RETRACTION POST DATE • 20 DECEMBER 2019 1SCIENCE sciencemag.org

The online Research Article “Insular cortex processes aversive somatosensory information and is crucial for threat learning” used optogenetic methods in mice to conclude that insular cortex is involved in auditory cued fear learning (1). A reanalysis of the data per-formed by the authors in October 2019 showed, however, that the mouse behavior data reported in Figs. 1C, 3C, 3F, and 6B, and the corresponding data in supplementary fig-ures, had been manipulated. The reanalysis showed that data points from many individu-al mice had been moved, with the effect that the difference between optogenetic silencing

groups and control groups became larger than in the real data. Thus, in the reanalyzed data, the statistical significance disappears for many datasets of Figs. 1, 3, and 6, and these experiments need to be reestablished in future work. The first author, who performed these measurements, has admitted to having committed the data falsification. No other coauthors were involved in the data manipulation, and thus their data (Figs. 2 and 5 and supplementary figs. S2, S4–S6, S11, and S15–S18) remain valid. Because the data manipulations affect important conclusions of the paper, the authors retract the Research Article. We apologize to the readership of Science.

Emmanuelle Berret, Michael Kintscher, Shriya Palchaudhuri, Wei Tang, Denys Osypenko, Olexiy Kochubey, Ralf Schneggenburger*

Laboratory of Synaptic Mechanisms, Brain Mind Institute, School of Life Science, École Polytechnique Fédérale de Lausanne (EPFL), 1015 Lausanne, Switzerland.*Corresponding author: Email: ralf.schneggenburger@epfl.ch

REFERENCES AND NOTES

1. E. Berret et al., Science 364, eaaw0474 (2019).

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RESEARCH ARTICLE SUMMARY◥

NEUROSCIENCE

Insular cortex processes aversivesomatosensory information and iscrucial for threat learningEmmanuelle Berret, Michael Kintscher, Shriya Palchaudhuri, Wei Tang,Denys Osypenko, Olexiy Kochubey, Ralf Schneggenburger*

INTRODUCTION:Animals and humans needto learn about potential dangers in the envi-ronment to guarantee survival. This threatlearning can be studied in laboratory animalswith fear conditioning paradigms, in which aninnocuous sensory event such as a tone [theconditioned stimulus (CS)] comes to predict apotentially harmful event such as a footshock[the unconditioned stimulus (US)]. Duringthreat learning, an association between thesensory representations of the CS and US isformed in a brain area called the amygdala,especially in the lateral amygdala (LA). How-ever, the synaptic pathways that carry informa-tion about a footshock to the LA are unknown.More generally, it has not been addressedwhich brain area(s) upstream of the amyg-dala process aversive somatosensory eventsand conduct this information to the amygdala.

RATIONALE: To address this question, wetook advantage of optogenetic approaches inbehaving mice. We concentrated on the in-sular cortex, which is known to send axons tothe amygdala and to respond to tones andsomatosensory stimulation. Mice were trainedto acquire a fear response, assessed as im-mobility (freezing), when a tone was pairedwith amild footshock. Our principal approachwas to use an inhibitory optogenetic proteinto suppress action potential (AP) firing ininsula neurons or in the axons connectingthe insula with the amygdala. We hypothe-sized that if the insula sends information aboutthe US to the amygdala, then this manipula-tion should suppress threat learning.

RESULTS: Silencing AP activity in the pos-terior insular cortex suppressed acute fear

behavior and strongly impaired the forma-tion of threat memories 1 day later, whentones were applied alone. Anatomical tracingand ex vivo electrophysiological experimentsthen showed that two largely separate neuronpopulations in the insular cortex form strongexcitatory synapses with neurons in the LAor the central amygdala (CeA). Silencing theprojection from the insular cortex to the CeAduring the US reduced acute fear behavior,

whereas silencing the pro-jection to the LA impairedthe formation of a threatmemory 1 day later butleft acute fear behaviorunchanged. Complemen-tary experiments with an

excitatory optogenetic protein showed thatactivation of the insular neurons that tar-get the CeA (CeA projectors) rapidly initiatedimmobility, but this manipulation did notresult in an aversive memory. Conversely, opto-genetic activation of the LA projectors pairedwith a tone led to strong aversive behaviorsand, on the next day, to escape-like behaviorswhen the tone was presented alone, showingthat stimulation of LA projectors creates anaversive memory. In vivo recordings showedthat about one-quarter of the neurons in theposterior insular cortex responded to foot-shocks (the US). A similar but not completelyoverlapping neuron population acquired aresponse to the tones when these were re-inforced by footshocks during the threat learn-ing paradigm. Finally, silencing the posteriorinsular cortex during tone presentation onthe retrieval day revealed a contribution ofthe insula to threat memory retrieval.

CONCLUSION: The insular cortex is intri-cately involved in processing aversive somato-sensory information. Silencing the posteriorinsular cortex largely removes the aversivequality of footshock stimulation, thus sup-pressing an essential drive for learning aboutsuch harmful events. The insular cortexroutes information to specific amygdalarsubdivisions and can thus drive temporallyseparate components of fear behavior. Fur-thermore, the insula forms associations be-tween innocuous and harmful sensory eventsand, together with the LA, is necessary forthe retrieval of threat memories. Taken to-gether, the posterior insula processes aver-sive somatosensory events and contributes toelaborate their negative valence.▪

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Berret et al., Science 364, 850 (2019) 31 May 2019 1 of 1

Laboratory of Synaptic Mechanisms, Brain MindInstitute, School of Life Science, École PolytechniqueFédérale de Lausanne (EPFL), 1015 Lausanne,Switzerland.*Corresponding author: Email: ralf.schneggenburger@epfl.chCite this article as E. Berret et al., Science 364, eaaw0474(2019). DOI: 10.1126/science.aaw0474

Optogenetic studies to determine aversive signaling in brain areas upstream of theamygdala. Optogenetic silencing of the insula during the footshock leads to reduced threatlearning (left). Largely separate neuron populations of the insular cortex project to theLA (red neurons) and the CeA (green neurons) (middle). Selective silencing of the projection toeither the CeA or LA impairs different phases of threat learning (right).

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RESEARCH ARTICLE◥

NEUROSCIENCE

Insular cortex processes aversivesomatosensory information and iscrucial for threat learningEmmanuelle Berret, Michael Kintscher, Shriya Palchaudhuri, Wei Tang,Denys Osypenko, Olexiy Kochubey, Ralf Schneggenburger*

Learning about threats is essential for survival. During threat learning, an innocuoussensory percept such as a tone acquires an emotional meaning when paired with anaversive stimulus such as a mild footshock. The amygdala is critical for threat memoryformation, but little is known about upstream brain areas that process aversivesomatosensory information. Using optogenetic techniques in mice, we found that silencingof the posterior insula during footshock reduced acute fear behavior and impaired 1-daythreat memory. Insular cortex neurons respond to footshocks, acquire responses totones during threat learning, and project to distinct amygdala divisions to driveacute fear versus threat memory formation. Thus, the posterior insula conveys aversivefootshock information to the amygdala and is crucial for learning about potentialdangers in the environment.

Animals and humans need to learn aboutpotential dangers in the environment.Strong mechanisms of threat learningare therefore present in most animalspecies (1, 2). In classical fear condition-

ing (3), animals learn to associate an innocuoussensory event such as a tone [the conditionedstimulus (CS)] with an inherently aversiveevent such as a mild electrical footshock [theunconditioned stimulus (US)]. After threatlearning, the CS acquires an emotional valueand then induces a defensive response suchas freezing behavior (immobility) (4, 5). Thelateral amygdala (LA) has been identified as abrain structure that is critical for threat learn-ing and in which an association between theCS and the US takes place (5–10). Auditory in-formation reaches the LA from the auditorythalamus and cortex (5, 11, 12). However, thebrain areas upstream of the amygdala thatprocess information about aversive somato-sensory events and transfer this information tothe amygdala have remained elusive (13–15).The insular cortex processes multisensory

information, including visceral (16), gustatory(17, 18), somatosensory (19), and auditory mo-dalities (19–21). It provides output to the auto-nomic nervous system (22), and the gustatoryarea of the insular cortex mediates approachor avoidance behavior in response to differenttastants (18, 23–25). Functional imaging in hu-mans suggests that the insula is involved in

multimodal sensory processing and some cog-nitive functions (26). The human insular cortexalso becomes activated by painful stimuli aspart of a larger pain network (27), and patientswith stroke-induced lesions that include theinsular cortex suffer from pain asymbolia, theabsence of emotional response to painful stimuli(28). Nevertheless, whether the insular cortexprovides information about aversive somato-sensory events to the amygdala during threatlearning has remained unclear (13, 29).

