© 2016 Nature America, Inc. All rights reserved. Massachusetts Institute Massachusetts of Technology, Cambridge, USA. Massachusetts, should Correspondence be addressed to S.T. ( Institute Massachusetts of Technology, Cambridge, USA. Massachusetts, 1 RNA-associated of modifications molecular exploiting or proteins models mammalian in RNA-associated epitope-tagged expressing ectopically involved have strategies profiling RNA Genetics-based Identification of BLA genetic markers RESULTS by stimuli. elicited negative and behaviors positive antagonistic underlying circuits neural the identifying for foundation a provide will neurons BLA positive and negative for markers genetic guishing of distin identification neurons. Inthe to and turn, negative positive molecular profiles of BLA neurons may reveal genetic markers unique a using by obtained be could BLA the in neurons positive and negative putative the experience capable of driving a behavioral response consistent with the valence of activity-dependent gene identified. be to yet has behaviors nature of emotional antagonistic the subserving circuit ral distinct, let alone genetically distinguishable structurally are neurons) positive and neurons (negative behaviors positive and negative to contribute that neurons pyramidal BLA the whether established not is it behaviors, emotional in BLA the of role behaviors positive and negative of regulation drive opposing therefore, behaviors; the BLA may be a key site for the associations and iors behav emotional in participates and stimuli emotional positive and genetically defined interneurons of populations with intermingled and pBLA) (aBLA, BLA posterior and anterior the into respectively segregated parvocellular, and lular magnocel neurons, pyramidal excitatory organized of nonlaminarly types two of consisting structure brain cortex-like a is BLA The juxtaposed nuclei: the lateral nucleus and basolateral intimately nucleus two of (BLA) consists amygdala the of complex basolateral The of emotional behaviors and memories. connected through mutual inhibition. These results identify a genetically defined neural circuit for the antagonistic control segregated populations of excitatory neurons in the mouse BLA that participate in behaviors valence-specific and are and positive in representations the basolateral amygdala are unknown. Here we identify two genetically distinct, spatially and However,associations. the neural substrate for negative and positive behaviors and relationship between negative The basolateral amygdala (BLA) is a site of convergence of negative and positive stimuli and is critical for emotional behaviors Joshua Kim basolateral amygdala Antagonistic negative and positive neurons of the nature nature Received 27 June; accepted 9 September; published online 17 October 2016; RIKEN-MIT Center for Neural Circuit Genetics at the Picower Institute for Learning and Memory, Department of Biology and Department of Brain and Cognitive Sciences, Recent studies demonstrated that BLA neurons, which express the the express which neurons, BLA that demonstrated studies Recent NEUR Fos 9 OSCI -based genetic expression system. Activity-dependent Activity-dependent system. expression genetic -based 1 , 1 , , Michele Pignatelli 5 . Therefore, we reasoned that molecular profiles of the EN 9– C 1 E 6 Fos

. Recent studies showed that BLA neurons neurons BLA that showed studies Recent . advance online publication online advance during a negative or positive stimulus, are 1 , 3– 8 . The BLA is activated by negative 1 , Sangyu , XuSangyu 17 9 , 1 , 1 8 5 . . Furthermore, a neu . Despite the critical critical the Despite . 1 , , Shigeyoshi Itohara

2 Brain Brain Science Institute, RIKEN, Saitama, Japan. 1 , doi:10.1038/nn.441 2 - - - - . substrates BLA ( BLA of neurons the and positive negative for putative the markers genetic for identifying screen of the basis the as used were and group female ential gene expression profiles were compared the between shock and differ Methods), (Online normalization Suite 5 (MAS5) Microarray Affymetrix or (RMA) average multi-array robust After chip. 430A cDNA and underwent to microarray analysis using an Affymetrix reverse-transcribed Mouse was RNA Isolated groups. female and shock the from Flag against antibodies using immunoprecipitation RNA performed we Therefore, system. genetic the of dependency activity ( group female or shock the to compare Flag were or kept on a Dox diet ( bers were greater than those in mice that were kept in their home cages were neurons BLA Flag on back of a numbers placed Dox Similar for diet 2 d before sacrifice. mice were exposed to footshocks or a female mouse, then immediately for 2 d, diet Dox the from removed Once on kept a diet. Dox mice in Fos-tTA AAVand ing and mice male to a footshocks respectively. mouse, female AAV and body ( protein beads magnetic A/G–coated then can be isolated via immunoprecipitation using an anti-Flag anti which mRNA, of tails poly(A) PABPthe binds endogenous and with binds drives the expression of tTA. In the absence of doxycycline (Dox), tTA element containing of promoter activity-dependent the of control the under tTA factor transcription tetracycline-based (PABP-Flag) tag Flag C-terminal a with protein poly(A)-binding protein, binding RNA epitope-tagged an expressing ectopically involving strategy a The The putative negative and positive neurons were targeted by expos TRE Fig. 1 Fig. TRE 2 + 19– to induce the expression of PABP-Flag. PABP-Flag competes & Susumu Tonegawa in the mice that underwent kainic acid–induced seizures seizures acid–induced kainic underwent that mice the in Pabpc1-Flag k (AAV 2 2 4 and and 3 2 . TwoAAV . . To obtain transcriptional profiles, we implemented implemented we profiles, transcriptional obtain To . Supplementary Fig. 1 Fig. Supplementary 9 9 -TRE-Pabpc1-Flag were introduced into the BLA BLA the into introduced were -TRE-Pabpc1-Flag -TRE-Pabpc1-Flag). Activation of the Fig. Fig. 1b under the control of the response tetracycline + in the shock and female groups; these num these groups; and in female shock the 9 constructs were used, one containing the the containing one used, were constructs [email protected] – e , g 3 Fos Howard Howard Hughes Medical Institute, – j ). ). In more contrast, BLA neurons (AAV 1 – 3 Fig. 1b Fig. ). 9 -Fos-tTA) and the other other the and -Fos-tTA) ). , Fig. Fig. 1 f – j t r a ). This affirms the the affirms This ). a ).

Fos

C I promoter

e l s 9  - - - - -

© 2016 Nature America, Inc. All rights reserved. female group (red) ( (red) group female and (green) group shock in enriched markers genetic candidate ( ANOVA fold, (>1.25 genes enriched represent ( shock from purified RNA of analysis microarray from values expression RNA 25 bars: Scale ( shock the for shown are (DAPI) marker nuclear ( shock ( Dox On of BLA the in expression ( neuron. BLA a of varicosities and soma in expression Flag **** ** comparisons, multiple for Significance group). ANOVA, (one-way seizure or female, shock, Dox), (Off cage home to exposed and diet Dox a off taken or Dox) (On diet Dox a on kept mice in BLA the in expression ( beads. A/G–coated Protein and antibodies anti-Flag using immunoprecipitated RNA (Dox), doxycycline of absence binds turn in profiling. ( 1 Figure t r a  mean show Results genes. l a a k

) Quantification of of Quantification ) ) Viral-based genetic scheme for activity-dependent transcriptional transcriptional activity-dependent for scheme genetic Viral-based )

