Abstract
Animals can learn about sources of danger while minimizing their own risk by observing how others respond to threats. However, the distinct neural mechanisms by which threats are learned through social observation (known as observational fear learning1,2,3,4 (OFL)) to generate behavioural responses specific to such threats remain poorly understood. The dorsomedial prefrontal cortex (dmPFC) performs several key functions that may underlie OFL, including processing of social information and disambiguation of threat cues5,6,7,8,9,10,11. Here we show that dmPFC is recruited and required for OFL in mice. Using cellular-resolution microendoscopic calcium imaging, we demonstrate that dmPFC neurons code for observational fear and do so in a manner that is distinct from direct experience. We find that dmPFC neuronal activity predicts upcoming switches between freezing and moving state elicited by threat. By combining neuronal circuit mapping, calcium imaging, electrophysiological recordings and optogenetics, we show that dmPFC projections to the midbrain periaqueductal grey (PAG) constrain observer freezing, and that amygdalar and hippocampal inputs to dmPFC opposingly modulate observer freezing. Together our findings reveal that dmPFC neurons compute a distinct code for observational fear and coordinate long-range neural circuits to select behavioural responses.
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Data availability
The data that support the findings of this study are available in the Source Data file and from the corresponding authors upon reasonable request. Source data are provided with this paper.
Code availability
Custom codes used to analyse data from this study are available upon reasonable request from the corresponding authors.
References
Kondrakiewicz, K., Kostecki, M., Szadzinska, W. & Knapska, E. Ecological validity of social interaction tests in rats and mice. Genes Brain Behav. 18, e12525 (2019).
Olsson, A., Knapska, E. & Lindstrom, B. The neural and computational systems of social learning. Nat. Rev. Neurosci. 21, 197–212 (2020).
Burgos-Robles, A., Gothard, K. M., Monfils, M. H., Morozov, A. & Vicentic, A. Conserved features of anterior cingulate networks support observational learning across species. Neurosci. Biobehav. Rev. 107, 215–228 (2019).
Keum, S. & Shin, H. S. Rodent models for studying empathy. Neurobiol. Learn. Mem. 135, 22–26 (2016).
Blanchard, D. C., Griebel, G., Pobbe, R. & Blanchard, R. J. Risk assessment as an evolved threat detection and analysis process. Neurosci. Biobehav. Rev. 35, 991–998 (2011).
Qi, S. et al. How cognitive and reactive fear circuits optimize escape decisions in humans. Proc. Natl Acad. Sci. USA 115, 3186–3191 (2018).
Fanselow, M. S. Neural organization of the defensive behavior system responsible for fear. Psychon. Bull. Rev. 1, 429–438 (1994).
LeDoux, J. E. & Pine, D. S. Using neuroscience to help understand fear and anxiety: a two-system framework. Am. J. Psychiatry 173, 1083–1093 (2016).
Jercog, D. et al. Dynamical prefrontal population coding during defensive behaviours. Nature 595, 690–694 (2021).
Mobbs, D. The ethological deconstruction of fear(s). Curr. Opin. Behav. Sci. 24, 32–37 (2018).
Sharpe, M. J. & Killcross, S. Modulation of attention and action in the medial prefrontal cortex of rats. Psychol. Rev. 125, 822–843 (2018).
DSM-5. Diagnostic and Statistical Manual of Mental Disorders, 4th edn (APA Press, 2013).
Kim, A., Keum, S. & Shin, H. S. Observational fear behavior in rodents as a model for empathy. Genes Brain Behav. 18, e12521 (2019).
Preston, S. D. & de Waal, F. B. Empathy: its ultimate and proximate bases. Behav. Brain Sci. 25, 1–20 (2002).
Cummings, K. A. & Clem, R. L. Prefrontal somatostatin interneurons encode fear memory. Nat. Neurosci. 23, 61–74 (2020).
Yizhar, O. & Levy, D. R. The social dilemma: prefrontal control of mammalian sociability. Curr. Opin. Neurobiol. 68, 67–75 (2021).
Chen, P. & Hong, W. Neural circuit mechanisms of social behavior. Neuron 98, 16–30 (2018).