Silencing the posterior insular cortexsuppresses threat learning

We used in vivo optogenetic methods in behav-ing mice to test whether the insular cortexmightprocess footshock information relevant for threatlearning.Micewere exposed to six tone blocks onday 1 in a habituation session (Fig. 1A). On day 2(training day), a 1-s footshock was given aftereach tone block, which induced freezing (4). Onday 3, four tone blocks were given without afootshock in a different context, to test the re-trieval of the auditory-cued threat memory (5)(Fig. 1A). Because we hypothesized that the in-sular cortex might code for footshock informa-tion, we aimed to inactivate the insular cortexselectively during each footshock. For this, weused an adeno-associated virus (AAV) driving theexpression of the light-sensitive Cl− transporterhalorhodopsin (eNpHR3.0) (30). The AAV wasinjected bilaterally in the posterior insular cortices4 weeks before behavioral testing, and an opticalfiber was implanted over each injection site (Fig.1B and fig. S1). On the training day, yellow lightwas applied for 3 s beginning 1 s before the foot-shock (Fig. 1A);mice expressing enhanced green

fluorescent protein (eGFP) served as control ani-mals (see the Materials and methods section).Control experiments showed that yellow lightstrongly hyperpolarizedhalorhodopsin-expressingneurons in the insular cortex, whereas in naïveinsula neurons without halorhodopsin expres-sion, yellow light did not influence the electri-cal signaling (fig. S2).In control mice, an increasing freezing re-

sponse with each tone presentation was ob-served during the training (day 2). During threatmemory retrieval (day 3), the mice showed pro-nounced freezing in response to the tone (Fig. 1C,black data). In contrast, in the halorhodopsin-expressing group, freezing was significantly re-duced [analysis of variance (ANOVA); P < 0.001;F1,176 = 140.4]. A post hoc Bonferroni test showedthat freezing was significantly reduced in thehalorhodopsin group compared with the controlgroup during the third to sixth tone–footshockpairing on the training day and during all fourtone presentations on the retrieval day (Fig. 1C;P < 0.05 and 0.001). Control mice displayedprolonged freezing during the fifth and sixthtone–footshock pairing on the training day,whereas the halorhodopsin-expressing micemoved normally and continued to explore theenvironment at the corresponding times, withonly brief bouts of freezing (movie S1; pooledanalysis of the last two CS–US pairings, P <0.001, t test; Fig. 1D, left). Similarly, the averagefreezing level in response to the n = 4 CS pre-sentations on the retrieval day was significantlyreduced in the halorhodopsin group comparedwith the control group (Fig. 1D, P < 0.001, t test;see fig. S3 for freezing levels for individual mice).On the other hand, mice in the halorhodopsingroup displayed unchanged acute jumping inresponse to the footshock (fig. S3), indicatingthat not all aspects of the painful stimulus wereblocked by silencing the posterior insular cortex.Post hoc mapping of the injection sites and ofthe optical fiber placement sites confirmed thata region in the granular part of the posteriorinsular cortex was silenced (Fig. 1E).

Insular cortex makes functionalconnections with LA and CeA

Given the role of insular cortex activity for threatlearning shown in Fig. 1, we mapped the poten-tial output connections from the insular cortex tothe amygdala (31–33), both anatomically and func-tionally (Fig. 2). Anterograde tracing using AAV8:hSyn:Chronos-eGFP injected into the posterior in-sular cortex (seeMaterials andmethods) showedfibers in the anterior part of the LA and the basalamygdala (BA), in the anterior part of the centralamygdala (CeA), as well as in the posterior partof the BA (Fig. 2, A and B). To validate that theseoutput fibers are functional and to assess the neu-rotransmitter employed at these putative connec-tions, weused an ex vivo optogenetic approach formapping long-range connections (34). Specifically,wemade recordings in amygdala slices 4 weeksafter the expression of Chronos-eGFP in the insu-lar cortex (fig. S4). In all neurons recorded in theanterior LA and anterior CeA, local stimulation

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Laboratory of Synaptic Mechanisms, Brain Mind Institute,School of Life Science, École Polytechnique Fédérale deLausanne (EPFL), 1015 Lausanne, Switzerland.*Corresponding author: Email: ralf.schneggenburger@epfl.ch

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by blue light caused excitatory postsynaptic cur-rents (EPSCs)with a fast, NBQX (2,3-dihydroxy-6-nitro-7-sulfamoylbenzo[f]quinoxaline)–sensitivecomponent at negative holding potentials. Atpositive potentials, an additional slow APV(D,L-2-amino-5-phosphonovaleric acid)–sensitivecomponent was observed, which identifies theconnections from the posterior insular cortex tothe LA andCeA as glutamatergic (Fig. 2, C andD).Control experiments at the insula-to-LA con-nection showed that EPSCs were blocked bytetrodotoxin (1 mM) and partially recoveredwhenthe K+ channel blocker 4-aminopyridine (1 mM)was coapplied with tetrodotoxin, showing thatthis connection is monosynaptic (fig. S4). EPSCamplitudes increased gradually with stimulusstrength, indicative of compound EPSCs com-posed of smaller unitary EPSCs (fig. S4). In the LA,postsynaptic neurons included CaMKII-positivepyramidal cells, which were targeted for record-ing by the use of CaMKIICre x tdTomato mice.Furthermore, EPSCs with similar amplitudeswere observed when Chronos-eGFP expressionwas targeted to CaMKII-positive neurons inthe insular cortex or when Chronos-eGFP wasexpressed nonselectively (Fig. 2C), suggestingthat many presynaptic neurons in the insula

were CaMKII-positive. In the CeA, a brain struc-ture rich in inhibitory neurons (35), EPSCs withapproximately similar amplitudes were observedin somatostatin-positive (SOM+) and somatostatin-negative (SOM−) neurons, as revealed by SOMCre xtdTomato mice (fig. S4), in agreement with a re-cent study (24). Taken together, neurons in the pos-terior insular cortex make strong glutamatergicoutput synapses in both the CeA and the LA.We next analyzed the degree of overlap of the

neuron populations in the posterior insula thatproject to the LA and to the CeA. We employeddouble-retrograde labeling experiments withinjections of green- or red-labeled choleratoxinB (CTB-A488 or CTB-A647) into the CeA and LA(Fig. 2E). This revealed two largely segregatedpopulations of LA and CeA projectors (Fig. 2, Fand G, and figs. S5 and S6; n = 3 mice). LA pro-jectors were found in the secondary somato-sensory cortex (S2, ventral part) and in thegranular and dysgranular part of the posteriorinsular cortex. On the other hand, CeA projec-tors were localized in various layers of the dys-granular and agranular posterior insular cortexbut could also be sparsely found in the deeperlayers of the granular posterior insular cortex(Fig. 2, F and G, and fig. S6). The overlap be-

tween the two populations was weak, especiallyin S2 and the granular posterior insular cortex(<1% for S2; <1, 5, and 13% for granular, dys-granular, and agranular posterior insular cortex,respectively) (Fig. 2G).

