Somatostatin interneurons in the prefrontal cortex control affective state discrimination in mice

Abstract

The prefrontal cortex (PFC) is implicated in processing of the affective state of others through non-verbal communication. This social cognitive function is thought to rely on an intact cortical neuronal excitatory and inhibitory balance. Here combining in vivo electrophysiology with a behavioral task for affective state discrimination in mice, we show a differential activation of medial PFC (mPFC) neurons during social exploration that depends on the affective state of the conspecific. Optogenetic manipulations revealed a double dissociation between the role of interneurons in social cognition. Specifically, inhibition of mPFC somatostatin (SOM+), but not of parvalbumin (PV+) interneurons, abolishes affective state discrimination. Accordingly, synchronized activation of mPFC SOM+ interneurons selectively induces social discrimination. As visualized by in vivo single-cell microendoscopic Ca2+ imaging, an increased synchronous activity of mPFC SOM+ interneurons, guiding inhibition of pyramidal neurons, is associated with affective state discrimination. Our findings provide new insights into the neurobiological mechanisms of affective state discrimination.

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Fig. 1: Mice can discriminate conspecifics based on their affective state.
Fig. 2: Enhanced neuronal activity during exploration of conspecifics in an altered affective state.
Fig. 3: Photoinhibition of PV+ interneurons does not affect affective state discrimination.
Fig. 4: Photoinhibition of mPFC SOM+ interneurons abolishes affective state discrimination.
Fig. 5: Photostimulation of SOM+ interneurons in the mPFC guides social discrimination.
Fig. 6: Photoinhibition of pyramidal cells in the mPFC does not abolish affective state discrimination.
Fig. 7: Concurrent activation of SOM+ interneurons in the mPFC inhibits pyramidal neurons and drives affective state discrimination.

Data availability

The data sets generated and analyzed in the current study are available from the corresponding author on reasonable request.

Code availability

All custom-written analysis code is available from the corresponding author on reasonable request.

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Acknowledgements

We thank M. Morini, D. Cantatore, R. Navone, G. Pruzzo, H. Huang, F. Torri, B. Chiarenza, A. Parodi, A. Monteforte, and C. Chiabrera at Istituto Italiano di Tecnologia and J. Zapata at Inscopix for technical support. The manuscript is certified by the Nature Research Editing Service for English copy editing. This work was supported by funding from the Istituto Italiano di Tecnologia and the Brain and Behavior Research Foundation (2015 NARSAD grant number 23234 to F.P. and 2018 NARSAD grant number 27829 to O.Y.).

Author information

D.S, F. Managò, and F.P. were responsible for the conceptualization of the study. D.S., F. Managò, F. Maltese, S.B., D.D., M.N., P.L., G. Contarini, M.G., L.M.R., V.F., G. Castellani, D.M., A.B., G. Carmignoto, O.Y., and F.P. were responsible for the methodology and investigation. F.P. provided the resources. D.S., F. Managò, O.Y., and F.P. wrote the manuscript. D.S., F. Managò, F. Maltese, S.B., D.D., P.L., M.G.-G., and F.P. were responsible for visualization and analysis. F.P. supervised the study. All the authors revised the manuscript.

Correspondence to Francesco Papaleo.

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Competing interests

The authors declare no competing interests.

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Peer review information Nature Neuroscience thanks Jaideep Bains, Meaghan Creed, 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 Mice can discriminate conspecifics based on the affective state.