Liang, B. et al. Distinct and dynamic ON and OFF neural ensembles in the prefrontal cortex code social exploration. Neuron 100, 700–714.e709 (2018).
Wu, Y. E. et al. Neural control of affiliative touch in prosocial interaction. Nature 599, 262–267 (2021).
Padilla-Coreano, N. et al. Cortical ensembles orchestrate social competition through hypothalamic outputs. Nature 603, 667–671 (2022).
Ito, W., Palmer, A. J. & Morozov, A. Social synchronization of conditioned fear in mice requires ventral hippocampus input to the amygdala. Biol. Psychiatry 93, 322–330 (2023).
Scheggia, D. et al. Somatostatin interneurons in the prefrontal cortex control affective state discrimination in mice. Nat. Neurosci. 23, 47–60 (2020).
Piva, M. et al. The dorsomedial prefrontal cortex computes task-invariant relative subjective value for self and other. eLife 8, e44939 (2019).
Wang, F. et al. Bidirectional control of social hierarchy by synaptic efficacy in medial prefrontal cortex. Science 334, 693–697 (2011).
Murugan, M. et al. Combined social and spatial coding in a descending projection from the prefrontal cortex. Cell 171, 1663–1677.e1616 (2017).
Yusufishaq, S. & Rosenkranz, J. A. Post-weaning social isolation impairs observational fear conditioning. Behav. Brain Res. 242, 142–149 (2013).
Allsop, S. A. et al. Corticoamygdala transfer of socially derived information gates observational learning. Cell 173, 1329–1342.e1318 (2018).
Jeon, D. et al. Observational fear learning involves affective pain system and Cav1.2 Ca2+ channels in ACC. Nat. Neurosci. 13, 482–488 (2010).
Keum, S. & Shin, H. S. Neural basis of observational fear learning: a potential model of affective empathy. Neuron 104, 78–86 (2019).
Paradiso, E., Gazzola, V. & Keysers, C. Neural mechanisms necessary for empathy-related phenomena across species. Curr. Opin. Neurobiol. 68, 107–115 (2021).
Smith, M. L., Asada, N. & Malenka, R. C. Anterior cingulate inputs to nucleus accumbens control the social transfer of pain and analgesia. Science 371, 153–159 (2021).
Fillinger, C., Yalcin, I., Barrot, M. & Veinante, P. Efferents of anterior cingulate areas 24a and 24b and midcingulate areas 24a′ and 24b′ in the mouse. Brain Struct. Funct. 223, 1747–1778 (2018).
Sierra-Mercado, D., Padilla-Coreano, N. & Quirk, G. J. Dissociable roles of prelimbic and infralimbic cortices, ventral hippocampus, and basolateral amygdala in the expression and extinction of conditioned fear. Neuropsychopharmacology 36, 529–538 (2011).
Halladay, L. R. & Blair, H. T. Distinct ensembles of medial prefrontal cortex neurons are activated by threatening stimuli that elicit excitation vs. inhibition of movement. J Neurophysiol. 114, 793–807 (2015).
Padilla-Coreano, N., Tye, K. M. & Zelikowsky, M. Dynamic influences on the neural encoding of social valence. Nat. Rev. Neurosci. 23, 535–550 (2022).
Huang, Z. et al. Ventromedial prefrontal neurons represent self-states shaped by vicarious fear in male mice. Nat. Commun. 14, 3458 (2023).
Franklin, T. B. et al. Prefrontal cortical control of a brainstem social behavior circuit. Nat. Neurosci. 20, 260–270 (2017).
Siciliano, C. A. et al. A cortical–brainstem circuit predicts and governs compulsive alcohol drinking. Science 366, 1008–1012 (2019).
Vander Weele, C. M. et al. Dopamine enhances signal-to-noise ratio in cortical-brainstem encoding of aversive stimuli. Nature 563, 397–401 (2018).
Assareh, N., Bagley, E. E., Carrive, P. & McNally, G. P. Brief optogenetic inhibition of rat lateral or ventrolateral periaqueductal gray augments the acquisition of Pavlovian fear conditioning. Behav. Neurosci. 131, 454–459 (2017).
Courtin, J. et al. Prefrontal parvalbumin interneurons shape neuronal activity to drive fear expression. Nature 505, 92–96 (2014).