Insula–amygdalar connectionsroute information for acute fearversus threat memory

We next investigated whether the output con-nections of insular cortex neurons to either theCeA or the LA have different functions duringacute fear behavior versus 1-day threat memory.We silenced the output fibers of the insula spe-cifically in either the CeA or the LA at the time offootshock presentation during training (Fig. 3A),after virally expressing halorhodopsin in theposterior insular cortex (see fig. S7 for thehistological validation of fiber placements).Silencing of the insula output fibers in the CeA(Fig. 3B) caused significantly reduced freezingin the halorhodopsin group compared with thecontrol group (Fig. 3C; ANOVA, P< 0.001, F1,240 =25.66; fig. S8). A post hoc Bonferroni test showeda significant reduction of acute fear behavior onthe training day (P < 0.01 and 0.001 for the fifthand sixth pairing) (Fig. 3C; see also Fig. 3D, left,

Berret et al., Science 364, eaaw0474 (2019) 31 May 2019 2 of 11

Fig. 1. The posterior insular cortex is necessary for threat learningand acute fear behavior. (A) Threat learning protocol (left) and timingof halorhodopsin-mediated silencing of insular cortex neurons duringthe US (right). (B) Scheme of bilateral injection of an AAV1 vector thatdrives expression of halorhodopsin or a control construct, followed bybilateral placement of optical fibers over the injection sites in the insularcortex. pInsCx, posterior insular cortex. (C) Time course of the freezinglevel across the 3 days of threat learning for the control group (black)and the halorhodopsin (Halo)–expressing group (red). Significance wastested by ANOVA and Bonferroni post hoc analysis (Materials andmethods). n, number of mice. (D) Average freezing levels during thetraining day in a pooled analysis (Fear; freezing levels pooled from thefifth and sixth pairings on day 2) and during retrieval on day 3 (Retrieval)

for both the control group and the halorhodopsin group (black and red,respectively). Statistical significance was computed by Student’s t test.(E) Side-view map of the mouse brain according to (49), displaying thelocation of the insular cortex and its adjacent cortical areas. The bottompanel is a zoomed-in view of the posterior insular cortex with granular (GI),dysgranular (DI), and agranular (AIP) areas. The locations of opticalfibers in the halorhodopsin-expressing mice are indicated by dots, and theestimated areas of silenced brain tissue (55) are depicted by the greenshaded areas (see also fig. S1). AI: agranular insular cortex; AID and AIV:dorsal and ventral parts of the AI; S1 and S2: primary and secondarysomatosensory cortex; Au1: primary auditory cortex; AuV and AuD:secondary auditory cortex, ventral and dorsal parts; V1 and V2: primaryand secondary visual cortex; Ect: ectorhinal cortex; PRh: perirhinal cortex.

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pooled analysis, P < 0.001; t test). However,threat memory retrieval was not significantlydifferent in the halorhodopsin and control groups(Fig. 3C; P > 0.05, Bonferroni post hoc test; Fig.3D, right, pooled data, P = 0.71, t test).When we placed the optical fibers bilaterally

over the LA (Fig. 3, E to G, and fig. S9), there wasalso a significant difference in freezing in thehalorhodopsin group compared with the con-trol group (Fig. 3F; ANOVA, P < 0.001, F1,240 =45.3; fig. S10). However, the post hoc Bonferronitest indicated no significant difference in acutefear behavior on day 2 (Fig. 3F, P > 0.05 for each

of the six pairings; Fig. 3G, left; P > 0.05). Con-versely, 1-day threat memory was significantlyreduced in the halorhodopsin group (Fig. 3F, P <0.01 or 0.001, Bonferroni post hoc test), as was alsorevealed by the pooled analysis for the retrievalday (Fig. 3G, right,P<0.001, t test). Reduced threatmemory retrieval but unchanged acute fearbehavior were also observed when LA neuronsthemselves were silenced during the footshockson the training day (fig. S11) (36). Therefore, LAprojectors of the insular cortex probably driveLA neuron depolarization during the footshock,an activity necessary for forming an associative

memory in the LA. However, the activity of LAneurons during the footshock was not neces-sary for acute fear behavior on the training day.

Separate insula neuron poolsdrive freezing versus aversiveadaptive behaviors

To further investigate the roles of CeA and LAprojectors in the posterior insula, we activatedeach projector neuron population separately.We injected an AAV vector that is taken upretrogradely and that drives Chronos-eGFPexpression (AAVretro:hSyn:Chronos-eGFP) (37),

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Bregma -1.22 mm Bregma -1.82 mm

Fig. 2. The posterior insular cortex makes robust excitatoryprojections to both the LA and CeA by largely separate projectorpopulations. (A) Coronal slice of a mouse brain showing the expressionof Chronos-eGFP in the posterior insula 3 weeks after injection with aAAV8:hSyn:Chronos-eGFP virus. Scale bar, 500 mm. (B) Images ofeGFP-positive fibers in the anterior (left) and posterior (right) amygdalacomplex. Scale bars, 250 mm. CeL, CeM, and CeC: lateral, medial, andcapsular parts of the CeA; LAv, LAd, and LAm: ventral, dorsal, and medialparts of the LA; BAl and BAm: lateral and medial parts of the BA. (C) Schemeof experimental approach (left) and optogenetically evoked EPSC(middle) in a CaMKII-positive LA neuron under control conditions (black)and after application of 50 mM APV (red) at two different holding potentials(−70 and +50 mV). The bar graph on the right shows the average EPSCamplitude (AMPA component, −70 mV), separated for recordings afterCre-dependent expression of Chronos in CaMKII-positive neurons (n = 24recordings) or after Cre-independent expression of Chronos in the insula

(n = 18 recordings). (D) Same as in (C) but for a recording of a SOM+

neuron in the CeA. The fast component of the EPSC was blocked byNBQX (5 mM; green), and the remaining slow EPSC at +40 mV wassensitive to APV (blue). (E) Brain section at bregma −0.94 mm (right)showing the injection of the retrograde labels CTB-647 (red) andCTB-488 (green) centered in the CeA and LA, as shown in the scheme(left). (F) Coronal mouse brain section on the level of the posteriorinsular cortex (bregma −0.9 mm) with projection neurons labeledby CTB-647 (red, LA projectors) and CTB-488 (green, CeA projectors).Yellow rectangle, ROI used for the cell quantification shown in (G).(G) Quantification of LA projectors (red), CeA projectors (green), andneurons labeled by both retrograde labels (yellow) over the thickness ofthe insular cortex (x axis) and for four separate regions (S2, GI, DI,and AIP). The inset bar graphs show quantifications over the entire corticaldepth (average ± SEM of neuron numbers from n = 3 subsequentsections). Data in (C) and (D) are average ± SD.

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bilaterally into the CeA or into the LA, and placedoptical fibers bilaterally over the posterior insularcortex (fig. S12). Four weeks later, mice under-went a modified threat learning paradigm inwhich the footshocks on the training day werereplaced by optogenetic stimulation of eitherCeA or LA projectors (1-ms stimuli at 40 Hz for10 s) (Fig. 4A). Stimulation of the CeA projec-tors stopped any movement of the mice (Fig. 4,B and D, andmovie S2). After cessation of blue-light stimulation, the mice swiftly returned totheir normal exploratory behavior. Presentingthe mice with tones on the next day evokedneither head shaking nor freezing (Fig. 4, Cand D; fig. S13; P < 0.05 and 0.01, respectively;n = 4 mice), and the time spent grooming didnot differ between the 2 days (Fig. 4D; P = 0.94).Thus, CeA projectors in the posterior insular cor-tex initiate freezing behavior, but their activitydoes not leave an aversive memory trace.We then stimulated LA projectors in an anal-

ogous approach (Fig. 4, E to G). During andafter the 10-s optogenetic stimulation blocks, themice displayed a sequence of strongly aversive

behaviors, including head shaking and backwardmoving, followed by rising on the hindpaws,movement of forepaws in the air with closedeyes, and flattened ears (Fig. 4E andmovie S3).After cessation of light stimulation, mice slowlyrecovered and often showed freezing, excessivegrooming, or escape-like exploratory behavior(Fig. 4E and fig. S14; n = 5 mice). Varying thelight intensity showed that aversive adaptivebehaviors started to appear at ~10 to 30% ofmaximal blue-light intensity, but no qualitativelydifferent behaviors were observed at lower lightintensities (fig. S14; n = 3mice). We hypothesizethat the strong intensity of aversive behaviors iscaused by the stimulation of a large number of LAprojectors, which might be equivalent to a stronginternal representation of various painful sensa-tions. One day later, exposing mice to the firsttone block in a different context caused escape-like exploratory behavior, with occasional boutsof other defensive behaviors that continued overthe entire retrieval session, regardless of thetiming of subsequent tone blocks (Fig. 4, F andG, and fig. S14; n = 5 mice).

