A competitive inhibitory circuit for selection of active and passive fear responses

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Abstract

When faced with threat, the survival of an organism is contingent upon the selection of appropriate active or passive behavioural responses1,2,3. Freezing is an evolutionarily conserved passive fear response that has been used extensively to study the neuronal mechanisms of fear and fear conditioning in rodents4. However, rodents also exhibit active responses such as flight under natural conditions2. The central amygdala (CEA) is a forebrain structure vital for the acquisition and expression of conditioned fear responses, and the role of specific neuronal sub-populations of the CEA in freezing behaviour is well-established1,5,6,7. Whether the CEA is also involved in flight behaviour, and how neuronal circuits for active and passive fear behaviour interact within the CEA, are not yet understood. Here, using in vivo optogenetics and extracellular recordings of identified cell types in a behavioural model in which mice switch between conditioned freezing and flight, we show that active and passive fear responses are mediated by distinct and mutually inhibitory CEA neurons. Cells expressing corticotropin-releasing factor (CRF+) mediate conditioned flight, and activation of somatostatin-positive (SOM+) neurons initiates passive freezing behaviour. Moreover, we find that the balance between conditioned flight and freezing behaviour is regulated by means of local inhibitory connections between CRF+ and SOM+ neurons, indicating that the selection of appropriate behavioural responses to threat is based on competitive interactions between two defined populations of inhibitory neurons, a circuit motif allowing for rapid and flexible action selection.

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Figure 1: Conditioned flight paradigm.
Figure 2: CRF+ neurons mediate conditioned flight.
Figure 3: Neuronal activity patterns of CRF+ and SOM+ cells.
Figure 4: CRF+ and SOM+ neurons form a reciprocal inhibitory circuit controlling defensive behaviour.

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Acknowledgements

We thank N. Whittle for comments on the manuscript. C. Ramakrishnan and K. Deisseroth provided intersectional viral vectors. M. S. Esposito and S. Arber provided flex-synaptophysin-GFP viral vectors. Z. Josh Huang provided the SOM-Flp mouse line. We thank J. Eglinger for providing overlap analysis scripts and S. Bourke for imaging advice. J.P.F. was funded by an EMBO LTF (952-2011) and a NARSAD Young Investigator Fellowship. C.X. was funded by EMBO ALTF (1579-2010). S.K., L.M. and P.T. were funded by a NARSAD Young Investigator Fellowship. All authors were supported by the National Center of Competences in Research: ‘SYNAPSY — The Synaptic Bases of Mental Diseases’ (financed by the Swiss National Science Foundation), a SNSF core grant (to A.L.), an ERC Advanced Grant (to A.L.), and the Novartis Research Foundation.

Author information

J.P.F. conceived, designed, performed and analysed most of the experiments and wrote the manuscript. S.K. conceived, designed, performed and analysed all in vitro experiments. M.M. contributed to project conceptualization, and performed and analysed rabies tracings. J.C. analysed single-unit data in relation to behaviour. C.X. generated rabies tracing tools, and performed and analysed rabies tracing experiments. L.M. performed the overlap analysis. P.B. performed experiments. K.B., C.M. and A.K. performed experiments and analysed histology. P.T. and A.L. conceptualized the project and wrote the manuscript. All authors contributed to the experimental design and commented on the manuscript.

Correspondence to Philip Tovote or Andreas Lüthi.

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Competing interests

The authors declare no competing financial interests.

Additional information

Reviewer Information Nature thanks K. Tye and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Figure 1 Data from all mice presented in Fig. 1, and single CS conditioned flight and no-shock control groups.

