 7 microdialysis males; *p < 0.05; **p < 0.001; ****p < 0.0001. Data are represented as mean ± SEM. DRN = dorsal raphe nucleus, MRN = median raphe nucleus, RM = repeated measures, ANOVA = analysis of variance.

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Extended Data Fig. 8 5-HT2CR antagonism alone does not alter fear conditioning.

(a) Schematic of procedures. Following habituation to training and testing contexts, cannulated TpH2-ChR2- mice received a bilateral infusion of the 5-HT2CR antagonist, RS102221, or aCSF into the adBNST prior to auditory fear conditioning (5 CS-US pairings). Mice were tested to tones and training context in the absence of shock or drug. (b) Histological verification of cannulae placements. Infusion of RS102221 into the adBNST without optogenetic stimulation of serotonin did not affect freezing to the tone during conditioning in (c) females (two-way RM ANOVA, antagonist, F(1,16) = 0.47, p = 0.50) or (f) males (two-way RM ANOVA, antagonist, F(1,12) = 1.20, p = 0.30. It also did not affect freezing during the tone test in (d) females (two-way RM ANOVA, antagonist, F(1,16) = 0.16, p = 0.69) or (g) males (two-way RM ANOVA, antagonist, F(1,12) = 1.83, p = 0.20). The antagonist did not affect recall of contextual fear memory in (e) females (two-tailed unpaired t test, t(16) = 0.89, p = 0.39) or (h) males (two-tailed unpaired t test, t(12) = 0.11, p = 0.91). n = 9 aCSF females; n = 9 RS102221 females; n = 9 aCSF males; n = 5 RS102221 males. Data presented as mean ± SEM. aCSF = artificial cerebrospinal fluid, RM = repeated measures, ANOVA = analysis of variance.

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Extended Data Fig. 9 Serotonin does not change BNST-to-CeA low gamma (30-50 Hz) communication in either sex.

(a) (Left) Tone-evoked CeA power spectra showing normalized low gamma in ChR2- (grey) and ChR2+ (blue) female mice. (Right) Quantification of normalized low gamma power in the CeA (Mann-Whitney, p = 0.53) of females, n = 5 ChR2-; n = 7 ChR2 + . (b) (Left) Tone-evoked adBNST power spectra showing normalized low gamma in ChR2- (grey) and ChR2+ (blue) female mice. (Right) Quantification of normalized low gamma power in the adBNST (Mann-Whitney, p = 0.37), n = 8 ChR2-;n = 9 ChR2 + . (c) (Left) average normalized spectrum of adBNST-CeA low gamma coherence in ChR2- (grey) and ChR2+ (blue) female mice. (Right) Quantification of normalized low gamma adBNST-CeA coherence in female mice (Mann Whitney, p = 0.93), n = 5 ChR2-; n = 6 ChR2 + . (d) (Left) Tone-evoked CeA power spectra showing normalized low gamma in ChR2- (grey) and ChR2+ (blue) male mice. (Right) Quantification of normalized low gamma power in the CeA of male mice (Mann-Whitney, p = 0.91), n = 5 ChR2-; n = 8 ChR2 + . (e) (Left) Tone-evoked adBNST power spectra showing normalized low gamma in ChR2- (grey) and ChR2+ (blue) male mice. (Right) Quantification of normalized low gamma power in the of adBNST of male mice (Mann-Whitney, p = 0.41), n = 7 ChR2-; n = 9 ChR2 + . (f) (Left) Average normalized spectrum of adBNST-CeA low gamma coherence in ChR2- (grey) and ChR2+ (blue) male mice. (Right) Quantification of normalized low gamma adBNST-CeA coherence in male mice (Mann-Whitney, p = 0.53), n = 4 ChR2-; n = 7 ChR2 + . (g) Change in tone-evoked high gamma power from pre-tone levels in the adBNST of (left) females (ChR2-, Wilcoxon t-test, p = 0.012; ChR2 + , Wilcoxon t-test, p = 0.43) and (right) males (ChR2-, Wilcoxon t-test, p = 0.039; ChR2 + , Wilcoxon t-test, p = 0.02). n = 9 females/genotype, n = 8 ChR2- males, n = 9 ChR2+ males. (h) Change in tone-evoked low gamma power from pre-tone levels in the adBNST of (left) females (ChR2-, Wilcoxon t-test, p = 0.039; ChR2 + , Wilcoxon t-test, p = 0.039) and (right) males (ChR2-, Wilcoxon t-test, p = 0.078; ChR2 + , Wilcoxon t-test, p = 0.008). n = 8 ChR2- females, n = 9 ChR2+ females, n = 7 ChR2- males, n = 9 ChR2+ males. For power spectra, SEM is shown in shaded regions.

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Extended Data Fig. 10 Schematic illustrating the proposed raphe-adBNST-CeA circuit mediating the enhancing effects of serotonin on tone recall in females.

We propose that increasing serotonergic input to the adBNST in females leads to greater activation of 5-HT2CRs located on inhibitory interneurons. This leads to greater inhibition of GABAergic projection neurons to the CeM, resulting in disinhibition of CeM outputs to the vlPAG. In addition, serotonin-mediated plasticity in a different subset of GABAergic adBNST neurons increases adBNST-to-CeL communication, which also results in disinhibition of CeM. The consequent increase in GABAergic output from the CeM to the vlPAG increases tone-evoked freezing during recall. Created in BioRender.

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Ravenelle, R., Lee, J., Fernandes-Henriques, C. et al. Serotonergic modulation of the BNST–CeA pathway reveals sex differences in fear learning. Nat Neurosci 28, 1897–1909 (2025). https://doi.org/10.1038/s41593-025-02025-x

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