Shock group (log ) h e g d c 2 Magnetic 12 14 10

AAV bead P 4 6 8 < 0.0001; not significant, N.S. ( N.S. significant, not 0.0001; < g AAV BLA C I ,

h Activity-dependent transcriptional profiling of BLA neurons. neurons. BLA of profiling transcriptional Activity-dependent Fos ), and female-exposed ( female-exposed and ), Anti-Flag n 8 6 4 Fos e l promoter activity drives the expression of tTA, which which tTA, of expression the drives activity promoter TRE = 3) and female ( female and 3) = AG Rspo2 TRE Dox µ Flag Flag Neural activity m ( m and drives the expression of PABP-Flag in the the in PABP-Flag of expression the drives and s Female group(log c n in situ in ), 250 250 ), = 3 mice per group); black, positive control control positive black, group); per mice 3 = Pabp1c tTA BLA hybridization of BLA expression of of expression BLA of hybridization ± µ s.e.m. ( s.e.m. F m ( m 4,25 RNA immunoprecipitation Flag n 10 DAPI d Flag Flag = 3) groups. Red and green points points green and Red groups. 3) = = 131.0, 131.0, = d 2 i – Ppp1r1b ) , ), Off Dox ( Dox Off ), g j ) groups. Flag expression and and expression Flag groups. ) , b i j i ), 80 80 ), , l 12 ). P = 0.9948). ( 0.9948). = RNA µ BLA P BLA m ( m < 0.0001, 0.0001, < 14 e P ), seizure ( seizure ), l h < 0.05, log 0.05, < h Ppp1r1b Camk2a , Neurl1a ) and female ( female and ) Gpr137 Gabra1 Gabra2 P Acvr1c j Rspo2 Zfpm Gpr39 ). ( ). Flag Flag Gipc1 Ntng2 Nptx2 = 0.0016, 0.0016, = Grin1 Htr2c Esrra Thy1 Aig1 b b k Labeling (%) ) PABP-Flag PABP-Flag ) c 2 ) RMA-normalized RMA-normalized ) 100 ) PABP- ) f Percent labeling(% 20 40 60 80 5 0 On Dox 0 n f = 6 per per 6 = ), ),

d Off Dox 2 ** –

scale). scale). BLA j ) Flag Flag )

****

Shock j 0

) groups. groups. ) Female

**** N.S.

Seizure

DAPI Flag Flag

100 ) the the role of more detailed expression pattern of Cre within the BLA, nor examined we had not further characterized this transgenic mouse with respect to the using line mouse transgenic Cre BLA-specific expression, we had previously generated a BLA-restricted BLA, with little expression in other brain areas. In fact, because of this ( neurons in the shock group ( ( of these candidate genes were expressed in a all virtually BLA neurons Quantification of gene expression( in theBLA BLA revealedthe that of the majority plane (AP) anterior-posterior mm −1.6 to mm −1.0 the in signals quantifiable a yielded probes 16 fluorescence single-label for genes 37 were individually screened on Allen Mouse Brain Atlas in expressionand shock the groupsgenes was female whose enriched pal neurons. First, independently of statistical significance, hundreds of the gene markers would label a subpopulation (<100%) of BLA princi and positive neuron populations. As a corollary, this posits that each of gene candidate, markersone a forsingle ofputative each the negative therefore, we sought to select from our potential list of candidateputative genetic negative and positive BLA neurons would be non-overlapping; Rspo2 neurons than 11 0 yielded qPCR single-cell neurons, parvocellular 38 of 10 yielded qPCR single-cell rons, neu magnocellular 37 Of neurons. BLA recorded patch-clamped of harvest from cytoplasmic PCR (qPCR) quantitative of use single-cell identity, genetic ( respectively neurons, BLA parvocellular and ings. and neurons. pyramidal BLA of entirety the define collectively and Camk2a the both for probes pBLA neurons of the pyramidal to the parvocellular neurons corresponded pyramidal neurons magnocellular of the aBLA. In contrast, Ppp1r1b Gad1 marker neuron pyramidal Camk2a the with colabeled were neurons BLA of BLA neurons were ( of neurons population segregated spatially that (−0.8 revealed bregma) BLA from mm the −2.8 to of axis AP the across quantification and (smFISH) BLA neuronspositive characterization. for further overlapping. Therefore, we selected non- are that neurons of sets in expressed be may markers two these basis of the distribution pattern of neurons.of on 50% Furthermore, BLA the than less gene that labeled DARPP-32) encodes which 1B, subunit inhibitor tory group, female-exposed the to belonging genes candidate the Among neurons. BLA of 50%, than neurons. marker for anatomical further and studies offunctional negative BLA Therefore, we selected Fig. 1 On the basis of previous basis observations the On The electrophysiological and morphological properties of of properties morphological and electrophysiological The fluorescence single-molecule Double-label Ppp1r1b Supplementary Supplementary Fig. 3 Ppp1r1b Rspo2 ( 1 + l , neurons than than neurons and 2 i. 2d Fig. 6 + and non-overlapping with the inhibitory neuron marker marker neuron inhibitory the with non-overlapping and . Double smFISH with a a with smFISH Double . Rspo2 BLA neurons expressed either either expressed neurons BLA Fig. 1 Fig. advance online publication online advance Rspo2 + + Supplementary Fig. 2 and and neurons ( neurons + Ppp1r1b neurons were examined using patch clamp record clamp patch using examined were neurons – + l -expressing BLA neurons in valence-related behavior. ). Furthermore, Furthermore, ). BLA neurons constituted less than 100%, but greater g Rspo2 Ppp1r1b and and Fig. 1 Rspo2 Ppp1r1b Rspo2 + Rspo2 + Fig. 2 Fig. neurons; membrane resistance was in smaller Table and k ) and was expressed in less than 100% of BLA + ). ). Therefore, + were targeted by patching magnocellular magnocellular patching by targeted were and and Ppp1r1b Ppp1r1b as a candidate for a negative BLA neuron Ppp1r1b i ). Soma diameter was larger in in larger was diameter Soma ). + 1 neurons; membrane capacitance was was capacitance membrane neurons; ). ). Rspo2 Ppp1r1b ). Rspo2 Rspo2 Camk2a Rspo2 Rspo2 Ppp1r1b + (protein phosphatase 1 regula 1 phosphatase (protein + Rspo2 ( neurons were identified by the neurons were identified in situ in expression was specific to the the to specific was expression Table Rspo2 and 9 + , 1 + neurons corresponded to to corresponded neurons (R-spondin 2) was enriched Rspo2 Rspo2 5 showed that virtually all all virtually that showed

and 0 0 and , we hypothesized that the , we hypothesized probe and the combined combined the and probe gene promoter, although promoter,although gene as a marker potential for nature nature Ppp1r1b Fig. 2a Fig. Supplementary Fig. 2 Fig. Supplementary hybridization, of which which of hybridization, 1 + ). and Fig. 2 Fig. and and in situ in or or Rspo2 Ppp1r1b Ppp1r1b Ppp1r1b – Ppp1r1b , it appeared that c NEUR h ). Less than 1% than Less ). + 2 hybridization hybridization ). To ascertain To ). ascertain 2 5 and 4 was the only only the was . . We selected Rspo2 + OSCI + neurons; neurons; Ppp1r1b ( neurons labeled labeled Table Rspo2 Rspo2 + EN and and C ). ). 1 + + + + E - - - -

© 2016 Nature America, Inc. All rights reserved. (green, (green, ( ( (**** diameter soma Rspo2 ( units. fluorescence relative rfu, shown; are gene either for occurred amplification which in traces qPCR neurons. BLA (bottom) parvocellular and (top) magnocellular ( 50 bars, scale neuron; BLA (bottom) parvocellular and (top) 500 in micrograph Larger BLA. and ( BLA. the of of smFISH double of view Coronal of smFISH double of mm) 3.4 mm, 3.2 midline from distance (ML views sagittal ( mice individual represent traces means; represent Bars BLA. the of mm) −2.8 to mm −0.8 bregma from distance (coronal of ( neurons. pyramidal BLA of populations 2 Figure ( water and water sucrose between as well as water, no sure to quinine water (which expo elicited between little expression water c-Fos drinking) relative compared in to observed was difference received no water or quinine water ( the pBLA in response to water and sucrose water compared to mice that sucrose (sweet)—relative c-Fos expression was significantly greater in response to valence-specific gustatory stimuli —quinine (bitter), water, peanut oil compared to exposure to benzaldehyde or TMT ( to response in pBLA the in greater significantly expressionwas c-Fos exposure to the neutral odor benzaldehyde or peanut oil, while relative was significantly greater in the aBLA in response to TMT compared to trimethyl-3-thiazoline (TMT) or peanut oil—relative c-Fos expression ( compared to exposure shock or control conditions of a neutral context mice response toin female pBLA greater the in significantly sion was ( context a in stimulus no delivered that conditions control or mouse female a to exposure to responsecompared toin footshocks aBLA greater the in significantly in the aBLA or pBLA as a percentage of total c-Fos relative c-Fos expression, bymeasured number the of c-Fos intervals across the AP axis ( c-Fos of number total the measuring by ries) separately in the aBLA and pBLA (defined by cytoarchitectural bounda mice—and were sacrificed 90 min later. female c-Fosor markers—shocks gene BLA identify to used stimuli the to iors may differentially activate the aBLA and pBLA. Mice were exposed of the BLA, respectively, then stimuli that elicit valence-specific behav If stimuli valence-specific by activation BLA types. cell distinct electrophysiologically cally, and morphologi genetically, segregated, spatially defined neurons BLA ( respectively neurons, parvocellular and magnocellular unconfirmed from ferent confirmed in larger nature nature mean show Results positive positive behaviors (female, water, sucrose, peanut oil). is iorspBLA recruited bythe TMT), (shocks, while stimuli that elicits behav negative elicits that stimuli by recruited is aBLA the Overall, C R i i. 3 Fig. ) Single-cell qPCR traces of of traces qPCR ) Single-cell Rspo2 m m Rspo2 ; ; **** ; ; ** Ppp1r1b µ m m ( + P (top) and and (top) d n +