Related to Fig. 1. a-f, Data derived from n=8 mice. a, Top, Experimental design of the ADT. One demonstrator was given water access for 1 hour before the test, after 23 hours of water deprivation (‘relief’, yellow), while the other demonstrator had ad libitum water access (‘neutral’, grey). Increased exploration (sniffing, in seconds) to the relieved compared to neutral demonstrator (left, showed in 120-seconds beams, two-tailed multiple t-test, Bonferroni correction, 2 min: t=3.85, df=14, p=0.005; right, showed in 60-seconds beams, 60s: t=3.6, df=14, p=0.017, 120s: t=2.77, df=14, p=0.014). b, No difference in average number of visits to each zone (two-tailed multiple t-test, Bonferroni correction, t=0.22, df=14, p>0.999) c, Longer visits of observers in the zone related to the relief demonstrators (two-tailed multiple t-test, Bonferroni correction, 2 min: t=3.89, df=14, p=0.004). d, Average distance of observers’ head to the relieved and the neutral demonstrators did not differ during ADT (two-tailed paired t-test, t=1.22, df=7, p=0.261). e, In female mice increased sniffing to the relieved compared to neutral, sex-matched, demonstrator (two-tailed multiple t-test, Bonferroni correction, 2 min: t=3.32, df=18, p=0.011; n=10 mice). f, No difference in grooming (two-tailed multiple t-test, Bonferroni correction, t=0.45, df=14, p=0.657) and rearing behaviors (t=0.00, df=14, p=0.998), and locomotor activity (one-way ANOVA, F(2,21)=1.59, p=0.226), displayed by the observers during the ADT with the neutral and the relieved demonstrators. g-l, Data derived from n=7 mice. g, Top, In the stress protocol one demonstrator (‘stress’, purple) was subjected to restraint stress test for 15 minutes culminating in the beginning of ADT. The other demonstrator (‘neutral’, grey) waited undisturbed in his home-cage. Bottom, mice showed increased sniffing to the stressed compared to neutral demonstrator (left, showed in 120-seconds beams, two-tailed multiple t-test, Bonferroni correction, 2 min: t=2.11, df=12, p=0.05; right, showed in 60-seconds beams, 60s: t=3.67, df=10, p=0.02, 120s: t=2.12, df=10, p=0.05; n=6 mice). h, Average number of visits to each zone did not differ (two-tailed multiple t-test, Bonferroni correction, t=0.81, df=12, p>0.999). i, Observers made longer visits in the zone related to the stressed demonstrators (two-tailed multiple t-test, Bonferroni correction, 2 min: t=3.46, df=12, p=0.017). j, Shorter average head distance of observer mice to the stressed demonstrator compared to neutral during the ADT (two-tailed paired t-test, t=5.31, df=7, p=0.001). k, Female mice showed increased sniffing to the stressed compared to neutral, sex-matched, demonstrators (two-tailed multiple t-test, Bonferroni correction, 2 min: t=2.69, df=18, p=0.044; n=11 mice). l, No difference in grooming (two-tailed multiple t-test, Bonferroni correction, t=0.84, df=12, p=0.708) and rearing behaviors (t=1.20, df=12, p=0.578), and locomotor activity (one-way ANOVA, F(2,18)=0.72, p=0.498) displayed by the observers during the ADT with the neutral and the stressed demonstrators. m, No correlation between discrimination index, calculated for the first two minutes of ADT, and grooming behavior of the stressed demonstrators (n=16, Pearson’s r=0.017, linear regression showed no significant deviation from zero, two-tailed p=0.949). n, No difference of average plasma corticosterone levels after ADT with two neutral demonstrators (gray), one relieved and one neutral (yellow), one stressed and one neutral demonstrator (purple) (one-way ANOVA, F(2,13)=1.08, p=0.367). o, First and second testing in the same ADT (‘relief’) showed similar behavioral pattern with increased sniffing towards the relieved demonstrator compared to the neutral (ADT 1: two-tailed multiple t-test, Bonferroni correction, 2 min: t=2.25, df=20, p=0.035; ADT 2: t=3.99, df=20, p=0.002; n=11). p, For each observer tested in the ADT with both protocol (relief and stress) a discrimination index was calculated to compared performance on ADT 1 (red) and ADT 2 (blue; discrimination index = exploration of “relief”/”stress” - exploration of “neutral” / total time of exploration). Positive index means discrimination between “affectively-altered” and “neutral”. Of 41 mice tested in ADT 1 and ADT 2 only 6 did not show a positive discrimination index on second testing. Average discrimination index did not differ between ADT 1 and ADT 2 (two-tailed unpaired t-test, t=1.73, df=80, p=0.089; n=41 mice). Bar and line graphs show mean ± s.e.m. * p<0.05, ** p<0.005. n.s. not significant.

Extended Data Fig. 2 Affective state discrimination is enhanced between familiar conspecifics.