Ozawa, T. et al. A feedback neural circuit for calibrating aversive memory strength. Nat. Neurosci. 20, 90–97 (2017).
Haaker, J., Yi, J., Petrovic, P. & Olsson, A. Endogenous opioids regulate social threat learning in humans. Nat. Commun. 8, 15495 (2017).
Huang, J. et al. A neuronal circuit for activating descending modulation of neuropathic pain. Nat. Neurosci. 22, 1659–1668 (2019).
Rozeske, R. R. et al. Prefrontal–periaqueductal gray-projecting neurons mediate context fear discrimination. Neuron 97, 898–910.e896 (2018).
Anastasiades, P. G., Marlin, J. J. & Carter, A. G. Cell-type specificity of callosally evoked excitation and feedforward inhibition in the prefrontal cortex. Cell Rep. 22, 679–692 (2018).
Senn, V. et al. Long-range connectivity defines behavioral specificity of amygdala neurons. Neuron 81, 428–437 (2014).
Sotres-Bayon, F., Sierra-Mercado, D., Pardilla-Delgado, E. & Quirk, G. J. Gating of fear in prelimbic cortex by hippocampal and amygdala inputs. Neuron 76, 804–812 (2012).
Hagihara, K. M. et al. Intercalated amygdala clusters orchestrate a switch in fear state. Nature 594, 403–407 (2021).
McGarry, L. M. & Carter, A. G. Inhibitory gating of basolateral amygdala inputs to the prefrontal cortex. J. Neurosci. 36, 9391–9406 (2016).
Anderson, S. W., Bechara, A., Damasio, H., Tranel, D. & Damasio, A. R. Impairment of social and moral behavior related to early damage in human prefrontal cortex. Nat. Neurosci. 2, 1032–1037 (1999).
Shin, L. M., Rauch, S. L. & Pitman, R. K. Amygdala, medial prefrontal cortex, and hippocampal function in PTSD. Ann. NY Acad. Sci. 1071, 67–79 (2006).
Jeon, D. & Shin, H. S. in Current Protocols in Neuroscience (eds. Crawley, J. N. et al.) Ch. 8, Unit 8 27 (2011).
Holmes, A. & Rodgers, R. J. Prior exposure to the elevated plus-maze sensitizes mice to the acute behavioral effects of fluoxetine and phenelzine. Eur. J. Pharmacol. 459, 221–230 (2003).
Karlsson, R. M., Tanaka, K., Heilig, M. & Holmes, A. Loss of glial glutamate and aspartate transporter (excitatory amino acid transporter 1) causes locomotor hyperactivity and exaggerated responses to psychotomimetics: rescue by haloperidol and metabotropic glutamate 2/3 agonist. Biol. Psychiatry 64, 810–814 (2008).
Feyder, M. et al. Association of mouse Dlg4 (PSD-95) gene deletion and human DLG4 gene variation with phenotypes relevant to autism spectrum disorders and Williams’ syndrome. Am. J. Psychiatry 167, 1508–1517 (2010).
Gunduz-Cinar, O. et al. A cortico-amygdala neural substrate for endocannabinoid modulation of fear extinction. Neuron 111, 3053–-3067.e10 (2023).
Halladay, L. R. et al. Prefrontal regulation of punished ethanol self-administration. Biol. Psychiatry 87, 967–978 (2020).
Eastwood, B. S. et al. Whole mouse brain reconstruction and registration to a reference atlas with standard histochemical processing of coronal sections. J. Comp. Neurol. 527, 2170–2178 (2019).
Ahrlund-Richter, S. et al. A whole-brain atlas of monosynaptic input targeting four different cell types in the medial prefrontal cortex of the mouse. Nat. Neurosci. 22, 657–668 (2019).
Sengupta, A. & Holmes, A. A discrete dorsal raphe to basal amygdala 5-HT circuit calibrates aversive memory. Neuron 103, 489–505.e487 (2019).