= 0.0016, = 0.0016, = 11) and and = 11) (green) and and (green) . n epne o aec-pcfc latr stimuli—2,3,5- olfactory valence-specific to response In ). NEUR and Rspo2 b P Rspo2 ), 200 200 ), < 0.0001, < 0.0001, Rspo2 Rspo2 d ( – Ppp1r1b e Table g OSCI ), ), + ) Double smFISH of of smFISH ) Double + and and and and neurons than than neurons µ Ppp1r1b Gad1 + m m ( ± and and Ppp1r1b j t P EN s.e.m. ( s.e.m. ) Electrophysiological response to current steps in a in steps current to response ) Electrophysiological (19) = 3.680), and membrane capacitance capacitance membrane and = 3.680), (19) Ppp1r1b Ppp1r1b 1 < 0.0001, < 0.0001, c Ppp1r1b t + and and ). Taken together, then, then, together, Taken ). (19) = 4.734) of qPCR-confirmed qPCR-confirmed of = 4.734) (19) ), 25 25 ), C neurons represent negative and positive neurons Ppp1r1b E +

Supplementary Figure 3 Figure Supplementary (bottom) BLA neuron. ( neuron. BLA (bottom) Fig. 3 Fig. Rspo2 advance online publication online advance + µ a Rspo2 Fig. 3a (red, (red, m m ( (red) expression across the AP axis axis AP the across expression (red) , + k with nuclear marker DAPI in the BLA ( BLA the in DAPI marker nuclear with BLA neurons define spatially segregated segregated spatially define neurons BLA ). d Ppp1r1b t d Rspo2 + ( (19) = 6.106), membrane resistance resistance membrane = 6.106), (19) – neurons were not significantly dif significantly not were neurons ). Conversely, relative c-Fos expres Conversely,c-Fos relative ). n f (green) and and (green) g Camk2a ) or ) or – = 12) neurons. Unpaired Unpaired neurons. = 12) ). ( ). Fig. 3 c and h a Gad1 and and ) Quantification of smFISH smFISH of ) Quantification ) Biocytin-filled magnocellular magnocellular ) Biocytin-filled + neurons ( neurons f Supplementary Fig. 4 and and ). In contrast, no significant Ppp1r1b and and + + neurons were quantified Ppp1r1b neurons per section at section per neurons Rspo2 Rspo2 . Scale bars: bars: . Scale Ppp1r1b k ) Comparison of mean mean of ) Comparison + BLA neurons, was across the AP axis axis AP the across Fig. 2j Fig. ( n + (red), from from (red), d = 3). ( = 3). Rspo2 and and ), ), ( µ Camk2a g m. m. ) in the the ) in , Fig. 3 t k

+ Ppp1r1b -test. -test.

+ b ). qPCR- ).

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xs lns ihn BA n pL, epciey ( 5 Fig. respectively pBLA, and aBLA within planes axis of of activation specific ( neurons in expression c-Fos increased ( in not but increased significantly Shocks experiments). behavioral subsequent in used be (to stimuli specific of i. 3g Fig. AP –1.6 ML 3. AP –2.4 AP –2.0 AP –1.2 a h c b Double smFISH was performed to directly assess the expression expression the assess directly to performed was smFISH Double Fos Ppp1r1b Labeling (%) 2 100 ). However, these analyses also revealed some heterogeneity of heterogeneity some revealed also analyses However, ). these in in 20 40 60 80 BLA BLA 0 BL BL , h A Rspo2 Fig. 3i Fig.

A –0.8 , m + Biocytin Biocytin pBLA cells were confirmed by further analyses of AP AP of analyses further by confirmed were cells pBLA Ppp1r1b Rspo2 Rspo2 Rspo2 Rspo2 ,

–1.0 n ). In contrast, administration of water significantly significantly water of administration contrast, In ). + , or or o i AP –1.6 ), compared to no water ( water no to compared ), AP –2.0 AP –1.2

AP –2.4 –1.2 6 6 Rspo2

Fluorescence (10 rfu) Fluorescence (10 rfu) Ppp1r1b Rspo2 2.0 4.0 6.0 2.0 4.0 6.0 + AP distancefrombregma(mm) 0 0 –1.4 ( neurons 2 0 2 0 BLA

BL BL Ppp1r1b Rspo2 Ppp1r1b Ppp1r1b Rspo2 BL PCR cycles PCR cycles A A A 4 0 4 0

–1.6 + Ppp1r1b Ppp1r1b Ppp1r1b Ppp1r1b aBLA cells and water-specific activation activation and water-specific cells aBLA + 6 0 6 0

BLA neurons in response to valence- to response in neurons BLA Ppp1r1b DAPI –1.8 Fos 8 0 8 0 i. 3h Fig. 0 0 AP –1.6 expression in in expression AP –2.4 AP –1.2 AP –2.0 ML 3.4

j –2.0 +

–2.2 ( Rspo2 Rspo2 Rspo2 Rspo2 BLA B B Fig. 3j Fig. B LA LA , LA l ), compared to context context to compared ),

–2.4 3i Fig.

Ppp1r1b Ppp1r1b Ppp1r1b Ppp1r1b ,

–2.6 p 0.2 0.2 ) but not in in not but ) Rspo2 t r a , j g f e d , s s q

–2.8 Supplementary Rspo2 , k 100 pA 20 mV 100 pA 20 mV r ). The shock- The ). Ppp1r1b + Rspo2 (

C (pF) R (MΩ) Diameter (µm) Ppp1r1b m m Ppp1r1b C I 100 200 300 400 100 150 200 250 Fig. 3g Fig. 50 10 15 20 Rspo2 Ppp1r1b Rspo2 0 0 0 5

Camk2a Rspo2 Camk2a e l **** ***

Gad1 Gad1 DAPI ** * , k s +  )

© 2016 Nature America, Inc. All rights reserved. Magnocellular ( Magnocellular Cell type Mean proportion (%) Total neurons ( Mean proportion (%) P unpaired for Significance cells. positive and of group) sucrose, vs. quinine peanut, vs. female, vs. Shock significant. not N.S., ANOVA, (one-way water sucrose ANOVA, (one-way oil TMT,to peanut or BA ANOVA, (one-way female or context shock, to response in pBLA and aBLA the in c-Fos of number ( (BA) benzaldehyde ( shock to response in (bottom) pBLA and (top) aBLA the of mm) −2.8 to mm −0.8 bregma 3 Figure neurons of populations defined genetically by represented is mation Fig. 5 more posteriorly within aBLA and pBLA, respectively ( and ( stimuli neutral the to relative pBLA, within water by activated rons Rspo2 and unpaired of allmagnocellularandparvocellularneuronsrecordedasubsetneurons,whichwereqPCR-confirmed morphological (somadiameter)andelectrophysiologicalproperties(restingmembranepotential( total numberofneuronscountedin and theircolocalizationwith ( Magnocellular Rspo2 ( Parvocellular Total neurons ( Mean proportion (%) Total neurons ( Total neurons ( Total neurons ( Total neurons ( Mean proportion (%) Total neurons ( Table 1 t r a  in (red) positive and (green) negative Rspo2 Magno- ( P Ppp1r1b Top, smFISHquantificationof ( Parvocellular Supplementary Fig. 5 -values