Related to Fig. 1. a-f, Data derived from n=7 mice. a, Top, Experimental design of the ADT with cagemates demonstrators. Observer and demonstrators were singly-housed 23 hours before testing. One demonstrator was given water access for 1 hour before the test, after 23 hours of water deprivation (“relief”, yellow), while the other demonstrator had ad libitum water access (“neutral”, gray). b, Increased sniffing to the relieved compared to neutral demonstrator (two-tailed multiple t-test, Bonferroni correction, 2 min: t=3.60, df=12, p=0.010, 4 min: t=3.35, df=12, p=0.017). c, Increased time spent in the zone related to the relieved demonstrator compared to the neutral (two-tailed multiple t-test, Bonferroni correction, 2 min: t=3.21, df=12, p=0.022, 4 min: t=2.37, df=12, p=0.035). d, Latency to make the first visit to the relieved demonstrator compared to the neutral (1.95±1.0 relief, 7.14±3.0, two-tailed paired t-test, t=1.37, df=6, p=0.13). e, Average number of visits to each zone did not differ (two-tailed multiple t-test, Bonferroni correction: t=0.63, df=12, p>0,999). f, When tested with cagemates, discrimination of relieved versus neutral demonstrators was longer as discrimination index was increased compared to mice tested with unfamiliar demonstrators (two-tailed multiple t-test, Bonferroni correction, 4 min: t=2.07, df=13, p=0.05). g-l, Data derived from n=7 mice. g, In the stress protocol using cage-mates, one demonstrator (“stress”, purple) was subjected to restraint stress test for 15 minutes culminating in the beginning of ADT. The other demonstrator (“neutral”, grey) waited undisturbed in his home-cage.. h, Increased sniffing to the stressed compared to neutral demonstrator (two-tailed multiple t-test, Bonferroni correction, 2 min: t=4.27, df=12, p=0.003; 6 min: t=5.16, df=12, p=0.0007). i, Increased time spent in the zone related to the stressed demonstrator compared to the neutral (two-tailed multiple t-test, Bonferroni correction, 6 min: t=6.13, df=12, p=0.0001). j, Shorter latency to make the first visit to the stressed demonstrator compared to the neutral (paired t-test, t=2.31, df=6, p=0.05). k, Average number of visits to each zone did not differ (two-tailed multiple t-test, Bonferroni correction: t=0.53, df=12, p>0,999). l, When tested with cage-mates, discrimination of the stressed versus the neutral demonstrators was longer as discrimination index was increased compared to mice tested with unfamiliar demonstrators (two-tailed multiple t-test, Bonferroni correction, 6 min: t=3.45, df=11, p=0.01). m, To rule out that social isolation 23 hours before testing (to allow water restriction of one cage-mate – “relief”) could have affected ADT with familiar mice, we tested singly-housed observers with unfamiliar demonstrators. Mice showed increased sniffing towards the relieved demonstrators (two-tailed multiple t-test, Bonferroni correction, 6 min: t=5.48, df=12, p=0.0004, n=7) and increased time spent in the related zone (two-tailed multiple t-test, Bonferroni correction, 6 min: t=2.86, df=12, p=0.041), only during the first 2 minutes of ADT, and not further, as showed in (b). Bar graphs show mean ± s.e.m. * p<0.05, ** p<0.005. *** p<0.0005.

Extended Data Fig. 3 Enhanced neuronal activity during exploration of conspecifics in an altered, but not neutral, affective state.