Acknowledgements
The authors thank C. Ramakrishnan for providing viral constructs and to the following (in alphabetical order) for valuable discussions and technical advice: G. Cui, N. Hájos, A. Lüthi and M. Xia. This work was supported by the NIAAA Intramural Research Program (1ZIAAA000411-19 to A.H.); NIMH Intramural Research Program (ZIAMH002497-34 to C.R.G. and 1ZIAMH002950 to M.A.P.); and the Bowles Center for Alcohol Studies and awards R01NS122230, R01AA019454 and 5P60AA011605 (T.L.K.).
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Contributions
S.E.S. and A.H. conceived and designed the project. S.E.S., O.B., D.P., J.A.S., L.Z, T.Y, J.J.O., A.G.L. and A.L. performed experiments. S.E.S., R.O., O.B., D.P., J.A.S., L.Z., T.Y., J.J.O., R.F.P., A.G.L., M.N., A.L. and C.R.G. analysed data. K.D. provided viral constructs. S.E.S. and A.H. prepared figures and wrote the manuscript. The other authors made valuable comments on drafts of the manuscript and figures. M.A.P. and T.L.K. supervised electrophysiology experiments. A.H. supervised the entire project.
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Nature thanks Ewelina Knapska, Scott Russo and Moriel Zelikowsky for their contribution to the peer review of this work. Peer review reports are available.
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Extended data figures and tables
Extended Data Fig. 1 OFL-related behavioural experiments.
a, Apparatus for OFL, with DEM present during conditioning. b, Trial-wise freezing in OBS and DEM during OFL conditioning. N = 17 OBS-DEM pairs. c, Higher CS-related freezing in DEM than OBS (*P < 0.0001) during OFL conditioning (left); more CS-related rearing (*P < 0.0001) and grooming (*P = 0.0024) in OBS than DEM during OFL conditioning (right). P values from unpaired t-tests. N = 10 OBS-DEM pairs. d, No increase in CS-related OBS freezing versus pre-CS when partition between OBS and DEM is opaque. N = 9 mice. e, Minimal increase in CS-related OBS freezing versus pre-CS (#P = 0.0312) during conditioning, not retrieval, when DEM is not shocked. P value from paired t-test. N = 9 mice. f, Anxiety-related behaviour and social interaction testing 1-2 days after OFL. g, Normal OBS performance, versus unconditioned controls, in the elevated plus-maze test. n = 6 mice/group. h, Normal OBS performance, versus unconditioned controls, in the novel open field test. n = 6 mice/group. i, Normal OBS social interaction with a novel conspecific mouse, versus unconditioned controls. n = 6 mice/group. j, Similarly higher CS-related freezing versus pre-CS in animals directly conditioned alone (conditioning #P = 0.0001, retrieval #P = 0.0087) or in the presence of a conspecific (conditioning #P = 0.001, retrieval #P = 0.0010). P values from Bonferroni post hoc tests following mixed-model ANOVA. n = 7 mice/group. Two-tailed statistical tests were used. Data shown as means ± SEM.
Extended Data Fig. 2 OFL-related neuronal activation and in vivo optogenetic manipulations.
a, More c-fos-positive neurons in BLA (*P = 0.0043), vHPC (*P = 0.0027) and cACC (*P = 0.0135), not S1 (P = 0.5022) in OBS than unconditioned controls (CON). P values from Bonferroni post hoc tests following mixed-model ANOVA. n = 12 OBS mice, n = 15 CON mice. b, Viral spread (green shading) and optic fibre tips (‘X’) directing light at eArch3.0-expressing dmPFC neurons during OFL conditioning. c, No difference in CS-related freezing during conditioning or retrieval between eArch3.0 and YFP controls when light was shone during retrieval (not conditioning). Higher CS-related freezing versus pre-CS in eArch3.0 group (conditioning #P = 0.0001, retrieval #P = 0.0058, n = 8 mice) and YFP controls (conditioning #P = 0.0011, retrieval #P = 0.0083, n = 12 mice). P values from Bonferroni post hoc tests following mixed-model ANOVA. d, Viral spread (green shading) and optic fibre tips (‘X’) directing light at eArch3.0-expressing dmPFC neurons during OFL retrieval. e, Location of GRIN lenses in dmPFC. f, Heat-map of calcium activity aligned to CS during OFL conditioning. g, Peak