= 0.5685, = 0.5685, ( ( parvocellular Ppp1r1b Rspo2 n Rspo2 = 11) Ppp1r1b Ppp1r1b + + ). Nevertheless, these data suggest that negative and positive infor + ( ( neurons activated by shock within aBLA and and aBLA within shock by activated neurons + t + n n n

C I -test comparisonsbetweenallmagnocellularandparvocellularBLAneurons,qPCR-confirmed ( ( + = 37) versus = 10) = 10) versus Genetic, anatomical, morphological and electrophysiological characterization Genetic, of anatomical, and morphological characterization electrophysiological Rspo2 Fos BLAneurons,qPCR-unconfirmedparvocellularand + n n ( = 10) = 11) P n < 0.0001; BA vs. peanut, peanut, vs. BA < 0.0001; + n n t ( e l n n n n n n n = 11) + (8) = 0.5946; ( = 0.5946; (8) Rspo2 n n = 38) = 27) versus j = 3) = 3) = 3) = 4) = 1) = 1) = 3) neurons by activated specifically water were distributed n , = 37) = 27) versus + p + = 38) and and , cells is represented for each coronal section of a unilateral BLA; micrographs in in micrographs BLA; a unilateral of section coronal each for represented is cells r s ) in response to water (W) or no water (NW) (AP axis analysis in in analysis axis (AP (NW) water no or (W) water to response ) in n + P = 7) or peanut oil ( oil peanut or = 7) (

Ppp1r1b Ppp1r1b < 0.0001; quinine vs. no water, water, no vs. quinine < 0.0001; g , ): Camk2a k , Rspo2 Rspo2 m

) or ) or j + ) ) n +

and intheBLA,

P animals.Bottom, BLA neurons are activated by valence-specific stimuli. ( stimuli. valence-specific by activated are neurons BLA F + Fos = 0.0013, = 0.0013, 2,19 neurons activated specifically by shock ( Rspo2 CTB-CeL/CeM Ppp1r1b Soma diameter( + Ppp1r1b = 33.91, = 33.91, a CTB-NAc CTB-CeC Soma Soma diameter – 96.2 96.2 12.8 12.8 13.1 13.1 j 30.7 30.7 5.58 59.9 59.9 + P P t ; ; 9.4 9.4 9.5 9.5 7 7 × 10 n -test ( -test Ppp1r1b 0.00002 Ppp1r1b < 0.0001; shock vs. context, context, vs. shock < 0.0001; = 0.0132; water vs. quinine, quinine, vs. water = 0.0132; Rspo2 Rspo2 Rspo2 = 6) ( = 6) Rspo2 2,361 3,611 F expressionintheBLA, 344 423 190 303 0.6 0.5 64 ± 2,16 ± ± ± ± t ± ± ± 0.945 (8) = 4.822. Scale bars, 125 125 bars, Scale = 4.822. (8) 0.2 0.2 0.3 0.5 + + + 3.53 1.74 1.28 Rspo2 Rspo2 ( g −17 Rspo2 P + and + = 16.61, = 16.61, (green) and and (green) + – h ) + b < 0.0001). Significance for multiple comparisons ( comparisons multiple for Significance < 0.0001). Rspo2 + j , ); and quinine water ( water quinine and ); ), ** ), Camk2 l m , n Ppp1r1b + + m) + ) in response to shock (S) or context (C). Double-label smFISH of of smFISH Double-label (C). context or (S) shock to response ) in and P Ppp1r1b + P < 0.01; N.S., not significant; ( significant; not N.S., < 0.01; + = 0.3500; sucrose vs. no water, water, no vs. sucrose = 0.3500; Supplementary Ppp1r1b P Ppp1r1b colocalizationwithCTBinCTB-injectedmice(CeC,CeL/CeM,andNAc),cellcountscorrespondstothe = 0.0001). ( = 0.0001). ( Ppp1r1b Rspo2 CTB-CeL/CeM + BLAneurons; CTB-NAc Rspo2 CTB-CeC CTB-CeC + 3.78 3.78 BLAneuronscorrespondtomagnocellularandparvocellularneurons.Quantificationof + 69.2 69.2 94.4 39.1 −60.9 −60.9 −55.6 −55.6 −62.5 −62.5 −57.1 −57.1 + Ppp1r1b Ppp1r1b 0.00003 V neu and 1,012 2,311 0.051 Gad1 Gad1 (red) in in (red) m 775 116 112 0.4 0.3 16 V (mV) ± n 0 P ± ± ± P + m 0.945 Ppp1r1b = 8), no water ( water no = 8), Ppp1r1b 3.53 1.74 1.14 ± ± ± ± = 0.0010; context vs. female, female, vs. context = 0.0010; < 0.0001; water vs. sucrose, sucrose, vs. water < 0.0001; f Ppp1r1b - - ) Relative c-Fos expression in response to quinine water, no water, water or water water, no water, quinine to response in expression c-Fos ) Relative + + + 0.9 0.8 1.5 2.1

Ppp1r1b ) µ n + − correspondstothenumberofneuronsfromatleast3micepergroup. m m ( Camk2 that these populations may be necessary for valence-specific behaviors; of activation Valence-specific behaviors valence-specific in BLA neurons, predominatewhich in representthe pBLA, valence. positive segregated: predominate in spatially the aBLA, represent negative valence, while are that BLA the in k – + k + r F colocalizationwith – . 2,19 V + Supplementary Fig. 5 Fig. Supplementary r ). Results show mean mean show Results ). + m ), membraneresistance( = 33.91, = 33.91, g 103.1 103.1 165.5 165.5 108.9 108.9 158.7 158.7 a ) ) 6 6 × 10 R n advance online publication online advance – 0.0016 P P = 8), water ( water = 8), m c n < 0.0001). ( < 0.0001). = 0.0098, = 0.0098, 0.5 0.5 Rspo2 (M R ) c-Fos expression across the AP axis (coronal distance from from distance (coronal axis AP the across expression ) c-Fos = 8), context ( context = 8), m ± ± ± ±  −11 6.5 4.7 9.5 9.6 P ) + < 0.0001). ( < 0.0001). and Gad1 Supplementary Figure 4 Figure Supplementary Rspo2 d 197.6 197.6 102.2 102.2 190.3 190.3 Rspo2 Ppp1r1b – intheBLA,combinedprobesfor 99.5 99.5 t 1 1 × 10 n P f (8) = 3.371; ( = 3.371; (8) ); ); 0.0009 C ), * ), g P = 6) or sucrose water ( water sucrose or = 6) = .9998; water vs. no water, water, no vs. water = .9998; ± m – + R = 0.0013; TMT vs. BA, BA, vs. TMT = 0.0013; 0.6 0.7 y C s.e.m. ( s.e.m. r (pF) ( and m n -axis values denote percentage of double- of percentage denote values -axis + ) Double-label smFISH ( smFISH ) Double-label Rspo2+Ppp1r1b P m ± ± ± ± and ), capacitance( = 8) or female ( female or = 8) 4.1 10 5.9 19.1 < 0.05, ** < 0.05, −11 + BLAneurons,qPCR-unconfirmedmagnocellular Rspo2 e Rspo2 ) Relative c-Fos expression in response response in expression c-Fos ) Relative 0.970 0.970 Ppp1r1b Ppp1r1b Ppp1r1b Rspo2 a – Spike threshold(mV) + j + ). Colors: valence of the stimuli stimuli the of valence Colors: ). h 54 Ppp1r1b and 0 0 0 Spike Spike threshold ± Fos + ) ) Gad1 + 0.191 + −37.7 −37.7 −35.5 −35.5 −34.8 −34.8 −37.6 P P Gad1 BLAneurons. C < 0.01, **** < 0.01, = 0.2222, = 0.2222, and and + 0.0008 . . ( ) m BLA neurons Ppp1r1b + 0.06