Related to Fig. 2. a, Up left, mice implanted with chronic recording electrodes showed increased exploration of the relieved demonstrator compared to the neutral one, during the first 2 minutes of testing (two-tailed unpaired t-test: t=2.33, df=10, p=0.0418; n=6 mice). Up right, electrode placement in the mPFC (Cg, cingulate; PL, prelimbic area; IL, infralimbic area). Bottom, increased average population (N=57) activity during the epoch before the exploration and the epoch in which the observer explored the relieved demonstrator (yellow), compared to the pre social exploration epoch (min2: p<0.0001; min4: p<0.0001; min6: p=0.000696) and compared to exploration of the neutral (gray, min2: p=0.020031, min4: p=0.016927; min6: p=0.47572; 3x2 RM ANOVA, exploration epoch and affective state, min2: F(2,112)=52.75, p<0.0001; min4: F(2,112) =54.75, p<0.0001; min6: F(2,112)=24.79, p<0.0001). The black arrows indicate the time of onset (upward arrow) and end (downward arrow) of significant separation of the activity between the two compared conditions. Increased average population activity during the end social exploration epoch of the relieved demonstrator compared to that during the following epoch (post social exploration epoch, min2: p=0.045877; min4: p=0.000218; min6: p=0.049170) and compared to the neutral (min2: p=0.000275; min4: p=0.001698; min6: p=0.001408 2x2 RM ANOVA, exploration epoch and affective state, min2: F(1,56)=7.07, p=0.010161; min4: F(1,56)=7.17, p=0.009716; min6: F(1,56)=4.7, p= 0.034324). b, Hierarchical clustering method was used to separate recorded cells, during the ADT with one relieved and one neutral demonstrator, into the following populations: wide spiking (putative pyramidal cell, green, N=33) or narrow spiking (putative interneurons; orange, N=24). c, Frequency distribution of preference indexes (PIs) for the relief or neutral affective state in the NS (orange) and WS (green) neuronal population during the exploration of conspecifics during all the test. The size of each circle is proportional to the number of single neurons (from N=1 to N=8). Independent-samples t-tests, min2: t=3.39015, p<0.002095; min4: t = 2.1174, p=0.04478; min6: 2.2808, p=0.031349. d, Up, mice showed more exploration of the stressed demonstrator than of the neutral demonstrator during the first 2 minutes of testing (n=7 mice, two-tailed unpaired t-test: t=4.12, df=12, p=0.0014). Bottom, increased average population (n=83) activity during the start social exploration epoch and the social exploration epoch towards the stressed demonstrator (purple), compared to the pre social exploration epoch (min2: p<0.0001; min4: p=0.000003; min6: p<0.0001) and compared to exploration of the neutral (gray, min2: p=0.006666; min4: p=0.001223; min6: p=0.000027; 3x2 RM ANOVA, exploration epoch and affective state, min2: F(2,164)=45.13, p<0.0001; min4: F(2,164)=60.42, p<0.0001; min6: F(2,164)=74.44, p<0.0001). Increased average population activity during end social exploration epoch of the stressed demonstrator compared to the following epoch (post social exploration epoch, min2: p<0.0001; min4: p=0.000002; min6: p=0.000001) and to the exploration of the neutral demonstrator (min2: p<0.0001; min4: p<0.0001; min6: p<0.0001; 2x2 RM ANOVA, exploration epoch and affective state, min2: F(1,82)=116.08, p<0.0001; min4: F(1,82)=48.95, p<0.0001; min6: F(1,82)=21.86, p<0.0001). e, Classification of recorded cells, during the ADT with one stressed and one neutral demonstrator, in NS (N=52) and WS as described in b. f, Frequency distribution of preference indexes (PIs) for the stress or neutral affective state in the NS (orange) and WS (green) neuronal population during the exploration of conspecifics during all the test. The size of each circle is proportional to the number of single neurons (from N=1 to N=13). Independent-samples two-tailed t-tests, min2: t = 2.62995, p=0.011206; min4: t = 4.55371, p=0.000029; min6: t = 3.43784, p=0.001137. g, Mice have been implanted with recording electrodes in the mPFC and tested in the ADT with two naïve “neutral” demonstrators. Mice equally explored the two demonstrator and did not show observable discrimination. h, Classification of the recorded cells (N=82) in NS (N=55) and WS cells (N=27) as described in b during the ADT with two neutral demonstrators. i, Top, population responses calculated as an average of the activity of NS neurons (N=55) during “Habituation”, “Pre-exploration”, “Exploration onset”, “Exploration offset”, and “Post-exploration” periods towards neutral demonstrator 1 (pink) and neutral demonstrator 2 (green) (mean ± s.e.m.). *p<0.05 versus exploration of the neutral mouse. Bottom, normalized population activity of NS neurons (n=55) during exploration of both neutral demonstrators was stronger compared to the entire pre-exploration period (min2: p=0.0417281; min4: p=0.016125; min6: p=0.047255) without any difference between the two mice (min2: p=0.135825; min4: p=0.789647; min6: p=0.666470; 3x2 RM ANOVA, exploration epoch and affective state, min2: F(2,108)=0.61, p=0.55; min4: F(2,108)=0.67, p=0.51; min6: F(2,108)=2.06, p=0.07). Other legends as in (c). j, Same analyses as in (i) but for WS (N=27). Top, population responses calculated as an average of the activity of WS neurons during “Habituation”, “Pre-exploration”, “Exploration onset”, “Exploration offset”, and “Post-exploration periods” towards two neutral demonstrators (mean ± s.e.m.). *p<0.05 versus exploration of the neutral mouse. Bottom, Normalized population activity of WS neurons (n=27) during exploration of both neutral demonstrators was stronger compared to the pre-exploration period (min2: p=0.028877; min4: p=0.0499772; min6: p=0.00326), without any difference between the two mice (min2: p=0.356287; min4: p=0.598312; min6: p=0.583072; 3x2 RM ANOVA, exploration epoch and affective state, min2: F(2,52)=0.9, p=0.4; min4: F(2,52)=0.8, p=0.45; min6: F(2,52)=2.8, p=0.07).