Z-score (left, P = 0.9121) and decay time constant (right, P = 0.1148) of calcium activity during Shock-to-DEM or Shock-to-OBS. P values from paired t-tests. h, Increased population calcium activity during Shock-to-OBS in a random subset of 100 neurons (compare with population trace from all neurons, depicted in Fig. 2a). This shows that different population responses to Observed and Direct (depicted in Fig. 2a) is unlikely to be due to the smaller % of observed than Direct-responsive neurons. i, Percent neurons responsive to Observed (Shock-to-DEM) and Direct (Shock-to-OBS). j, K-means clustering of neuronal subpopulations with affinity for both Observed, Direct, both or neither. Affinity defined as the strength of the correlation between an individual neuron’s activity and the population-average activity of Observed or Direct related neurons (as depicted in Fig. 2c). k, Microendoscope dmPFC neuronal calcium imaging in OBS during 2 OFL conditioning sessions, followed by a DFL session. l, Location of GRIN lenses in dmPFC. m, Percent neurons responsive to Observed (Shock-to-DEM) on two OFL episodes and to Direct (Shock-to-OBS) in the same animals. Adjusted mutual information estimation indicated a high degree of Shock-to-DEM neuronal population overlap across the two OFL episodes (0.0179, adjusted *P < 0.0001 versus chance), whereas there was less overlap between the Shock-to-DEM population on OFL session 1 and the Shock-to-OBS population (0.0023, adjusted P = 0.0680). n, Decoding Observed (OFL1) versus Direct (*P = 0.0059) and failure to decode two sessions of OFL (P = 0.1372). P values from paired t-tests versus rotated. N = 8 mice panels e-j, N = 5 mice panels l-n. Two-tailed statistical tests were used. Data shown as means ± SEM.
Extended Data Fig. 3 In vivo single cell resolution calcium recordings in dmPFC during OFL.
a, PC1 (upper) and PC2 (lower) neuronal trajectories associated with Shock-to-DEM and Shock-to-OBS. b, When freezing was equated across Observed and Direct conditioning by taking a subset of the 10 highest-freezing OFL trials (72 ± 12%) and the 10 lowest-freezing Direct trials (61 ± 11%) for each mouse (paired t-test, t(6) = 1.795, P = 0.1227), Observed and Direct related activity trajectories still occupy distinct places in low-dimensional state-space after PCA (compare with Fig. 2e,f). c, PC1 trajectory distance remained different between Observed and Direct when freezing was equated across OFL and DFL conditioning. Black line denotes periods that are significantly different from rotated data (Benjamini-Hochberg corrected permutation tests). d, Uniform manifold approximation and projection (UMAP) dimensionality reduction state-space visualization of neuronal data during shock and inter-shock periods on Shock-to-DEM and Shock-to-OBS test sessions. e, Percent neurons exhibiting changes in activity during freeze or move. f, Example of spatially intermingled freeze and move neurons within the field of view. Neither freeze-related (mean distance to cluster-centroid: 61.2 a.u.) nor move-related (mean distance to cluster-centroid: 61.9 a.u.) neurons were clustered to a greater or lesser extent than expected by chance in any mouse. The distance between the centroid of freeze-related and move-related clusters (11.3 a.u.) was not to a greater or lesser extent than expected by chance in any mouse. g, Freeze and move state-related activity trajectories in low-dimensional state-space following PCA. h, PC2 neuronal trajectories associated with freeze and move state. i, Weak relationship between neuronal activity related to stopping during pre-CS baseline and freezing during OFL, as measured by Pearson’s r and linear regression adjusted R2. j, PC1 + 2 neuronal trajectories differentially anticipate upcoming switches to freeze state during Observed (centre) and Direct (right) but not switches to stopping during pre-CS baseline (left). Black line denotes period significantly different from rotated data (Benjamini-Hochberg corrected permutation tests). Data shown in c and j are means ± SEM. N = 8 mice for all panels.