Insular neuron activity reflects threatlearning of an auditory CSThe posterior insular cortex plays a role inacute fear learning and in 1-day threat mem-ory (Figs. 1 to 3). We therefore sought to re-cord the response of posterior insular cortexneurons in vivo over the 3 successive days of thethreat learning protocol. We used four tetrodeelectrodes placed around a central optical fiber(Fig. 5A) (38). We injected an AAVretro drivingthe expression of mCherry-IRES-Cre into theLA of channelrhodopsin-2 (ChR2) reporter mice(Fig. 5, A and B), which resulted in the expres-sion of ChR2 in LA projectors of the posteriorinsular cortex (Fig. 5B). This, in turn, allowedus to make recordings from optogeneticallyidentified LA projectors (fig. S15) (38), as wellas from nonidentified units in the posteriorinsular cortex.Overall, we found n = 20 units in the pos-

terior insular cortex that responded to the foot-shock on day 2, out of a total of n = 78 unitsthat could be followed over all 3 days (n = 6mice). Figure 5, D and E, shows a unit thatresponded to the footshock. This unit was ini-tially unresponsive to tones but developed a re-sponse to the tone (CS) on the training day andretained this response during threat memoryretrieval on day 3 (Fig. 5, D and E). Such unitswere called US-responsive (US+) and CS-entrainedunits. In addition, we found other combinationsof responses, including units that were non-responsive to footshocks and did not show CSentrainment; units that showed CS entrainmentbut had no footshock response; and units thatresponded to footshocks but did not show CSentrainment (see fig. S16 for examples of theresponse types and fig. S17 for examples of spikewaveforms). Thus, there was some dissociationbetween US-responding neurons and the plas-ticity of the CS response, as found recently in invivo imaging of BA/LA neurons (10). Withinthe population of optogenetically identified LAprojectors, the summed categories of all US-responsive units were enriched (Fig. 5F; sumof red and pink categories, P = 0.026, Fisher’sexact test). On the other hand, the fraction ofCS entrained units was not significantly differ-ent for unidentified units (22%) and for theputative LA projectors (39%) (Fig. 5F; sum ofred and dark blue categories; P = 0.16; Fisher’sexact test).We averaged the responses to tones and foot-

shocks over all 3 days for both CS-entrained andnon–CS-entrained unitswithinmice (Fig. 5, G andH, and fig. S18) and then calculated the grandaverage across mice (Fig. 5I). This showed thatCS-entrained units developed a robust responseto the tone presentation on the training day andretained this response during the retrieval day(Fig. 5I).

The insular cortex contributes to threatmemory retrieval

The observation of tone-entrained units indi-cates that associative plasticity takes place in theinsular cortex. Therefore, activity in the insula

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Fig. 3. Differential roles of the insular cortex projections to the CeA and LA in acute fearbehavior versus threat memory. (A) We expressed halorhodopsin bilaterally in the posteriorinsular cortex and placed optical fibers over the output axons in either the CeA [(B) to (D)] or theLA [(E) to (G)]. We silenced the output fibers during the footshock presentation on the trainingday (A). (B to D) Freezing levels throughout the 3 days of behavior (C) and pooled analysis offreezing for both day 2 and day 3 [(D), left and right, respectively] for the experiments in which theposterior insula CeA synapse was silenced. Note the reduced acute fear behavior especially forthe fifth and sixth pairing on day 2, whereas freezing during the retrieval day was not significantlyreduced. (E to G) Results for the posterior insula LA synapse silencing. Acute fear behavior was notsignificantly altered, whereas threat memory retrieval on day 3 was significantly reduced [(F) and(G), right]. Data in (C), (D), (F), and (G) are mean ± SD.

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might also be necessary for the retrieval of threatmemory. To test this possibility, we silenced theposterior insular cortex during each tone pipon the retrieval day after bilateral expressionof eNpHR3.0-eYFP in the insula (Fig. 6A and fig.S19). This resulted in significantly reduced freez-ing in the halorhodopsin group compared withthe control group (Fig. 6B; ANOVA, P < 0.01,F1,100 = 8.59). Analysis with a post hoc Bonferronitest showed significantly reduced freezing dur-

ing two tone blocks on the retrieval day (Fig. 6B;P < 0.05). Similarly, pooled analysis showed sig-nificantly reduced freezing during retrieval (Fig.6C; P < 0.001) but no change during training asexpected (Fig. 6C, left, P = 0.8). Post hocmappingof the fiber positions confirmed a position in thegranular posterior insular cortex (Fig. 6D). Thus,tone-driven activity of the posterior insular cor-tex contributes to the retrieval of auditory-cuedthreat memory.

DiscussionThe posterior insular cortex plays an importantrole in threat learning. Silencing of neurons inthe posterior insular cortex during the footshocklargely removed the aversive qualities of the USand subsequently prevented the formation of1-day threatmemory (Fig. 1). Because a teachingsignal about an aversive event is thought todrive associative threat learning at CS-codingsynapses in the LA (5, 11, 15) and possibly in

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Fig. 4. Separate insula projectors drive freezing versus aversiveadaptive behaviors. (A) Protocol of the optogenetic stimulationused to activate either the CeA or LA projectors in the posterior insularcortex. On day 2, the optogenetic stimulation (a 10-s train of 40-Hzlight stimuli of 1-ms duration) coterminated with 30-s blocks of tonestimulation (left). On day 3, the tones were given alone (right). (B) (Left)Scheme of the targeting of CeA projectors. (Right) Scored behaviorof one example mouse. Mice exhibited stereotypic freezing behaviorselectively during the 10 s of optogenetic stimulus. (C) During the thirdday, tone stimuli alone did not evoke a notable behavioral response.(D) Quantification of the behaviors observed in four mice. During day 3,the tone evoked significantly less head shaking and freezing than on

day 2 (P < 0.05 and 0.01, respectively; t test), whereas the micespent significantly more time in exploration (P < 0.001; t test).(E) Optogenetic activation of LA projectors resulted in a range ofpronounced defensive behaviors (see key) that outlasted theoptogenetic stimulation. (F) During the third day, presentation of thefirst tone (in the absence of optogenetic stimulation) inducedescape-like behavior (dark gray), which outlasted the entire experimentsession. (G) Quantification of the behaviors observed in five mice.Note the significant shift from various aversive behaviors onday 2 toward escape-like exploration on day 3 (gray bar; P < 0.001).Most other behaviors also differed significantly from day 2 to day 3(*P < 0.05; **P < 0.01; ***P < 0.001).

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Fig. 5. Insula neurons respond to US stimulation and acquire CSresponsiveness. (A) Schematic showing the optrode placement in theposterior insular cortex and the injection of an AAVretro vector driving theexpression of Cre-recombinase and mCherry in a channelrhodopsin reportermouse. The scheme on the right shows the optrode with four tetrodes(T1 to T4). (B) Expression of mCherry in LA (left) and the retrogradelyexpressing neurons in the insular cortex (right). Note the position of theoptrode in the granular part (GI) of the posterior insular cortex (right).Scale bars, 250 mm. ec, external capsule. (C) Freezing level across the 3 daysof fear conditioning training for the example mouse. (D) Responses of aunit to tone (CS) presentations on the training day [top; average (av) z-scoreresponse to the n=300.1-s tones during each tone block] and to the footshockcoterminating with each tone block (bottom). PSTH, peristimulus timehistogram.The response on the far right is the average over n = 6 footshocks.