Extended Data Fig. 4 Photoinhibition of PV+ interneurons does not affect affective state discrimination.

Related to Fig. 3. a, Top, representative images of viral expression in the mPFC after injection with AAV-EF1a-DIO-eNpHR3.0-eYFP. Bottom, reconstruction of viral expression and location of optical fibers. Red areas represent the expression (higher expression = darker color) of AAV-EF1a-DIO-eNpHR3.0-eYFP in PV-cre mice. Findings were replicated in two independent experiments with similar results. Data derived from n=7 mice. b, PV-cre mice were tested in the ADT with one relieved and one neutral demonstrator. Photo-inhibition was performed for 2 minutes, from the beginning of the test, using continuous green light. c, No effect of PV+ photoinhibition on latency to made the first visit to the relieved demonstrator (two-way RM ANOVA, affective state (relief, neutral): F(1,12)=7.18, p=0.020). d, Optical inhibition of PV+ did not modify the number of visits to each demonstrator (two-way RM ANOVA, affective state (relief, neutral) x light (off,on): F(1,24)=0.38, p=0.541). e, PV-cre mice were tested in the ADT with one stressed and one neutral demonstrator. Photoinhibition was performed for 2 minutes, from the beginning of the test, using continuous green light. Data derived from n=7 mice. f, No effect of PV+ photoinhibition on latency to made the first visit to the stressed demonstrator (two-way RM ANOVA, affective state (stress, neutral): F(1,12)=12.75, p=0.003). g, Optical inhibition of PV+ did not modify the number of visits to each demonstrator (two-way RM ANOVA, affective state (stress, neutral) x light (off,on): F(1,24)=0.30, p=0.587). h and i, Optical inhibition of PV+ did not induce gross motor deficits during ADT in both relief (two-tailed multiple t-test, Bonferroni correction, distance travelled: t=0.35, df=12, p=0.730; average speed: t=1.10, df=12, p=0.290) and stress conditions (distance travelled: t=1.19, df=12, p=0.254; average speed: t=0.47, df=12, p=0.640). Bar and line graphs show mean ± s.e.m. * p<0.05.

Extended Data Fig. 5 Photoinhibition of SOM+ interneurons abolishes affective state discrimination.