Extended Data Fig. 4 OFL-related neuronal activation, dmPFC→l/vlPAG or dmPFC→BLA collaterals.
a, Assessing OFL-related neuronal activation in CTB-labelled dmPFC→l/vlPAG and dmPFC→BLA neurons. b, c-fos-positive and CTB-positive dmPFC→l/vlPAG (left) and dmPFC→BLA (right) neurons. c, Percent c-fos-positive/CTB-posiitive dmPFC→l/vlPAG (n = 5 mice) and dmPFC→BLA (n = 7 mice) neurons in OBS. d, Synaptophysin expression in dmPFC (left), l/vlPAG (centre, upper) and BLA (centre, lower). Regions exhibiting synaptophysin expression (right upper and lower). e, Regions receiving collaterals from dmPFC→l/vlPAG neurons. f, Regions receiving collaterals from dmPFC→BLA neurons. Abbreviations (based on Paxinos and Franklin atlas, 5th edition): AI; agranular insula, Aq; aqueduct, CL; centrolateral thalamus, CLA; claustrum, DLS; dorsolateral striatum, DMS; dorsomedial striatum, lPAG; lateral periaqueductal gray, LH; lateral hypothalamus, LHb; lateral habenula, MD; mediodorsal thalamus, MHb, medial habenula, PH; posterior hypothalamus, ZI; zona incerta, vlPAG; ventrolateral periaqueductal gray, V3; 3rd ventricle. Data shown in c are means ± SEM.
Extended Data Fig. 5 Fibre photometry calcium recordings and optogenetic manipulations.
a, Location of optic fibre tips (‘X’) for calcium recordings in dmPFC→l/vlPAG neurons during OFL. b, Location of optic fibre tips (‘X’) for calcium recordings in dmPFC→BLA neurons during OFL. c, Shock-to-DEM and CS-related calcium activity in OBS dmPFC→BLA neurons uncorrelated (Pearson’s r) with OBS freezing on early (first 5) or late (last 5) conditioning trials (compare with event-correlated activity in dmPFC→l/vlPAG neurons, depicted in Fig. 3h). n = 5-6 mice/group. d, Increased calcium activity in OBS dmPFC→l/vlPAG neurons during move versus freeze (*P = 0.0226, paired t-test). N = 5 mice. e, Freeze-related calcium activity in OBS dmPFC→l/vlPAG neurons anti-correlates (Pearson’s r) with OBS freezing during OFL. N = 5 mice. f, Optogenetic photoexcitation of dmPFC→l/vlPAG axons, via ChR2 (n = 11 mice), decreases CS-related freezing during Direct conditioned (*P < 0.0021), but not retrieval (P = 0.1806), versus YFP controls (n = 7 mice). Higher CS-related freezing versus pre-CS during conditioning in YFP (#P < 0.0001) and ChR2 (#P < 0.0001). P values from Bonferroni post hoc tests following mixed-model ANOVA. g, Location of optic fibre tips (‘X’) in l/vlPAG for dmPFC→l/vlPAG ChR2 manipulation during OFL. h, Location of optic fibre tips (‘X’) in l/vlPAG for dmPFC→l/vlPAG eArch3.0 manipulation during OFL. Two-tailed statistical tests were used. Data shown as means ± SEM. a.u.; arbitrary units.
Extended Data Fig. 6 Rabies virus labelling of inputs to dmPFC neurons.
a, Brain regions exhibiting rabies virus (RV) labelling from dmPFC→l/vlPAG transfected neurons. b, Brain regions exhibiting RV labelling from dmPFC→BLA transfected neurons. n = 5–7/group.
Extended Data Fig. 7 Quantification of rabies virus-labelling of inputs to dmPFC neurons.