a, The data from Fig. 1d (day 3, conditioning) represented as a line plot to illustrate changes in each mouse. Left, the number of escape jumps (n = 20, Friedman test, P < 0.0001, Dunn’s multiple comparisons test). Middle, flight scores for each mouse. The pre-SCS period is used to calculate the flight score and is therefore plotted as a value of 1 (n = 20, Wilcoxon matched-pairs signed-rank test). Right, freezing values (n = 20; one-way repeated-measures ANOVA, F = 56.82, P < 0.0001, Tukey’s multiple comparisons test). b, The data from Fig. 1e (day 4, extinction) represented as a line plot to illustrate changes in each mouse. Left, flight scores for each mouse. The pre-SCS period is used to calculate the flight score and is therefore plotted as a value of 1 (n = 12; Wilcoxon matched-pairs signed-rank test). Right, freezing values, including the pre-SCS period, during the first block of four trials of extinction (n = 12; Friedman test, P < 0.01, Dunn’s multiple comparisons test). c, The data from Fig. 1f (day 4, retrieval) represented as a line plot to illustrate changes in each mouse. Left, flight scores for each mouse. The pre-SCS period is used to calculate the flight score and is therefore plotted as a value of 1 (n = 8; two-tailed paired t-test). Right, freezing values, including the pre-SCS period (n = 8; one-way repeated-measures ANOVA, F = 49.55, P < 0.0001, Tukey’s multiple comparisons test). d, Schematic of the paradigm used to elicit flight in response to a single CS. e, Two groups of mice were subjected to the flight paradigm and either a pure tone (n = 10) or white noise (n = 10) was paired with a 1 s shock during conditioning. f, Flight scores for each group, across days. While active behaviour developed in both groups, the flight response was significantly greater in the white noise group on day 3 on the first two trials (two-way repeated-measures ANOVA, cue × trial interaction, F(17,153) = 1.81, P < 0.05, Bonferroni’s multiple comparison test). g, Freezing responses for each group, across days. Freezing in the pure tone or white noise group was similar on all trials (two-way repeated-measures ANOVA, cue × trial interaction, F(17,153) = 1.62, P = 0.06). h, Schematic of behavioural paradigm used to test for possible aversive nature of white noise. i, The SCS was presented but was never paired with footshock. j, Left, flight was not observed in the conditioning context in the absence of pairing with footshock (n = 10, two-tailed paired t-test). Right, freezing values remained at low baseline values in the absence of pairing with footshock (n = 10, Friedman test). k, Left, flight was not observed in the neutral context (n = 10, two-tailed paired t-test). Right, freezing values were decreased, not increased, in response to the SCS (n = 10, one-way ANOVA, F = 9.45, P < 0.01, Tukey’s multiple comparisons test). Horizontal lines denote the mean. Values in f and g are means ± s.e.m. *P < 0.05; **P < 0.01. Source data

Extended Data Figure 2 Data related to Fig. 2, and loss-of-function for PKCδ groups, effects of optical inhibition on naive mice, and overlap analysis.

a, Positions of fibre tips for loss-of-function experiments. Placements were determined using standard histological techniques and lesions were matched to a mouse brain atlas. Mouse brain figure reproduced with permission from ref. 31. b, Injection and implantation strategy for identified single-unit recordings used to demonstrate Arch-mediated inhibition. c, Single-unit recordings from identified CEl units in behaving mice. Units were isolated and identified as described (Fig. 3, Extended Data Fig. 3 and Methods). The light was switched on four times for 10 s. Shown for each group is an example raster above and the average peri-event histogram of firing rate below. The light inhibited neuronal activity in all three cell types. d, Expression of Arch in CEl PKCδ+ neurons. Bregma −1.34 mm caudal. Scale bar, 250 μm. e, Average speed traces of PKCδ–Arch mice (n = 8) in the conditioned flight paradigm, illustrating that there was no effect of the light on conditioned flight (see Fig. 2 for protocol). f, Average speed traces from naive mice given four, 10-s light stimulations (GFP control, n = 8;CRF–Arch, n = 7; SOM–Arch, n = 11; PKCδ–Arch, n = 11). g, Flight and freezing values for all mice shown in Fig. 2, plus the PKCδ–Arch group (flight scores: two-tailed paired t-test for GFP control, SOM–Arch, and PKCδ–Arch; Wilcoxon matched-pairs signed-rank test for CRF–Arch; freezing scores: Wilcoxon matched-pairs signed-rank test for GFP control and SOM–Arch; two-tailed paired t-test for CRF–Arch and PKCδ–Arch). Horizontal lines indicate the mean. h, Flight and freezing values for naive mice given four, 10-s light stimulations (Wilcoxon matched-pairs signed-rank test for freezing values). Horizontal lines indicate the mean. i, Example images from overlap analysis between CRF+ neurons and SOM+ or PKCδ+ neurons in the CEA. Arrowhead indicates a rare example of overlap between CRF+ and PKCδ+. Scale bar, 10 μm. j, Percentage of overlap. (PKCδ+, n = 4 sections from two mice; SOM+, n = 6 sections from three mice). Values are means ± s.e.m. *P < 0.05. Source data