), spikethreshold,andrheobase) 0.5 0.9 + Camk2 d n + ) Relative c-Fos expression expression c-Fos ) Relative nature nature = 6) ( = 6) + Rspo2 ± ± ± ± Rspo2 0.5 0.4 1.1 0.9 n = 8) ( = 8) P − Rspo2 n = 0.0377; TMT TMT = 0.0377; a + + = 5 in each each = 5 in ); TMT ( TMT ); P ( neurons suggests t neurons, which which neurons, P (10) = 1.302; ( = 1.302; (10) -values for i P c NEUR , < 0.0001; < 0.0001; o ). The total total The ). < 0.0001; < 0.0001; and , Rheobase (pA) 213.7 213.7 137.7 137.7 152.3 152.3 198.7 q ) or of of ) or Rheobase 8 8 × 10 Ppp1r1b OSCI n 0.06 Ppp1r1b 0.2 0.4 = 6), = 6),

± ± ± ±

6.7 10.5 15.2 18 Fos −8 EN

C i ) + E

© 2016 Nature America, Inc. All rights reserved. “Ppp1r1b.” The light-activated inhibitory ion channel eArch3.0 was was eArch3.0 channel ion using inhibitory to “Ppp1r1b.”light-activated The referred be will mice Cartpt-Cre virus-injected hereafter, protein ( (Cartpt-Cre) mice transcript driver Cre (Cartpt) amphetamine-regulated and cocaine- through respectively. mice, Cartpt-Cre and Rspo2-Cre using targeted genetically were neurons experiments. conditioning reward and fear in populations BLA these of inhibiting effects the we studied therefore, nature nature NEUR OSCI Ppp1r1b EN d a C

k + + l + + + + c-Fos cell count c-Fos cell count E pBLA Fos /total Fos aBLA Fos /total Fos 100 150

50 20 40 60 100 100 0 0 20 80 80 60 40 20 40 60 advance online publication online advance Shock Shock +

–1.6 –0.8 accessible genetically are neurons BLA Shock **

Female Context Shock –1.8 –1.0 ****

** –2.0 –1.2 Context Ppp1r1b

–2.2 6 Fig. Supplementary Rspo2 –1.4 Fos Fos e –2.4 –1.6 100 100 Female 60 40 80 20 20 40 60 80 Rspo2 m –2.6

n –1.8

–2.8 –2.0 Context Context Peanut BA TM * + **** and and T b * 100 20 40 60 25 50 75

0 Ppp1r1b AP distancefrombregma(mm)forpBLA AP distancefrombregma(mm)foraBLA 0

–1.6 –0.8 and ), Ppp1r1b f Rspo2 TM 100 100

80 60 40 20 20 40 60 80 –1.8 –1.0 0 Fos Fos B T +

–2.0 –1.2 Sucrose Water No water Quinine N.S. p A eYFP) ( eYFP) bilaterally targeted to the BLA during footshocks ( (AAV eArch3.0 lacking vector viral a received eYFP) and (Rspo2-eYFP, Ppp1r1b- mice Control respectively. mice, Rspo2-Cre Cartpt-Cre of BLA the to targeted bilaterally eArch3.0-eYFP) (AAVvector viral BLA neurons a using Cre-dependent in expressed o –2.2 –1.4 On On day 1 of contextual fear conditioning, mice received green light, Peanut ****

Water Water –2.4 –1.6 N.S. –2.6 –1.8 4a Fig.

–2.8 –2.0 Rspo2 , l , Ppp1r1b j h i g m + + + + c Rspo2 Fos /Ppp1r1b (%) Fos /Rspo2 (%) and and 20 40 60 100 + 10 15 20 10 15 20 0 Fos Fos 50 75 25 0 5 0 5 (Rspo2-Arch) and and (Rspo2-Arch) 0

–1.6 7 Fig. Supplementary S C S q –0.8 r N.S. ** –1.8

C –1.0 No water Quinine No Water No Water –2.0 –1.2

+ + –2.2 Fos+/Ppp1r1b+ (%) Fos /Rspo2 (%) –1.4 10 15 20 10 15 20 25 0 5

0 5 –2.4 –1.6 Sucrose Water Ppp1r1b N W Ppp1r1b N W –2.6

N.S. –1.8 Rspo2 **

Fos Fos –2.8 W

W –2.0 ). Fig. 4 + t r a (Ppp1r1b-Arch) (Ppp1r1b-Arch) b 5 ). Rspo2-Arch 5 -EF1 -EF1 C I α α -DIO- -DIO- e l s  © 2016 Nature America, Inc. All rights reserved. Ppp1r1b-Arch mice ( mice Ppp1r1b-Arch **** 0 0.001, t ( 0.0001, ( ( mice ChR2 0.0020, ( controls eYFP 2 than 1 and days on more ( test freezing optogenetic an pokes Ppp1r1b, ( Rspo2-Arch between difference no ( P ( controls eYFP 2 than 1 and days on less froze ( neurons. BLA 4 Figure similar made mice Rspo2-eYFP and Rspo2-ChR2 while mice, eYFP of a Ppp1r1b- than made number mice pokes greater Ppp1r1b-ChR2 ( port nose a into poked mouse the when administered was stimulation light blue which in chamber conditioning operant an in context. neutral a to conditioned be can not ( freezing of levels similar displayed mice Ppp1r1b-eYFP and Ppp1r1rb-ChR2 while mice, Rspo2-eYFP than of levels freezing greater displayed mice Rspo2-ChR2 shock. without measured was freezing and context the to returned were mice 2, day ( of levels freezing similar mice displayed Ppp1r1b-eYFP of levels and greater Ppp1r1rb-ChR2 while mice, displayed Rspo2-eYFP to compared freezing mice Rspo2-ChR2 Methods). Online ( stimulation light blue receiving while context neutral (AAV ChR2 lacking vector viral a received Ppp1r1b-eYFP) (Rspo2-eYFP, mice Control tively. respec mice, Cartpt-Cre and Rspo2-Cre of BLA the to targeted ally Cre-dependent viral vector (AAV and (Rspo2-ChR2) in was expressed ChR2 ion channel excitatory light-activated behavior. appetitive to stimulus conditioned a of association the for and behavior seeking Ppp1r1b mice ( compared to Ppp1r1b-eYFP reward association contrast, and cue– of pokes In nose levels reduced displayed mice Ppp1r1b-Arch Methods). Online see period; cue during port reward ( association cue–reward and pokes nose of levels similar displayed mice Rspo2-eYFP and Rspo2-Arch delivered into the BLA with simultaneously the presentation of water. ( cue light external an lowing fol poke nose a on contingent dispensed was water where chamber behavior. of to a freezing context association and for the stimuli not Ppp1r1bp ( mice Ppp1r1b-eYFP to compared mice in Ppp1r1b-Arch were of levels observed freezing similar while mice, Rspo2-GFP in than mice Rspo2-Arch in observed was freezing Less stimulation. or light shock without context the in were tested mice 2, day On mice. Ppp1r1b-eYFP to compared displayed freezing of mice levels similar Ppp1r1b-Arch mice. Rspo2-eYFP with compared footshocks to response in freezing of levels reduced displayed mice t r a  ( amygdala central (12) = 5.589. Significance for unpaired unpaired for Significance = 5.589. (12) n n e

< 0.0001, < 0.0001, ) Ppp1r1b-Arch mice ( mice ) Ppp1r1b-Arch = 7) mice displayed greater preference for light stimulation than eYFP controls ( controls eYFP than stimulation light for preference greater displayed mice = 7) 1 day Rspo2, mice. = 6) On day 1 of the optogenetic self-stimulation test, mice were mice test, placed On day 1 self-stimulation of the optogenetic a in placed were mice test, freezing optogenetic the of 1 day On Next we assessed the effects of activating these BLA neurons. The The neurons. BLA these activating of effects the assessed we Next conditioning operant an in place took conditioning Reward Ppp1r1b