a, Percent rabies virus (RV) labelled neurons within the cortical subplate. b, Percent RV labelled neurons within the hippocampal formation. c, Percent RV labelled neurons within isocortex regions and as a percent of whole-brain labelling. d, Percent RV labelled neurons within thalamic regions and as a percent of whole-brain labelling. e, Percent RV labelled neurons within olfactory areas and as a percent of whole-brain labelling. f, Percent RV labelled neurons within cerebral nuclei and as a percent of whole-brain labelling. g, Percent RV labelled neurons within the hypothalamus and as a percent of whole-brain labelling. h, Percent RV labelled neurons within the midbrain/hindbrain and as a percent of whole-brain labelling. Data shown as means ± SEM in all panels. dmPFC→l/vlPAG n = 5 mice, dmPFC→BLA n = 7 mice. Abbreviations (based on Allen Brain Atlas definitions) by panel: a, BLA; basolateral amygdala, BMA; basomedial amygdala, CLA; claustrum, EP; endopiriform, LA; lateral amygdala Misc; miscellaneous. b, ENT; entorhinal cortex, SUBd; subiculum dorsal part, SUBv; subiculum ventral part, Misc; miscellaneous. c, AI; agranular insular, ILA, infralimbic, GU; granular insular cortex, ECT; ectorhinal cortex, PERI; perirhinal cortex, TEA; temporal cortex, association area, VISC; visceral area, FRP; frontal association cortex, AUD; auditory cortex, PTLp; parietal association cortex, VIS; visual cortex, SS; somatosensory cortex, MOp; primary motor, RSP; retrosplenial cortex, ORBvl; ventral orbital cortex, ORBm; medial orbital cortex, ORBl; lateral orbital cortex. d, AD; anterodorsal nucleus, AM; anteromedial nucleus, CM; central mediodorsal thalamic nucleus, IAD; interanterodorsal thalamic nucleus, IMD; intermediodorsal thalamic nucleus, MD; mediodorsal thalamic nucleus, PF; parafascicular thalamic nucleus, PO; posterior thalamic nuclear group, PT; parataenial thalamic nucleus, PVT; paraventricular thalamic nucleus, RE; reuniens thalamic nucleus, RH; rhomboid thalamic nucleus, VAL; ventral anterior thalamic nucleus, VM; ventromedial thalamus, Misc; miscellaneous. e, AON; anterior olfactory nucleus, DP; dorsal peduncular, MOB; main olfactory bulb, NLOT; nucleus of lateral olfactory tract, PIR; piriform cortex, TT; dorsal taenia tecta, Misc; miscellaneous. f, AAA; anterior amygdaloid area, GP; globus pallidus, LSx; lateral septum, MS; medial septal nucleus, NDB; nucleus of the vertical limb of the diagonal band, OT; olfactory tubercle, SI; substantia inominata, Misc; miscellaneous. g, LHA; lateral hypothalamus, LPO; lateral preoptic nucleus, MBO; mammillary body, MPO; medial preoptic nucleus, PH; posterior hypothalamic area, VLPO; ventrolateral preoptic nucleus, ZI; zona incerta, Misc; miscellaneous. h, LDT; laterodorsal tegmental area, PAG; periaqueductal gray, PRN; pontine reticular nucleus, PRT; pretectal region, RAmb; midbrain raphe nuclei, MRN; median raphe nucleus, RPF; retroparafascicular, SC; superior colliculus, SNr; substantia nigra, pars reticularis, VTA; ventral tegmental area.
Extended Data Fig. 8 Rabies virus control experiments.
a, Omitting TVA and G injections, with all other injections and timings as in rabies virus (RV) labelling experiment (depicted in Fig. 4a–c). b, Omitting TVA and G produced no RV labelling in dmPFC, BLA or vHPC. c, Omission of G injection, with all other injections and timings as in RV labelling experiment. d, Omitting G produced no RV labelling in dmPFC, BLA or vHPC. Note autofluorescence at dmPFC injection site. e, Viral injections as in RV labelling experiment, but in wild-type, not Vglut1-Cre, mice. f, Viral injections in wild-type mice produced no RV labelling in dmPFC, BLA or vHPC. Note autofluorescence at dmPFC injection site.
Extended Data Fig. 9 Input-Output analysis of dmPFC-Pvalb neurons.