Extended Data Figure 3 Data related to Fig. 3, and neuronal responses of identified units to tone on day 3.

a, Positions of electrode lesions and fibre tips for data presented in Fig. 3. Electrolytic lesions were made at the tip of one wire (see Methods) and placements were determined using standard histological techniques. Lesions were matched to a mouse brain atlas. Mouse brain figure reproduced with permission from ref. 31. b, Raster plots and peri-event histograms of firing rate for example neurons from each condition illustrating Arch-mediated inhibition, or ChR2-mediated excitation, of CEl CRF+ and SOM+ cells (an example from the CRF–ChR2 group is presented in Fig. 3). c, Latency to activation/inhibition plotted against z scores for all CRF+ and SOM+ neurons. Average latency: CRF+ = 5.25 ± 0.88 ms; SOM+ = 4.6 ± 0.9 ms. d, Left, waveforms of action potentials were unchanged by light for all unidentified units recorded in Arch-expressing mice with identified units. Centre, firing rates of unidentified units before, during, and after light, illustrating that the light pulses had no effect on unidentified unit activity. Right, z-score transformation of unidentified unit activity during and after light pulses. ej, Normalized activity (z score, bottom) and example raster plots (top) of CEl CRF+ or SOM+ neurons to the tone component of the SCS. e, Neuronal response to the tone component for CRF+ neurons (n = 8) during pre-exposure (day 1). Pre-SCS firing rate: 4.5 ± 1.3 spikes per s. f, CRF+ neurons were not significantly activated by the pure tone on day 3 of the flight paradigm (n = 8). Pre-SCS firing rate: 5.5 ± 2 spikes per s. g, There was no significant activation of CRF+ cells to the tone in the neutral context (n = 7). Pre-SCS firing rate: 5.4 ± 2.3 spikes per s. h, SOM+ neuronal activity (n = 10) was not significantly changed in response to the tone during the pre-exposure session (day 1). Pre-SCS firing rate: 2.7 ± 0.6 spikes per s. i, Neuronal responses of SOM+ neurons to the tone on day 3 of the flight paradigm (n = 10). Pre-SCS firing rate: 5.8 ± 1.3 spikes per s. These higher firing rates are consistent with the pre-SCS contextual freezing observed on this day. j, SOM+ cells (n = 8) are excited by the tone in the neutral context. Pre-SCS firing rate: 2.1 ± 0.4 spikes per s. Values are means ± s.e.m. Source data

Extended Data Figure 4 Data related to Fig. 4, and effects of optical excitation on naive mice and CRF–ChR2 mice during retrieval.

a, Top, positions of fibre tips for CRF+ and SOM+ gain-of-function groups. Placements were determined using standard histological techniques and lesions were matched to a mouse brain atlas. Mouse brain figure reproduced with permission from ref. 31. b, Single-unit recordings from identified CEl units in behaving mice. Units were isolated and identified as described (see Fig. 3, Extended Data Fig. 3 and Methods). The light was switched on four times for 10 s (see Methods). Shown for each group is a raster plot of an example neuron above and a peri-event histogram of the average firing rate below. The light protocols were effective in exciting both neuronal subclasses. c, Average speed traces from naive mice given four, 10-s light stimulations (CRF control, n = 10; CRF–ChR2, n = 7; SOM control, n = 7; SOM–ChR2, n = 7). Blue light caused a decrease in speed in the SOM–ChR2 group. d, Flight and freezing values for naive mice given four, 10-s light stimulations. ChR2-mediated excitation of SOM+ cells induced freezing behaviour (two-tailed paired t-test, CRF–ChR2 and SOM–ChR2; Wilcoxon matched-pairs signed-rank test, CRF control and SOM control). Horizontal lines indicate the mean. e, Flight and freezing values for individual CRF control (left) and CRF–ChR2 (right) mice shown in Fig. 4d (two-tailed paired t-test for CRF control freezing, CRF–ChR2 flight, CRF–ChR2 freezing. Wilcoxon matched-pairs signed-rank test for CRF control flight). Horizontal lines indicate the mean. f, Flight and freezing values for individual SOM control (left) and SOM–ChR2 (right) mice shown in Fig. 4d (two-tailed paired t-test). Horizontal lines indicate the mean. g, The CRF–ChR2 (n = 7) and GFP control (n = 10) groups were subjected to the conditioned flight paradigm. They were placed into the neutral context on day 4 (retrieval) and the SCS was played eight times. On four of the trials, the blue light was turned on during the entire SCS. Left, flight and freezing values for individual mice in the CRF control group (flight scores: Wilcoxon matched pairs signed-rank test; freezing scores: two-tailed paired t-test). Middle, flight and freezing values for individual mice in the CRF–ChR2 group (two-tailed paired t-test). Horizontal lines indicate the mean. Right, average change in freezing and flight between light and no-light conditions. CRF+ excitation significantly reduced conditioned freezing (two-tailed unpaired t-test). Box and whisker plots indicate median, interquartile range, and 5th to 95th percentiles of the distribution. Crosses indicate means. All other values are means ± s.e.m., except as indicated. Source data