(E) Response of the same unit as in (D) to n = 4 tone blocks (CS) duringretrieval on day 3. Note the response to footshocks, the entrainment to toneson day 2, and the retained tone responsiveness on day 3. Correspondingly, thisunit was classified as a US+ CS-entrained unit. (F) Distribution of units withdifferent combinations of US responsiveness and CS entrainment, averagedover all units of the sample (78 units from six mice). (G and H) z-scoreresponses of all units measured in the sample mouse from (B) to (E) inresponse to the tone blocks (G) and to the footshocks on day 2 (H).The datawas separated for non–CS-entrained units (gray) versus CS-entrained units(pink), and the average response of each group is shown superimposed(red and black; average±SEM). (I) Grand average of responses to tone of non–CS-entrained units (black) and CS entrained units (red) for all units fromsix mice. In the figure, red values of n indicate CS-entrained units, whereasblack values denote units that were nonresponsive to the CS.

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other areas (39), suppression of a relevant rep-resentation of the aversive sensory event shouldimpair the formation of threat memory, as weobserved (Figs. 1 and 3). Furthermore, becauseoptogenetic stimulation of LA projectors in theinsula was strongly aversive for mice (Fig. 4),and because silencing of this connection duringthe footshock impaired threat memory forma-tion (Fig. 3F), the posterior insula likely carriesUS information to the LA during associativethreat learning. It has recently been shown thata neuron population in the BA codes for theaversive qualities of pain (40). Because we foundthat insular cortex activity is necessary for theaversive properties of footshocks, it is possiblethat insular cortex–amygdala networks cooper-ate in attributing aversive meaning to painfulstimuli. Affective pain information can also beconveyed from the parabrachial nucleus to theCeA (41), but the latter pathway cannot explainassociative plasticity in the LA.Beyond providing information about a US to

the amygdala, our study shows that the insularcortex itself substantially contributes to the for-mation of a 1-day threatmemory.Many neuronsin the posterior insular cortex, including LAprojectors, responded to the US. A partiallyoverlapping population of neurons relative tothese US responders developed a response tothe tone (CS) during threat learning and re-tained this response during retrieval 1 day later(Fig. 5). Consistent with the insular cortex’s role(besides LA) for storing threat memories, si-lencing the insula during the tone stimulationon the retrieval day reduced threat memory

(Fig. 6). Thus, 1-day auditory cued threat mem-ories might be stored in both the LA and theinsular cortex, whereas longer-lasting threatmemories become dependent on secondarysensory cortices (42). A functional magneticresonance imaging study of fear conditioningin rats using a visual CS has shown activationof the LA, granular insular cortex, and hypo-thalamus upon replay of the CS after fear con-ditioning (43), consistent with our finding thatthe insular cortex contributes to threat memoryretrieval. Because the posterior insular cortexis immediately upstream to the LA and makesstrong glutamatergic synapses in the LA (Fig. 2),it is possible that the increased activity of in-sular cortex neurons to the CS (Fig. 5) con-tributes to the increased tone responsiveness ofLA neurons after threat learning (7, 10). Theserial arrangement of the posterior insular cortexand LA evokes a model of memory storage inwhich associative plasticity takes place seriallyin connected brain structures, a mechanism thatmight amplify the response to the CS of themore downstream brain areas.The posterior insular cortex is also essential

for acute fear behavior during the training day(Fig. 1), with a clear contribution of the insularcortex to the CeA pathway (Fig. 3). In agreementwith this role, optogenetic stimulation of CeAprojectors in the insula caused strong freezing(Fig. 4), consistent with the previous demonstra-tion that optogenetic stimulation of CeA SOM+

neurons, or more nonselective stimulation in themedial CeA, drives freezing (35, 39). Amygdalarconnections from the gustatory part of the insular

cortex, which is located anterior to the posteriorinsular cortex studied here (16, 44, 45), havebeen shown to cause conditioned place aversionin response to bitter tastants (23–25), suggestingroles for both the somatosensory insular cortexand the gustatory insular cortex in aversive va-lence coding. Our finding that CeA projectors inthe insular cortex drive acute freezing behaviorwhereas LA neurons do not (Fig. 3 and fig. S11)reinforces previous evidence that brain structuresdifferent from the canonical LA andBA can accessthe CeA in fear behavior (46).In human stroke patients with lesions that

include the posterior insular cortex, an absenceof emotional and avoidance responses to painor threatening gestures was observed, a condi-tion termed pain asymbolia (28). This conditionis analogous to the behavioral deficits describedhere in which mild painful stimuli lose theiraversive meaning in mice with a silenced pos-terior insular cortex (Fig. 1). As we show here inmice, the rich interconnectivity between the in-sular cortex and amygdala (Fig. 2) (31–33) wasresponsible for these deficits. Thus, the insularcortex has a fundamental role in evaluatingaversive events and signaling them to the LA, inpreparing motor actions in response to dangervia its connections to the CeA and possibly toother structures, and in storing associative mem-ories about threats. Given the crucial functions ofthe insular cortex in threat learning, and giventhat insular cortex activity is altered in severalpsychiatric diseases (47, 48), a more detailedunderstanding of plasticity and circuit mecha-nisms in this brain areawill be of high relevance.

Materials and methodsAnimals

All experimental procedures on laboratory ani-mals (Mus musculus) were conducted in accord-ance with the veterinary office of the canton ofVaud, Switzerland (authorizations 2885.0, 3274.0).For behavior experiments we used male C57BL6/Jmice (6 to 8 weeks old), purchased from CharlesRiver Laboratories (France) and maintained in abreeding colony in the EPFL-SV animal facility.For some experiments (Fig. 5), ChR2 reportermice of the same age were used (Rosa26:lsl:ChR2-eYFP, Jackson Lab 024109, Ai32). For theex vivo experiments, SOMcre x tdTomato mice(Somatostatin-ires-Cremice; Jackson Lab 013044,crossedwithAi9 reporter lineRosa26:lsl:tdTomato;Jackson Lab 007909) and CaMKIIcre x tdTomato(CaMKII-Cre mice; Jackson Lab 005359, crossedwithAi9 reporter line) were used. Themiceweremaintained on a normal 12-hour light/dark cycle,and provided with food and water ad libitum.Mice were randomly assigned to different experi-mental groups.

Stereotaxic surgery for virus injectionand optical fiber implantation

Mice were anesthetized by inhalation of a 3% iso-flurane mix in O2 gas (produced by the CombiVetanimal gas anesthesia system;RothacherMedical,Switzerland) andmaintained under inhalation of1 to 1.5% isoflurane in O2. Mice were then placed

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Fig. 6. Tone-driven insular cortex activity is necessary for threat memory retrieval. (A) Protocol tooptogenetically silence the response of insula neurons to each tone on the retrieval day. (B) Time course ofthe freezing level across the 3 training days for control mice (black) and the halorhodopsin-expressingmice (red). (C) Freezing level during the training day (Fear; freezing averaged for the fifth and sixthtone–footshock pairing) and the retrieval day (all four presentations grouped) for the control and thehalorhodopsin-expressing animals. ns, not significant. (D) Side-view map displaying the position ofeach optical fiber in halorhodopsin-expressing mice. Data in (B) and (C) are mean ± SD.

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in a stereotactic apparatus (Kopf Instruments,model 940, USA) on a heating pad (HarvardInstruments, USA) and local subcutaneous anal-gesia using 2% lidocaine was applied. The coor-dinates of the craniotomy were determined bya mouse brain atlas (49). Following exposure ofthe skull, a hole was drilled on each side with a0.5-mm bore (Komet Dental, Germany) using aTech2000 drill handpiece (Ram Products Inc,USA). Viral suspension was microinjected using10- to 20-mm tip diameter glass pipettes (PCRcapillaries, Drummond Scientific, USA; pulledon a P-97 puller, Sutter Instruments, USA) and anoil hydraulic micromanipulator (MO-10, Narishige,Japan) at the following coordinates (AP, posteriorfrom the bregma/ML, ± lateral from themidline/DV, ventral from the skull surface at bregma, inmm): for insula: −0.9/±3.9/−3.8; for LA: −1.15/±3.45/−4.45; for CeA: −1.22/±2.65/−4.70. Forin vivo optogeneticmanipulations, we implantedoptical fibers bilaterally in the skull as describedby (50) using dental UV curing cement (TetricEvoFlow; Ivoclar Vivadent, Liechtenstein). The im-plants were inserted at the following stereotaxiccoordinates: posterior insula (AP:−0.95mm;ML:±3.80mm;DV:−3.40mm); LA (AP:−1.15mm;ML:±3.48 mm; DV: −3.60 mm); CeA (AP: −1.22 mm;ML: ±2.60mm;DV: −4.17 mm). To prevent inter-ference with the implants by other animals, themice were singly housed after the surgery. Be-havioral experimentswere performed3 to 5weekslater, to allow sufficient time for the injected AAVto express the opsins.