a, AAV1 transsynaptic anterograde labelling of vHPC synaptic inputs to dmPFC-Pvalb neurons (left). AAV1 expression and Pvalb immunohistochemical (IHC) staining in dmPFC. b, AAV1 transsynaptic anterograde labelling of vHPC synaptic inputs to dmPFC in 4 Cre-driver lines (left). AAV1 expression in dmPFC in each line (right). c, Rabies virus (RV) labelling of vHPC inputs to dmPFC-Pvalb neurons in Pvalb-Cre mice (left). TVA-mCherry and RV GFP expression in dmPFC (centre left), and RV expression in BLA (centre right) and vHPC (right). Abbreviations: EP; endopiriform, LA; lateral amygdala, CeA; central amygdala, ENT; entorhinal cortex. d, AAV1 transsynaptic anterograde labelling of BLA synaptic inputs to dmPFC-Pvalb neurons (left). Low and high (inset) magnification images of AAV1 expression and Pvalb immunohistochemical (IHC) staining in dmPFC (right). e, ChR2-driven dmPFC-Pvalb modulation of dmPFC→l/vlPAG neurons. f, Optically-evoked Pvalb-mediated postsynaptic response amplitude in dmPFC→l/vlPAG neurons. n = 5 cells, N = 2 mice. g, Optically-evoked Pvalb-mediated postsynaptic response latency in dmPFC→l/vlPAG neurons. n = 5 cells, N = 2 mice. h, Example recordings traces. i, Schematic cartoon placing dmPFC in a heuristic circuit model of OFL and DFL. During OFL, there is differential representation of Observed and Direct by dmPFC neuronal population activity which, in turn, is informed by vHPC input to dmPFC that encodes information about a threat targeted at another and not self. The intra-dmPFC microcircuitry through which vHPC input might support differential regulation of Observed and Direct remains to be clarified. During Direct experience, animals are in a high fear state encoded via a reciprocal functional loop between BLA and dmPFC. In this high fear state, animals primarily exhibit freezing behaviours. This behavioural response is proposed to be, in part, due to BLA-driven dmPFC-Pvalb-mediated inhibition of a dmPFC→l/vlPAG pathway – a pathway maintaining movement in response to threat. During observed threat, BLA-dmPFC-Pvalb engagement is posited to be lesser than during Direct experience, which could favour movement due to lesser Pvalb inhibition of dmPFC→l/vlPAG neuronal activity. A potential contribution of dmPFC-SST interneurons during OFL is likely, given recent findings show these cells are innervated by BLA and regulate fear and social cognition via input to dmPFC principal neurons (PN) and dmPFC-Pvalb interneurons 15,22, but this contribution remains to be determined. Data shown in panels f,g as means ± SEM.
Extended Data Fig. 10 Analyses of dmPFC inputs.
a, More c-fos-positive neurons in OBS (n = 5 mice) versus unconditioned controls (CON, n = 5 mice) in dmPFC-projecting neurons located in caudal anterior cingulate cortex (cACC) (*P = 0.0005), insular cortex (InsCtx) (*P = 0.0043) and claustrum (CLA) (*P = 0.0039). P values from paired t-tests. b, Similar numbers of dmPFC-projecting CTB-positive neurons in cACC, InsCtx, CLA, BLA, and vHPC of OBS versus CON. n = 5 mice/group. c, Post-OFL ex vivo slice neuronal recordings of optically-evoked vHPC-mediated postsynaptic responses in dmPFC neurons. d, Higher post-OFL optically-evoked E/I ratio in dmPFC neurons in OBS (n = 20 cells, n = 5 mice) than CON (n = 18 cells, n = 5 mice) (*P = 0.023, unpaired t-test). e, Higher post-OFL optically-evoked postsynaptic oIPSC than oEPSC latency (*P = 0.0001, main effect from mixed model ANOVA). n = 20 cells, CON n = 5 mice, OBS n = 18 cells, n = 5 mice. f, Example recordings traces. g, Location of optic fibre tips (‘X’) in dmPFC for vHPC→dmPFC ChR2 manipulation during OFL (left); ChR2 expression and fibre tip and tract position (right). h, Location of optic fibre tips (‘X’) in dmPFC for vHPC→dmPFC eArch3.0 manipulation during OFL (left); eArch3.0 expression and fibre tip and tract position (right). i, Location of optic fibre tips (‘X’) in dmPFC for BLA→dmPFC eArch3.0 manipulation during OFL (left); eArch3.0 expression and fibre tip and tract position (right). Data shown as means ± SEM.
Supplementary information
Supplementary Table 1
Summary table of statistical tests and results.
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Silverstein, S.E., O’Sullivan, R., Bukalo, O. et al. A distinct cortical code for socially learned threat. Nature 626, 1066–1072 (2024). https://doi.org/10.1038/s41586-023-07008-1
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DOI: https://doi.org/10.1038/s41586-023-07008-1
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