Extended Data Figure 5 In vitro slice recording data related to Fig. 4, and bassoon immunolabelling to determine monosynaptic connectivity between CRF+ and SOM+ cells.

a, Top, a light-evoked action potential recorded in cell-attached mode from an example ChR2-expressing CRF+ cell. The average latency to light-evoked spike was 3.3 ± 2.2 ms (n = 4). Bottom left, ChR2 expression in the example CRF+ cell. Bottom right, recorded neurons were filled with biocytin after recording. b, Whole-cell recordings from CEl target cells were performed while stimulating ChR2-expressing CRF+ cells with blue light. c, Top left, example histology showing ChR2–eYFP fibres from CRF+ cells and biocytin-filled cells from whole-cell patch-clamp recording. Top right, IPSCs evoked in a PKCδ+ neuron by brief photostimulation of the CRF+ network (individual traces from one cell in grey, corresponding average IPSC in black). Bottom, immunohistochemistry performed after electrophysiology recordings revealed PKCδ expression. d, Average IPSC amplitudes normalized to cellular capacitance as approximation for cell size. e, Average latency to IPSC peak from start of photostimulation. f, Average rise time of IPSCs (10–90%). For df, no significant differences were observed between SOM+ (n = 23), PKCδ+ (n = 8) or unidentified (n = 13) cells (Kruskal–Wallis test). g, Top, a light-evoked action potential recorded from a ChR2-expressing SOM+ cell. The average latency to light-evoked spike was 3.0 ± 1.1 ms (n = 3). Bottom left, ChR2 expression in the example SOM+ cell. Bottom right, recorded neurons were filled with biocytin after recording. h, Whole-cell recordings from CEl target cells were performed while stimulating ChR2-expressing SOM+ cells with blue light. i, Top left, example histology showing ChR2–eYFP fibres from SOM+ cells and biocytin-filled cells from whole-cell patch-clamp recordings. Top right, IPSCs evoked in a PKCδ+ cell by brief photostimulation of the SOM+ network. Bottom, immunohistochemistry performed after electrophysiology recordings revealed PKCδ expression. j, Average IPSC amplitudes normalized to cellular capacitance. No significant differences were observed between CRF+ (n = 24), PKCδ+ (n = 7) or unidentified (n = 8) cells (Kruskal–Wallis test). k, Average latency to IPSC peak from start of photostimulation (Kruskal–Wallis test P < 0.05; Dunn’s multiple comparisons test, no significant differences). l, Average rise time of IPSCs (10–90%). No significant differences were observed (Kruskal–Wallis test). m, Whole-cell recordings from CEl SOM+ cells were performed while stimulating ChR2-expressing CRF+ cells with blue light. n, Top, IPSCs evoked in a SOM+ neuron by brief photostimulation of the CRF+ network (individual traces from one cell in grey, corresponding average IPSC in black). Bottom, IPSCs were blocked by application of the GABAA-receptor antagonist picrotoxin. o, Whole-cell recordings from CEl CRF+ cells were performed while stimulating ChR2-expressing SOM+ cells with blue light. p, Top, IPSCs evoked in a CRF+ neuron by brief photostimulation of the SOM+ network (individual traces from one cell in grey, corresponding average IPSC in black). Bottom, IPSCs were blocked by application of the GABAA-receptor antagonist picrotoxin. q, The amplitude of light-evoked IPSCs was significantly reduced by application of picrotoxin for both CRF to SOM (n = 3; reduction to 3.9% of baseline amplitude) and SOM to CRF (n = 4; 2.8% of baseline) connectivity (two-tailed paired t-test). r, Left, mCherry label of a SOM+ cell which received inhibitory input from the CRF+ network. Middle, CRF–ChR2+ fibres come in close apposition (arrowheads) to the biocytin-filled SOM+ cell (maximum intensity projection). Right, single confocal planes of appositions labelled with arrowheads in middle panel. Co-immunolabelling with the presynaptic marker bassoon suggests monosynaptic connections between CRF+ and SOM+ neurons. s, Left, tdTomato labelling of a CRF+ cell which received inhibitory inputs from the SOM+ network. Middle, SOM–ChR2+ fibres come in close apposition (arrowheads) to the CRF+ cell. Right, single confocal planes of appositions labelled with arrowheads in middle panel. Co-immunolabelling with the presynaptic marker bassoon suggests monosynaptic connections between SOM+ and CRF+ neurons. Horizontal lines throughout denote the mean. ***P < 0.001. Source data