Viral vectors

To silence either the posterior insula neurons(Figs. 1 and 6), or the terminal areas of pos-terior insula projections to the CeA or the LA(Fig. 3), or else LA neurons (fig. S11), we used anAAV1:hSyn:eNpHR3.0-eYFP (Addgene). As con-trols in these experiments, we used anAAV1:hSyn:eGFP (Addgene). For the ex vivo optogenetic re-cordings (Fig. 2), an AAV8:hSyn:Chronos-eGFP(University of North Carolina vector core; UNC)was used to drive the expression of Chronos[a channelrhodopsin variant; see (51)]. In someof these experiments (see Fig. 2C), we used anAAV8:EF1a:FLEX:Chronos-eGFP (UNC) andCaMKIICre mice (see above), to achieve expres-sion of Chronos selectively in CaMKII-positiveneurons of the posterior insula cortex. To activateCeA or LA projectors in the posterior insular cor-tex (Fig. 4), we used a retrograde AAVretro:hSyn:Chronos-eGFP, and as control an AAVretro:hSyn:eGFP (both from Addgene). Usually, 200 nl ofviral suspension was injected per site; the viraltiter was adjusted to ~3.5·1012 ml−1. For optroderecordings, an injection of 200 nl of retrogradeAAVretro:Ef1a:mCherry-IRES-Cre (Addgene) wasmade in the LA, at a titer of 1.3·1013 ml−1.

Optrode recordings

For in vivo recording of extracellular AP activityof optogenetically identified neurons (Fig. 5) (38),we custom-built optrodes using four tetrodesplaced around a central optical fiber (200-mm/230-mm core/outer diameter, NA 0.5; Thorlabs,

USA; see Fig. 5A). Tetrodes were twisted frominsulated Pt/Ir wires (17-mm diameter; CaliforniaFine Wire, USA) and glued onto the externalsurface of the optical fiber that was glued insidethe ceramic ferrule (1.25-mm outer diameter;Thorlabs) and inserted into the movable part ofa microdrive (Axona Ltd, UK). The free ends ofthe tetrode wires were fixed to the pins of aNPD-18-VV-GSmicro-connector (Omnetics Corp.,USA) that was rigidly attached to the movablepart of the microdrive. To minimize the imped-ance of the recording and reference channels tovalues typically <100 kilohms at 1 kHz, platinumwas deposited on the tips of the tetrode wiresusing platinumblack plating solution (Neuralynx,UK) according tomanufacturer’smanual, and aniontophoretic amplifier (MVCS-01,NPIElectronic,Germany). The impedance for each channel wasmeasured in the phosphate-buffered saline so-lution (PBS) using the lock-in function of anEPC-10 patch-clamp amplifier (HEKAElektronik,Germany). The optrode implantation surgery intothe left insular cortex was similar to the proce-dure used to implant optical fibers; in addi-tion, a groundingmicroscrew (AntrinMiniatureSpecialties, USA) was implanted in the skull,to which a copper wire connected to a groundpin was presoldered.For the optrode recordings in freely behav-

ing mice, the fear conditioning chamber wassurrounded with a custom-built Faraday cage.Spiking activity was acquired with a 16-channelamplifier ME16-FAI-mPA under control of theMC_Rack software (both from Multi ChannelSystems) at 40-kHz sampling frequency; un-sorted spike events were visualized online afterband-pass filtering (0.6 to 6 kHz) and amplitude-based detection (threshold of −3 to −3.5 SD) inthe MC_Rack software (see also fig. S15A). Oneday before the start of the threat learning proto-col, the optrode was repositioned in the ventraldirection by ~100 to 300 mm using a microdrive(performed under ketamine anesthesia) untilthe target depth of 3.9 mm was reached. Wethen applied light pulses (1 to 3 ms, 0.8 to 3 mWat the fiber output, 2-Hz repetition rate) pro-duced by a 473-nm diode pumped solid state(DPSS) laser (MBL-FN-473-150mW; CNI Lasers,China) that was triggered with a Master-8 stim-ulator (A.M.P.I., Israel). The opto-tagged unitscould be detected by apparent light-evoked spik-ing activity within the expected latency windowof 2 to 8 ms after the light pulse onset (fig. S15B).During the 3 days of fear conditioning protocol(Fig. 1A), the spiking activity as well as the syn-chronization triggers indicating the timing ofthe sound CS and the footshock US stimuli (gen-erated by the VideoFreeze software; Med Asso-ciates Inc, USA) were continuously sampled withthe MC_Rack software. After each behavior ses-sion, light-evoked spikes were collected by ap-plying ~2000 light pulses at 2 Hz to identifythe opto-tagged cells post hoc during the dataanalysis.The raw data was converted from MC_Rack

into HDF5 format using Multi Channel DataManager software (MultiChannel Systems).

The data was then processed (except the spikeclustering) using custom-written routines in IGORPro 7 (WaveMetrics, USA). The voltage traceswere band-pass filtered (0.6 to 6 kHz; 4th-orderButterworth filter), and footshock stimulationartefacts (only from recordings on day 2) weredetected and blanked by zeroing under manualcontrol (see fig. S15A). In some mice, we observeda slow light-evoked artefact likely related to thelocal field potentials that could not be filteredout completely and thus prevented proper spikedetection. To minimize these artefacts in suchcases, we subtracted the per-channel averagetraces calculated over ~200 subsequent lightpulses. Negative amplitude spikes were thendetected using threshold method (typically setat −3.2 SD) on each channel, and the precisespike locations within each tetrode were dictatedby the timestamp of the largest amplitude event.The light-evoked spikes recorded after behaviorwere sampled in a time window 2 to 8 ms afterthe light pulse onset (see fig. S15B) and wereclustered separately from the spikes recordedduring behavior. Individual spike cutouts (fil-tered for clustering with a wider band-pass 4th-order Butterworth filter, 0.4 to 6 kHz) were thenexported into MATLAB (MathWorks, USA). TheMClust toolbox (Dr. David Redish; University ofMinnesota, USA) was used to cluster the tetrodespikes by an unsupervised clustering algorithm(KlustaKwik) (52), using the spike valley andthe principal components PCA1-PCA3 as theclustering parameters. The quality of clusteringwas manually controlled by checking the aver-age spike waveform similarity and visualizingthe cluster projections, and occasionally someof the clusters were fused together if they werenot well separated. Following this, the qualityof the spike cluster isolation was analyzed bycomputing the isolation distance and L-ratio, tocontrol for type I and type II errors (53). Onlyclusters with isolation distance larger than 24and L-ratio lower than 0.5 were retained forfurther analysis; this reduced the total numberof clusters in the sample from n = 179 to 78 (sumof unidentified units and LA projectors; n = 6mice). However, qualitatively similar results asthe ones shown in Fig. 5 were obtained whenthis quality control step was omitted.The clustered timestamp data were re-imported

into IGOR Pro for subsequent analyses such aswaveform matching, alignments to the stimuliand z-score calculations, averaging and display-ing. For each recording day in every animal,identification of the opto-tagged units was doneby matching the average waveforms of the clus-ters recorded during the behavior session withthose collected upon delivery of the light pulses.Similarly, this averagewaveformmatchingmethodwas used to follow the units across 3 experimen-tal days (fig. S15C). Waveform matching wasfacilitated by calculation of a metric for eachpair of units, which was the product of thesummed inverted root mean squared value ofthe point-to-point difference between the wave-forms, and of the inverse of unity complementof the average Pearson’s coefficient calculated