C I + t t Rspo2 (10) = 6.550. ( = 6.550. (10) 2 day = 3.926, (12) , , but not n t e l + = 6) made more nose pokes on days 1 and 2 than eYFP controls ( controls eYFP 2 than 1 and days on pokes nose more made = 6) (15) = 5.366, day 2 day = 5.366, (15) P , BLA neurons are capable of eliciting freezing, which which freezing, eliciting of capable are neurons BLA , b + < 0.0001; N.S., not significant; results show mean mean show results significant; not N.S., < 0.0001; + – , BLA neuronal activity is critical for freezing to for shock is freezing critical , activity BLA neuronal P and and e = 0.0192, = 0.0192, s m ) Scheme and results for Rspo2-Arch and Ppp1r1b-Arch mice in fear ( fear in mice Ppp1r1b-Arch and Rspo2-Arch for results and ) Scheme Rspo2 , o m Ppp1r1b Ppp1r1b ). Scale bars, 300 300 bars, Scale ). ). Expression of ChR2-eYFPin Rspo2-ChR2 mice ( mice Rspo2-ChR2 ChR2-eYFPin of Expression ). n k = 10) displayed lower total nose pokes and cue–reward association in nose port ( port nose in association cue–reward and pokes nose total lower displayed = 10) + ) Rspo2-ChR2 mice ( mice ) Rspo2-ChR2 , , BLA neuronal activity is critical for reward- P = 0.0528, = 0.0528, f 5 t , -EF1 (19) = 2.55, = 2.55, (19) + g + P BLA neurons participate in valence-specific behaviors. ( behaviors. valence-specific in participate neurons BLA (Ppp1r1b-ChR2) BLA neurons using a using neurons BLA (Ppp1r1b-ChR2) ), optogenetic self-stimulation test ( test self-stimulation optogenetic ), < 0.0001, < 0.0001, Fig. 4 Fig. P α 5 n < 0.0001, < 0.0001, -EF1 = 9) and Rspo2-eYFP ( Rspo2-eYFP and = 9) -DIO-eYFP) ( -DIO-eYFP) d t µ t (12) = 2.148, day 2 day = 2.148, (12) z ). Green light was bilaterally bilaterally was light Green ). α -test between experimental groups compared to corresponding eYFP controls, * controls, eYFP corresponding to compared groups experimental between -test m m ( n -score of time spent in the the in spent time of -score -DIO-ChR2-eYFP) -DIO-ChR2-eYFP) unilater z = 6); no difference between Ppp1r1b-ChR2 ( Ppp1r1b-ChR2 between difference no = 6); t (12) = 6.216; Ppp1r1b, day 1 day Ppp1r1b, = 6.216; (12) Fig. 4 Fig. -score -score Fig. 4 Fig. l – n o n t = 8); no difference between Ppp1r1b-Arch ( Ppp1r1b-Arch between difference no = 8); ). Colors: Rspo2-Cre mice (green) and Cartpt-Cre mice (red) ( (red) mice Cartpt-Cre and (green) mice Rspo2-Cre Colors: ). (15) = 10.24; Ppp1r1b, day 1 day Ppp1r1b, = 10.24; (15) = 11) displayed greater preference for light stimulation than eYFP controls ( controls eYFP than stimulation light for preference greater displayed = 11) c P g ). Thus, Rspo2 Thus, ). Fig. 4a Fig. ). Thus, Thus, ). = 0.0097, = 0.0097, Fig. Fig. 4 , n n Fig. Fig. 4 Rspo2 Fig. 4 Fig. = 8). Rspo2, pokes pokes Rspo2, = 8). P , o = 0.4256, = 0.4256, t ). e Fig. 4 Fig. (19) = 2.873. ( = 2.873. (19) ). ). Thus, Rspo2 g h ± + + f ). ). On , s.e.m. ( s.e.m. and and , but , , but , i ), and optogenetic place preference test ( test preference place optogenetic and ), h n ). ). + - - - ) and Ppp1r1b-ChR2 mice ( mice Ppp1r1b-ChR2 ) and

t P (12) = 0.8247; Ppp1r1b, day 1 day Ppp1r1b, = 0.8247; (12) = 0.5408, = 0.5408, c time in the light-stimulated side compared to corresponding con trols ( corresponding to compared side light-stimulated the in more time spent mice Ppp1r1b-ChR2 while compared controls, side corresponding light-stimulated to the in time less spent mice ChR2 shocks ( in foot the BLA during blue stimulation light bilateral received mice ChR2-expressing conditioning, fear contextual of 1 day On stimuli. Rspo2 pose, we examined the behavioral of effects activating optogenetically pur this For memories. and behaviors emotional of control onistic examined whether these two types of neurons contribute to the antag Rspo2 neurons BLA positive and negative between Antagonism preference. place while aversion place conditioning. reward support and self-stimulation eliciting while ( mice, Ppp1r1b-eYFP pokes of numbers than similar made mice Rspo2-eYFP and pokes Rspo2-ChR2 more made mice ChR2 Ppp1r1b- delivered. was stimulation light no chamber, which in tion numbers of pokes. On day 2, mice were returned to the operant condi interactions between these two neuronal populations. Therefore, the Therefore, populations. two neuronal these between interactions than by rather by direct circuits be result of downstream interference may antagonism behavioral valence, opposing the of stimuli by ited reward. a with stimulus tioned condi a of association the and behaviors reward-seeking disrupting association ( cue–reward and pokes nose of levels similar displayed to mice, Rspo2-eYFP while Ppp1r1b-ChR2 and Ppp1r1bp-eYFP mice compared association cue–reward and pokes nose of levels reduced ( reward delivery during stimulation footshocks. with stimulus contextual conditioned a of association the and footshocks to freezing disrupting of capable is neurons BLA mice ( than in Ppp1r1b-eYFP mice Ppp1r1b-ChR2 in observed was freezing less while mice, eYFP difference in freezing was between observed Rspo2-ChR2 and Rspo2- no 1, day on As stimulation. light or to footshock mice without context the returning by assessed was freezing conditioned 2, day On displayed lower levels of freezing than Ppp1r1b-eYFP mice ( levels of freezing in response to footshocks, while Ppp1r1b-ChR2 mice Fig. Fig. 4 Fig. 5e Fig. , e n P In a real-time optogenetic place preference test ( test preference place optogenetic real-time a In Although Although blue light received mice ChR2-expressing In conditioning, reward , f = 6); no difference between Rspo2-ChR2 ( Rspo2-ChR2 between difference no = 6); = 0.3700, = 0.3700, g – P , n k i = 0.6811, = 0.6811, , ) Scheme and results for Rspo2-ChR2 and Ppp1r1b-ChR2 mice in mice Ppp1r1b-ChR2 and Rspo2-ChR2 for results and ) Scheme = 7). Rspo2, Rspo2, = 7). Fig. Fig. 4 + + k i and or ). ). Thus, ). ( ). Fig. 5 , f a ). Thus, activation of of activation Thus, ). b l t advance online publication online advance Ppp1r1b – ) Optogenetically targeting targeting ) Optogenetically n (8) = 0.6388, day 2 day = 0.6388, (8) , k Ppp1r1b o c = 5) and Ppp1r1b-eYFP ( Ppp1r1b-eYFP and = 5) Rspo2 ) Expression of eArch-eYFP in Rspo2-Arch mice ( mice Rspo2-Arch in eArch-eYFP of ) Expression ) and reward ( reward ) and ). ). Therefore, a n t (12) = 0.9314, day 2 day = 0.9314, (12) ). Rspo2-ChR2 and Rspo2-eYFP mice displayed similar = 8) and Ppp1r1b-eYFP ( Ppp1r1b-eYFP and = 8) Ppp1r1b t (15) = 0.4191, = 0.4191, (15) + P + and < 0.0001, < 0.0001, + neurons during the presence of valence-specific of valence-specific presence the during neurons neurons drive opposing behaviors; therefore, we Ppp1r1b o ). Strong Ppp1r1b Strong ). + Ppp1r1b , , but not d Rspo2 , z e -score) compared to eYFP controls ( controls eYFP to compared -score) ) conditioning. ( ) conditioning. P c Fig. Fig. 5b = 0.0003, = 0.0003, + P , t e BLA neurons are capable of eliciting eliciting of capable are neurons BLA j = 0.4784, = 0.4784, (17) = 5.969; Ppp1r1b, Ppp1r1b, = 5.969; (17) + , z , k + BLA neurons are capable of eliciting g Rspo2 -score -score ). ( ). Rspo2 neurons antagonize behaviors elic , i , P k Rspo2 n g = 0.8268, = 0.8268, , Fig. Fig. 5 , = 5) mice. Rspo2, day 1 day Rspo2, mice. = 5) c ) Rspo2-ChR2 mice ( mice ) Rspo2-ChR2 l – ). Thus, activation ). of Thus, activation + o n P + BLA neurons is capable of of capable is neurons BLA P ). + = 6) mice. Rspo2, day 1 day Rspo2, mice. = 6) = 0.8510, = 0.8510, , , BLA neurons are capable of t fibers are found in the the in found are fibers < 0.05, ** < 0.05, (10) = 5.479, day 2 day = 5.479, (10) +