Extended Data Figure 6 Input structures of CRF+ and SOM+ neurons, and axonal targets of CRF+ cells.

a, Pseudotyped-rabies tracing approach used to identify input areas to the CEA CRF+ or SOM+ population. A Cre-conditional AAV encoding the rabies glycoprotein and a G-deleted EnVA-pseudotyped rabies were injected into the CEA of mice. Mouse brain figure reproduced with permission from ref. 31. b, Left, merged image of glycoprotein and rabies expression localized to CRF+ cells of the CEA. Arrowheads indicate starter cells, which co-express rabies and glycoprotein. Inset, an example starter cell expressing both rabies and glycoprotein. Top right, rabies expression in the CEA. Bottom right, neurons expressing glycoprotein. c, Example regions providing monosynaptic input to the CRF+ network. vHIPP, ventral hippocampus; BLP, basolateral amygdaloid nucleus, posterior part; PVT, paraventricular thalamic nucleus. d, Comparison of the number of presynaptic cells to CRF+ and SOM+ cells, normalized to the total number of presynaptic cells in the defined regions. While there are a similar number of presynaptic neurons across brain regions projecting to SOM+ cells (Friedman test, P = 0.32), there are significantly different numbers of presynaptic neurons projecting to CRF+ cells (repeated-measures one-way ANOVA F = 14.39, P < 0.05). APir, amygdalopiriform transition area; BLA, basolateral amygdala; IC, insular cortex. e, Table listing all raw values from rabies-tracing experiments. f, Injection strategy used to locate terminal fields of CEA CRF+ axonal projections. A Cre-conditional AAV encoding synaptophysin conjugated to GFP was injected into the CEA of CRF-ires-cre mice. Mouse brain figure reproduced with permission from ref. 31. g, Expression of GFP in the CEA illustrating transfected somata and GFP+ puncta representing local axon terminals. h, CRF+ neurons project to multiple structures in the hypothalamus. PH, posterior hypothalamic area; LH, lateral hypothalamic area; PeF, perifornical nucleus; DM, dorsomedial hypothalamic nucleus; MTu, medial tuberal nucleus; VMH, ventromedial hypothalamic nucleus; Arc, arcuate hypothalamic nucleus. i, Demonstration of CRF+ terminals within the VMH. j, CEA CRF+ neurons also project to the peri-aqueductal grey (PAG). dmPAG, dorsomedial PAG; dl/lPAG, dorsolateral/lateral PAG; vlPAG, ventrolateral PAG. k, CEA CRF+ cells project widely throughout PAG columns. Source data

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Fadok, J., Krabbe, S., Markovic, M. et al. A competitive inhibitory circuit for selection of active and passive fear responses. Nature 542, 96–100 (2017) doi:10.1038/nature21047

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