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between the waveforms for four channels of atetrode. The experimenterwas blind to the spikingpattern of individual units during the behavior ses-sions until the identity of units (opto-tagged orunidentified, and across days) was determined.The recorded units were classified into four

categories, based on their responses to sensorystimuli, according to the following criteria. Ingeneral, a z-score of 2 (1 SD above baselinenoise) was considered a response. (i) The unitwas considered US-responsive if the z-scoreduring the 1-s-long footshock, calculated fromthe average of the n = 6 footshock presentationson day 2, exceeded a value of 2. (ii) For theclassification of CS responses, average z-scoresover 100-ms-long tones were calculated from30 aligned and averaged tone response (on day2, only 29 CS repetitions were averaged to avoidthe influence by the US response that immedi-ately followed the 30th tone). For the animalFT9742, the z-score was calculated over the first40 ms after the tone onset because of the briefresponse characteristics in this mouse. A givenunit was considered CS entrained if, first, thez-score did not exceed 2 for at least five out ofsix CS presentations on day 1 (criterion that theunit was not innately responsive to tones), and,second, if out of the last three tone presentationson day 2, at least two resulted in a summedz-score larger than 4 (criterion for increasingtone response on day 2) or if at least two tonepresentations on day 3 resulted in a summedz-score larger than 4 (criterion for the mainte-nance of the tone response on day 3).

Fear conditioning

Before the start of fear conditioning, the micewere habituated to head tethering with thefiber-optic patch cord. Each mouse underwentone habituation phase daily for a total of 5 to6 days before starting the behavioral training.Fear conditioning was conducted on 3 con-

secutive days in a rectangular conditioningchamber placed in a sound- and light-attenuatedbox (63.5 cm wide, 35.5 cm high, 76 cm deep;NIR-022MD, Med Associates) equipped with aspeaker in a sidewall, and a CMOS video camera(30 frames/s) with a near-infrared filter for con-tinuous video recordings of mouse behavior.On day 1, mice were subjected to a habituation

session in the fear conditioning chamber (con-text A), in which six tone blocks (CS) of 7 kHz at80 dB were presented (each block composedof 100-ms pips repeated 30 times at 1 Hz). Thefreezing level of the mice during CS presentationwas analyzed as an estimate of baseline behavior.On the training day (day 2), mice were placed

in the conditioning chamber (context A) with astainless-steel grid floor connected to a shockgenerator (ENV-414S, Med Associates). Micewere submitted to a fear conditioning paradigmby pairing the conditioning sound stimulus (CS)with a mild electric footshock (US) (AC 0.6 mA,1-s duration). The onset of the US coincidedwith the offset of the last tone pip in the CS. TheCS-US pairing was repeated six times at a var-iable intertrial interval of 60 to 90 s.

Threatmemory retrieval consisted of the tonerecall on day 3. The mice were placed in the con-ditioning chamber with a new environment(context B) and were reexposed to four presen-tations of the CS tone without footshock. Thefreezing behavior was monitored during CS toevaluate the retrieval of cued fear memory. ThecontextBwasmodified fromtheoriginal condition-ing chamber to minimize context generalization.During optogenetic behavioral experiments,

yellow (561 nm) or blue (473 nm) light wasdelivered from a DPSS laser (MGL-FN-561-AOM-100mW or MBL-FN-473-150mW, respec-tively; CNI Lasers). To avoid resting leak of lightthrough an AOM module of the yellow laser,an additional mechanical shutter (SHB05T;Thorlabs) was introduced at the output beforethe fiber. The shutter was closed at all timesexcept when the laser light had to be applied,for silencing during fear conditioning day 2:3-s duration, starting 1 s before the footshock;for silencing during retrieval day 3: 250-msecduration, starting 50 msec before each pip; foractivation during fear conditioning day 2: 10-sduration, coterminating with the tone. The laserpower was adjusted for each animal in accord-ance with the attenuation value of the given op-tical fiber implant so that the total light outputpower was 10 mW at each fiber tip.Freezing was analyzed using Video Freeze

software (Med Associates) and expressed as thepercentage of time mice spent freezing duringthe CS presentation. We found that the opticalfiber connected to the mouse head on the day ofoptogenetic silencing (day 2 in Figs. 1C, 3C, 3F;day 3 in Fig. 6B) caused movement artefacts inthe quantification of freezing with the VideoFreeze software. To compensate for this, we useddifferent threshold levels on day 2 (versus days1 and 3) for the data in Figs. 1C, 3C, 3F; for thisreason, the absolute values of freezing are notdirectly comparable between days 2 and 3 inthese datasets. For the dataset in Fig. 6B, inwhich the cable artefact interferes with the cor-rect quantification of the freezing behavior duringretrieval, we hand-scored the behavior of all micein the control and halorhodopsin group on day 2and day 3, similar as shown in Fig. 4. Duringhand-scoring, the analyzing person was blindedto the identity of the mouse with respect to con-trol versus halorhodopsin group. The fraction oftime spent freezing during the tone blocks wasthen computed from the scored behavior data.

Ex vivo electrophysiological recordingsand optogenetic circuit mapping

For ex vivo electrophysiology, adult mice of thesame age range as used for behavior were em-ployed. Precautions were made to obtain viableslice preparations suitable for recordings, in-cluding the use of low-Na+ dissection buffer (seebelow) (54). Mice used for electrophysiologicalexperiments were anaesthetized with isoflurane,decapitated, and their brains were quickly re-moved and placed in ice-cold dissection buffer(in mM): 110 NMDG (N-methyl-D-glutamine),110 HCl, 2.5 KCl, 1.2 NaH2PO4, 20 HEPES, 25 Glu-

cose, 5 sodium ascorbate, 2 Thiourea, 3 sodiumpyruvate, 10 MgCl2, 0.5 CaCl2, saturated withcarbogen gas (95% O2 and 5% CO2). Coronalslices (300 mm) containing the amygdala (withLA or CeA) complex were cut in dissection bufferusing aMicrotome VT1200S (LeicaMicrosystems,Germany), and were subsequently transferredto a storage chamber containing aHEPES basedstorage solution (in mM): 92 NaCl, 2.5 KCl,30 NaHCO3, 1.2 NaH2PO4, 20HEPES, 25 glucose,5 sodium ascorbate, 2 Thiourea, 3 sodium pyru-vate, 2 MgCl2 and 2 CaCl2, at 34C°, pH7.4, sat-urated with carbogen, and allowed to cool downto room temperature. After at least 40 min ofrecovery time, slices were transferred to therecording chamber and constantly perfused withACSF (in mM): 125 NaCl, 2.5 KCl, 25 NaHCO3,1.2 NaH2PO4, 25 glucose, 0.4 sodium ascorbate,3 Myo-Inositol, 2 sodium pyruvate, 1 MgCl2 and2 CaCl2, pH7.4, saturated with carbogen. Record-ings were done either at room temperature (20 to22°C; Fig. 2 and fig. S4, F to H) or at 35°C using abath heater (Warner Instruments; model TC-334B; for the data shown in fig. S2).To test the functional connectivity between

posterior insular cortex and CeA or LA struc-tures, an AAV8:hsyn:Chronos-eGFP virus wasinjected into posterior insula of SOMCre x tdTmice and CaMKIICre x tdT mice. After brainslicing, whole-cell patch clamp recording wasperformed in genetically identified cells basedon their tdTomato fluorescence, as well as intdTomato-negative cell in case of CeA record-ings. Whole-cell currents were recorded usingEPC-10 patch-clamp amplifier (HEKA Elektronik)under control of PatchMaster software (HEKAElektronik). The patch pipette solution contained(in mM): 140 Cs-gluconate, 10 HEPES, 8 TEA-Cl,5 Na-phosphocreatine, 4 Mg-ATP, 0.3 Na-GTP,5 EGTA, pH 7.2 adjusted with CsOH.For optogenetic activation of Chronos-expressing

insula projections to LA or CeA, 1-ms-long blue-light pulses were applied from a high-powerLED (CREE XP-E2, emission spectrum centeredaroundl =480nm;Cree Inc,USA)drivenbyaLEDcontroller (Cyclops LED Driver, www.open-ephys.org/cyclops). The LED light source was coupledinto the epifluorescence port (TILLphotonics,Germany) of a BX51WI microscope (Olympus,Japan) equipped with the 60X/0.9 NA objec-tive, thus focusing the light onto a region ofinterest. Single-light pulses were delivered at20-s intervals and repeated at least 10 times.To identify the currents evoked by the stim-

ulation of posterior insula fiber terminals, weapplied gabazine (GABAA receptor inhibitor;5 mM; at −70-mV holding), NBQX (AMPA recep-tor inhibitor; 5 mM; at −70-mV holding) and AP-5(2-amino-5-phosphonopentanoic acid; NMDAreceptor inhibitor; 50 mM; at +40-mV holdingvoltage) (all from BIOTREND, Switzerland).