t d and and n (8) = 0.7435. ( = 0.7435. (8) c ). Rspo2-ChR2 displayed displayed ). Rspo2-ChR2 = 8) and Rspo2-eYFP Rspo2-eYFP and = 8) n nature nature ) Rspo2-Arch mice ( mice ) Rspo2-Arch = 8); Ppp1r1b-ChR2 Ppp1r1b-ChR2 = 8); Ppp1r1b t (12) = 0.2237. = 0.2237. (12) t P (15) = 0.1911; = 0.1911; (15) Fig. 4 Fig. NEUR < 0.01, *** < 0.01, P + = 0.0001, = 0.0001,

n

i ) ) Ppp1r1b- = 7) froze froze = 7) l OSCI j ) and ) and Fig. 5b Ppp1r1b ), Rspo2- ), P n P = 11); = 11); < =

n EN

= 9) P

< , c C ). + E ------

© 2016 Nature America, Inc. All rights reserved. Ppp1r1b-eYFP mice ( mice Ppp1r1b-eYFP in footshocks, of presence the in stimulation light was measured using stimuli valence-specific to response in other the of activation the on effect of optogenetic activation of one of the two neuronal populations nature nature Ppp1r1b NEUR + neurons and decreased in in decreased and neurons OSCI EN Fos g f a

C Freezing (%) Rspo2-cr Fig. 5g Fig. E l . . In mice, which Ppp1r1b-ChR2 received blue 20 40 60 80 eAr Ca 0 AA

r ChR2 ch3.0 tpt-cr V advance online publication online advance 5 -Ef1a-DIO- e miceor ** e mice , -eYFP or -eYFP or h eYFP Da , i Da ). In water-deprived Rspo2-ChR2 Rspo2-ChR2 water-deprived In ). y y 1 N.S 1 473 nm . Rspo2 b c

20 40 60 80 Freezing (%) 0 Rspo2-ChR2 10 20 40 60 80 0 0 + **** neurons compared to to compared neurons m **** Rspo2-Ar Da Da Fos Da y Da y 2 y 2 y N.S was increased increased was 1 1 Rspo2-eYFP N.S 532 nm chT . .

i h

Nose pokes Rspo2-eYFP 10 200 15 10 20 40 60 80 50 0 0 0 0 0 Da y Ppp1r1b-ChR2 **** N.S 2 Da Da . Da y the activity elicited by valence-specific stimuli in the opposite opposite the in stimuli valence-specific population. neuronal by elicited activity the Thus, Ppp1r1b consump the water, of during tion stimulation light blue received which mice, n 1 y y Ppp1r1b-Ar 473 nm 2 1 N.S *** . Ppp1r1b + neurons compared to that in mice Rspo2-eYFP ( Ppp1r1b-eYFP 20 15 10 d chT e 0 5 Nose pokes (×102) Da 10 0 2 4 6 8 Fos y N.S 2 Ppp1r1b-eYFP + and and was increased in in increased was . N.S Da Da y . 2 **** y 1 Rspo2 532 nm o * k j

Difference score (s) + neurons are capable of reducing reducing of capable are neurons –7 –5

50 z-score –2 473 nm 5 0 0 0 2 4 6 8 Rspo2 **** N.S Da . y + 1 neurons and decreased in in decreased and neurons **** ** t r a Fig. Fig. 5j C I e l , k , l s ). ).  -

© 2016 Nature America, Inc. All rights reserved. ( ( white) or (red positive and ( (red) mice Cartpt-Cre 300 bars, Scale ( Unpaired ( stimulation. light blue with simultaneously water Ppp1r1b blue ( with stimulation. simultaneously light shock received that mice Ppp1r1b-eYFP and of smFISH z 0.0002, controls. eYFP Unpaired mice. ( Ppp1r1b-ChR2 ( between controls eYFP difference to compared association cue–reward and ( mice. ( of conditioning. reward during mice Ppp1r1b-ChR2 and ( 2.705. = Ppp1r1b P ( Rspo2-ChR2 ( ( ( Ppp1r1b-eYFP Ppp1r1b-ChR2 in 2 day and 1 day during ( 1). freezing (day of shocks during mice Ppp1r1b-ChR2 and ChR2 ( behaviors. specific 5 Figure t r a  significant. not N.S., b j g n g c b a -score -score

= 0.9684, 0.9684, = ) Left Left ) i h ) Left Left ) , = 8) froze less than eYFP controls ( controls eYFP than less froze 8) =

z Freezing (%) Freezing (%) c 10 -score of poking in Rspo2-ChR2 ( Rspo2-ChR2 in poking of -score 10 , + + 20 40 60 80 20 40 60 80 e

Fos /Ppp1r1b (%) 0 0 0 0 , 10 f 20 40 60 80 473 nm f , P P ) Rspo2-ChR2 mice ( mice Rspo2-ChR2 ) 0 0 Shoc g C I P N.S.

+ 1 day , Rspo2-ChR = 0.0013, 0.0013, = t , = 0.9986, 0.9986, = = 0.0002, 0.0002, = Rspo2 neurons in Rspo2-ChR2 and Rspo2-eYFP mice that received received that mice Rspo2-eYFP and Rspo2-ChR2 in neurons (9) = 6.209; 6.209; = (9) j d ). * ). Day Fos k ) Scheme of activation of BLA neurons in Rspo2-ChR2 Rspo2-ChR2 in neurons BLA of activation of Scheme ) e l *** Day t 1 P (10) = 0.04056, day 2 2 day 0.04056, = (10) n Shoc ** in in Rspo2 < 0.05, ** 0.05, < = 6) and Rspo2-eYFP ( Rspo2-eYFP and 6) = + n t 1 P µ and and -test between experimental groups and corresponding corresponding and groups experimental between -test 2 s = 8) mice. ( mice. 8) = = 0.0055, 0.0055, = k m ( m Ppp1r1b 10 j 20 40 60 80 – 0 0 a l t t Rspo2-eYFP t ) Quantification of smFISH of of smFISH of Quantification ) h ) Scheme of activation of BLA neurons in Rspo2- in neurons BLA of activation of Scheme ) : pokes pokes : (12) = 4.180; right right 4.180; = (12)

(14) = 0.001757. ( 0.001757. = (14) + + Ppp1r1b (12) = 5.377; right right 5.377; = (12)