Related to Fig. 4. a, Top, representative images of viral expression in the mPFC (in rostro-caudal order) after injection with AAV-EF1a-DIO-eNpHR3.0-eYFP. Bottom, reconstruction of viral expression and location of optical fibers. Red areas represent the expression (higher expression = darker color) of AAV-EF1a-DIO-eNpHR3.0-eYFP in SOM-cre mice. Findings were replicated in four independent experiments with similar results. b, Increased exploration toward the odor of the relieved demonstrators compared to the neutral in light off and light on conditions (two-way RM ANOVA, affective state (relief, neutral): F(1,14)=12.65, p<0.005, n=8 mice), and no effects of SOM+ photo-inhibition (F(1,14)=0, p=0.99). c, Avoidance of the odor of the stressed demonstrators in in light off and light on conditions (two-way RM ANOVA, affective state (stress, neutral): F(1,10)=76.21, p<0.0001, n=6 mice), which SOM+ photo-inhibition did not change (F(1,10)=0.33, p=0.576). d and e, SOM+ photoinhibition with continuous green light for two minutes did not induce any gross motor change in both relief (two-tailed multiple t-test, Bonferroni correction, distance travelled: t=0.57, df=12, p=0.576; average speed: t=0.50, df=12, p=0.625) and stress conditions (distance travelled: t=0.69, df=12, p=0.498; average speed: t=0.45, df=12, p=0.658). Data derived from n=7 mice in each condition (relief, stress). f, Exploration of the relieved demonstrator was paired to SOM+ photo-inhibition throughout the test (n=8 mice). g, No preference to spend more time with the relieved demonstrator during photo-inhibition of SOM+, on the first two minutes of ADT (two-tailed multiple t-test, Bonferroni correction: t=0.47, df=14, p>0.999). h, No change of number of visits to each demonstrator during photoinhibition of SOM+ (two-tailed multiple t-test, Bonferroni correction: t=0.88, df=14, p>0.999). i, SOM+ photoinhibition paired to exploration of the relieved demonstrators did not induce any gross motor changes. j, Exploration of the relieved demonstrator was paired to SOM+ photo-inhibition throughout the test (n=9 mice). k, No difference in time spent with the two demonstrators during photoinhibition of SOM+ (two-tailed multiple t-test, Bonferroni correction: t=0.19, df=16, p>0.999). l, No difference of number of visits to stressed and neutral demonstrator during inhibition of SOM+ (two-tailed multiple t-test, Bonferroni correction: t=0.11, df=16, p>0.999). m, SOM+ photoinhibition paired to exploration of the stressed demonstrators did not induce any gross motor changes. n, Exploration of one naïve “neutral” demonstrator (“neutral 1”) was paired to SOM+ photoinhibition throughout the ADT (counterbalanced, left or right, across observers, continuous green light). Data derived from n=8 mice. SOM+ photoinhibition did not induce social discrimination or avoidance conditions (two-way RM ANOVA, time x light (on,off): F(359,2513)=0.21, p>0.999). No discrimination of the two neutral demonstrators without light stimulation (“No light”, two-way RM ANOVA, time x light (on,off): F(359,2513)=0.19, p>0.999). Bar and line graphs show mean ± s.e.m. * p<0.05.

Extended Data Fig. 6 Optogenetic light pulses inhibit AP firing in YFP-eNpHR3.0-expressing SOM+ and PV+ interneurons.

a, Left, AP firing induced in an YFP-eNpHR3.0-expressing PV+ interneuron from a brain slice by a steady-state depolarizing current pulse (800 ms duration). Right, representative experiment showing the APs successfully evoked by 4 ms current pulses (injected at 2 Hz) in the absence of 532 nm light (black trace) and the depolarizations evoked by 4 ms current pulses during the 120 s 532 nm light pulse (green line) which fail to reach AP threshold (red trace). Inset, enlarged time-scale showing an AP (black trace) in the absence and a subthreshold depolarization (red trace) in the presence of green light. b, Same as in (a), but from an YFP-eNpHR3.0-expressing SOM+ interneuron. c, Summary of the laser power (mW) that inhibits (as in a, b) or fails to inhibit the AP firing for each interneuron (n=10 SOM+ cells from n=2 mice, n=6 PV+ cells from n=2 mice). The bar chart shows the mean (+/- s.e.m.) laser power inhibiting AP firing from the recorded cells (SOM+ cells, 27.8 ± 4.45; PV+ cells, 30.25 ± 7.55; Mann-Whitney Rank Sum Test, p=0.708). Error bars show s.e.m. d, Optogenetic-assisted tetrode recordings in the mPFC in AAV-EF1a-DIO-eNpHR3.0-eYFP injected SOM-cre mice; representative example (one neuron with 11 stimulations) of a peristimulus time histogram showing the firing rate (mean +/- s.e.m.) of an inhibited putative SOM interneuron, before during and after the delivery of the green light pulse (for 120 seconds).

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Scheggia, D., Managò, F., Maltese, F. et al. Somatostatin interneurons in the prefrontal cortex control affective state discrimination in mice. Nat Neurosci 23, 47–60 (2020). https://doi.org/10.1038/s41593-019-0551-8

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