Immunohistochemistry andimmunofluorescence microscopy

After virus injection or behavior, mice wereperfused transcardially with 4% paraformal-dehyde (PFA) solution in PBS. Afterward, the

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brainswere extracted, kept in 4%PFA overnightfor postfixation and then dehydrated in 30%sucrose solution for 2 days. Frozen brains werecut into 40-mm-thick coronal slices with a slidingfreezing microtome Hyrax S30 or HM450 (CarlZeiss, Germany). Sections were washed with PBSand mounted on slides in Dako fluorescencemounting medium (Dako, Switzerland).For assessing the viral expression and the

placement of optical fibers, images of seriallymounted slices were taken with a slide scannermicroscope VS120-L100 (Olympus) with 10X/0.4NA air objective or an upright LSM 700 confocalmicroscope (Carl Zeiss) with 20X/0.8 NA air and40X/1.3 NA oil-immersion objectives (Bioimagingand Optics Platform, BIOP, EPFL). Cell quan-tification following CTB injection in LA andCeA (Fig. 2 and figs. S5 and S6) was performedusing an automated ImageJ plugin (developedby Mr. Olivier Burri, BIOP, EPFL). For this anal-ysis, the cells were counted in 20-mm bins withinregions of interest (ROIs) of 250×1100 mmorientedperpendicularly to the pia.Verification of the optical fiber placement

from series of histological sections (40 mm) wasdone with a custom-written image analysisroutine in Igor Pro 7 (available upon request).This routine allowed manual alignment of amodel optical fiber with a fiber tract in 3D, thustaking into account the tilt introduced duringtrimming and sectioning. After the alignment, alight cone volume [calculated according to (55)]was mapped onto the tissue below the fiber toassess the brain areas that were illuminated invivo (see figs. S1, S7, S9, and S18 for the mappedfiber tips and light cones shown in red and blue,respectively).The histological sections were aligned to the

reference mouse brain atlas (49). The bregmaposition of the anatomical plates that corre-spond to a given section is indicated on thehistological sections. The abbreviations of namesof brain areas follow the reference brain atlas(49) (see Figs. 2 and 5 and figs. S1, S4, S6, S7, S9,S12, andS19).A side-viewmapof the mouse brain(Figs. 1E and 6D) was constructed by projectingthe edges of the individual cortical subareas fromeach coronal section of the reference mouse brainatlas (49), centering on the insular cortex and itsdorsal and posterior neighboring cortical areas.

Statistical analysis

Data are expressed as mean values ± standarddeviation of the mean (mean ± SD). Error barsin the figures represent SD, unless specified.Statistical analysis was performed using PRISMstatistical software (GraphPad, USA). Differencesin the freezing time courses between the exper-imental groups (optogenetic actuators vs. control)were analyzed using a two-way ANOVA (referredto as “ANOVA” in the main text), followed by aBonferroni post hoc test to compare replicatemeans by row (group effect), assuming the datawere normally distributed for each time point.We additionally performed a pooled analysis ofthe freezing data by pooling the freezing levelsof the 5th and 6th tone–footshock stimulation

for the training day, and the n = 4 tone blocksfrom the retrieval day (Figs. 1D, 3D, 3G, and 6C).The statistical significance of this pooled anal-ysis was tested with a Student’s t test (referredto as “t test” in the main text). This was doneeither after a Shapiro-Wilk normality test sug-gested normality of the respective distributions,or else after assuming the normal distribution ifthe sample size was not large enough for thenormality test.We also tested the homogeneity of variancewith

theF-test (www.statisticshowto.datasciencecentral.com/) for the behavioral data in Figs. 1D, 3D,3G, and 6C on days 2 and days 3. For a total of40 time points in this data [resulting from 4×(6 + 4) time points in these datasets], we foundthat n = 33 time points had a homogeneousvariance. Violation of the variance homogeneityin the remaining n = 7 time points should betolerable because of almost identical samplenumbers in the control group versus the halo-rhodopsin group.P values <0.05 were considered to be sig-

nificant. * indicates P < 0.05, ** indicates P <0.01, and *** indicates P < 0.001.

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ACKNOWLEDGMENTS

We thank H. Murray, B. Lecrinier, and T. Baticle for technicalassistance and help with virus constructs; Y. Sych (University of

Zürich, Switzerland) for advice on the construction of optrodes;and O. Burri for custom codes for image analysis. Imageacquisition was carried out in the Bioimaging and Optics Platformof EPFL (BIOP). Funding: This work was supported by the SwissNational Science Foundation (SNSF) (31003A_176332/1 to R.S.),the SNSF National Competence Center for Research Synapsy“The Synaptic Bases of Mental Diseases” (project 28 to R.S.), theGerman Research Foundation (DFG Priority Program 1608/SCHN451/5-2 to R.S.), and an EMBO fellowship (ALTF 224-2015 to M.K.).Author contributions: E.B., M.K., O.K., and R.S. designed theresearch. E.B., W.T., and M.K. performed in vivo optogeneticexperiments. S.P. and E.B. performed ex vivo optogenetic mappingexperiments with slice electrophysiology. M.K. performedanatomical experiments and image analysis. O.K. designedmeasurement and analysis routines for in vivo optrode recordings;O.K. and D.O. performed in vivo optrode recordings. R.S., M.K.,E.B., and O.K. wrote the paper. Competing interests: Theauthors declare no competing interests. Data and materialsavailability: All data needed to evaluate the conclusions ofthe study are present in the paper or the supplementary materials.AAV vectors were obtained under material transfer agreementswith Stanford University, the Massachusetts Institute ofTechnology, and the University of North Carolina.

SUPPLEMENTARY MATERIALS

science.sciencemag.org/content/364/6443/eaaw0474/suppl/DC1Figs. S1 to S19ReferencesMovies S1 to S3

13 November 2018; accepted 18 April 2019Published online 16 May 201910.1126/science.aaw0474

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learningInsular cortex processes aversive somatosensory information and is crucial for threat

SchneggenburgerEmmanuelle Berret, Michael Kintscher, Shriya Palchaudhuri, Wei Tang, Denys Osypenko, Olexiy Kochubey and Ralf

originally published online May 16, 2019DOI: 10.1126/science.aaw0474 (6443), eaaw0474.364Science 

, this issue p. eaaw0474Scienceretrieval. Multisensory integration in the insula thus contributes to the storage and retrieval of threat memories.their response patterns to conditioned stimulus presentation during fear conditioning and maintained this pattern atamygdala and drive threat learning and fear-associated responses, respectively. Neurons in the posterior insula adapted

found that independent subpopulations of neurons in the insular cortex project either to the lateral or the centralal.etformation of threat memories. How unconditioned stimulus information reaches the amygdala remains unclear. Berret

Threat learning is important to avoid dangers in the environment. The amygdala is a brain structure involved in theFear behavior and the insular cortex

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MATERIALSSUPPLEMENTARY http://science.sciencemag.org/content/suppl/2019/05/15/science.aaw0474.DC1

CONTENTRELATED http://science.sciencemag.org/content/sci/366/6472/1460.1.full

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