, Fos /Rspo2 (%) b N.S. Ppp1r1b i 10 20 30 Ppp1r1b-eYFP Ppp1r1b-ChR , , D 0 k c ay , , Day P l e + ). Colors: Rspo2-Cre mice (green) and and (green) mice Rspo2-Cre Colors: ). 2 h – and and < 0.01, *** 0.01, < g n , 2 P t * i ** c , = 6) displayed lower total nose pokes pokes nose total lower displayed 6) = (14) = 3.275, day 2 2 day 3.275, = (14) , + = 0.0003, 0.0003, = j ) On day 1 and day 2, Ppp1r1b-ChR2 Ppp1r1b-ChR2 2, day and 1 day On ) k ) or valence of stimuli negative (green) (green) negative stimuli of valence or ) BLA neurons antagonize valence- antagonize neurons BLA : pokes pokes : Rspo2 , l P 2 ). Results show mean mean show Results ). pp1r1b-ChR f 2 n l k d e j

n Nose pokes (×10 ) = 9) and Ppp1r1b-eYFP ( Ppp1r1b-eYFP and 9) = P 10 n

= 6) and Rspo2-eYFP ( Rspo2-eYFP and 6) = z-score + + + n 2 4 6 8 0 = 0.5777, 0.5777, = = 8), no difference between between difference no 8), = neurons in Ppp1r1b-ChR2 Ppp1r1b-ChR2 in neurons –5 15 10

P Fos /Rspo2 (%) P = 6) mice. mice. 6) = 0 5 100 = 0.5990, 0.5990, = g < 0 0.001, **** 0.001, 0 < P 20 40 60 80 P 0 t – *** 0 2 < 0.0001, 0.0001, < 473 nm (9) = 5.604; 5.604; = (9) = 0.0035, 0.0035, = i Water ) Quantification of of Quantification ) Cu Ppp1r1b-eYF Cu e ** N.S. 5 e t P Fos (10) = 0.5755; 0.5755; = (10) Rspo2 = 0.0171, 0.0171, = Time (s t (14) = 0.5381; 0.5381; = (14) n

z-score 10 e t in in t = 8) and and 8) = 10 15 (12) = 6.012. 6.012. = (12) b ± ) Time course course Time ) P (12) = 3.616. 3.616. = (12) + + 0 5 n z ) Time course course Time ) s.e.m. s.e.m. Fos /Ppp1r1b (%) -score -score = 5), no no 5), = ) Rspo2 , day 1 day , 10 20 30 40 P 0 t < 0.0001; 0.0001; < -test ( -test 15 ** Rspo2-eYFP Rspo2-ChR n = 5) 5) = n **** t P + = 7) 7) = (14) (14) and and = N.S. g 20 , j ). ). 2

inhibitory and excitatory ( excitatory and inhibitory of connections of 17% and neurons ( Ppp1r1b potentials ( Rspo2-ChR2 of stimulation ChR2 of ( properties physiological electro and soma size, intrinsic of position, combination anatomical Therefore, the postsynaptic cell target was recognized on the basis of a ( neurons properties intrinsic physiological distinct revealed ( axons cell-type-specific of stimulation optogenetic with recordings clamp Rspo2 tion neurons. of The relationship valence-specific functional between stimula optogenetic with recording clamp patch by combining level ioral and cial for negative behaviors and associations, while while associations, and behaviors negative for cial behaviors. positive elicit that stimuli while behaviors, negative function. neuronal predicted and neurons BLA of populations distinct for ers mark transcriptionally genetic revealed approach This in BLA. neurons to active profile strategy genetic forward a employed we Here DISCUSSION both Ppp1r1b that suggest fibers tion projec of characterization anterograde and tracing retrograde CTB ( PL or CeC the NAc in not but IL and CeM, CeL, or IL ( NAc, in CeC, were found the and CeM, PL, but not fibers in CeL, the in Rspo2-ChR2 and Ppp1r1b-ChR2 mice. In Rspo2-ChR2 mice, dense Ppp1r1b CTB of distributions segregated neurons spatially labeled in the BLA—PL- labeled CTB targeted to the prelimbic (PL) and infralimbic (IL) cortex posterior the end of spanning the aBLA to end the of posterior the pBLA ( BLA, the of side medial the along tributed dis neurons labeled (NAc) accumbens nucleus the to targeted CTB ( pBLA the of side lateral the along distributed neurons lateral and medial nucleus of the central amygdala( labeled (CeL/CeM) aBLA the in primarily neurons labeled (CeC) amygdala central the of nucleus capsular the to geted tar CTB b (CTB). subunit toxin cholera using examined was targets tive behaviors. Therefore, retrograde tracing from putative projection may divergent reveal that brain structures mediate negative and posi The distinct projection targets of the the of targets projection distinct The neurons BLA positive and negative from Projections inhibition. mutual through predominantly interact populations two and and associations. and behaviors positive for crucial are neurons 94% and Rspo2 CeC-CTB that revealed mice CTB-injected ( pBLA the in Fig. 6i Fig. Antagonism of valence-specific BLA neurons observed at the behav For anterograde characterization, we examined ChR2-eYFP Rspo2 Ppp1r1b + Rspo2 neurons primarily in the aBLA, IL-CTB aBLA, the in primarily neurons + + + Fig. Fig. 7 fibers and recordings of of recordings and fibers and 4% 4% and and and Fig. 6a Fig. , j + + + + ), of which 25% of connections of of connections of 25% which of ), Fos neurons in response to optogenetic stimulation of Ppp1r1b- and vice versa were 100% and 100% inhibitory, respectively and were versa and vice 100% inhibitory, 100% respectively ( Ppp1r1b neurons distinctly project to the CeL, CeM, and IL, and and IL, and CeM, CeL, the to project distinctly neurons advance online publication online advance Rspo2 Fig. 7k Fig. Fig. Fig. 6e + o and and Ppp1r1b activation level was further examined at the microcircuit + ). ). In Ppp1r1b-ChR2 mice, dense fibers were found in the neurons are antagonistic at the behavioral, neuronal neuronal behavioral, the at antagonistic are neurons Fig. 7b Fig. – Ppp1r1b d + Ppp1r1b ). Patch clamp recordings of of recordings clamp Patch ). BLA neurons are activated by stimuli that elicit elicit that stimuli by activated are neurons BLA – + – ; NAc CTB NAc ; h n , , , + k , neurons were examined by combining patch patch combining by examined were neurons Table i , , l j Rspo2 ). ). The probability of connections of + ). smFISH with with smFISH ). ; CeL/CeM-CTB ; Fig. Fig. 6m + BLA neurons both project to the NAc. the to project both neurons BLA Ppp1r1b Fig. 6i Fig. + 1 fibers showed inhibitory postsynaptic postsynaptic inhibitory showed fibers and and + + distinctly project to the CeC and PL, PL, and CeC the to project distinctly neurons were 30% 30% were neurons , n Ppp1r1b Fig. 7a Fig. Supplementary Fig. 8 Fig. Supplementary , ). Electrophysiological recordings ). Electrophysiological j + ). These data suggest that these these that suggest data These ). BLA neurons are activated by by activated are neurons BLA Rspo2 Rspo2 Rspo2 Rspo2 ,

+ c + neurons were 6% 6% were neurons , + neurons in response to to response in neurons d Ppp1r1b + nature nature + + BLA neurons were 96% 96% were neurons BLA and and ). CTB targeted to the the to targeted CTB ). BLA neurons are cru are neurons BLA to to Rspo2 or or + Ppp1r1b neurons primarily primarily neurons Fig. 7 Fig. Fig. Fig. 7a Ppp1r1b Ppp1r1b Rspo2 + NEUR + Ppp1r1b to to and and p Fig. 7a Fig. Rspo2 , ). + ). Together,). g + were both both were OSCI + , and 70% 70% and Ppp1r1b Rspo2 probe in in probe h neurons neurons Table ). ). Dual- + Rspo2 Rspo2 + fibers + BLA BLA EN , BLA BLA e + , 1 to C f ). ). ). ). + + + E ------

© 2016 Nature America, Inc. All rights reserved. examination of of examination within within neurons or neurons of subsets distinct functionally may reveal studies and these neurons, BLA distinct genetically other of role the examine to performed be could studies study, further but this in pursued not were screen our on candidates as found markers genetic Other types. two cell of each these within diversity structural or and genetic, functional, further be may there neurons, pyramidal Although of respectively. behaviors, positive participation and negative in pBLA and aBLA the the manipulations, cell-type-specific for markers conditioning reward conditioning fear contextual in aBLA the of tion respectively. behaviors, positive and negative Ppp1r1b inhibitory mutually which in model these a support Collectively, results inhibition. feedforward reciprocal through act inter and neurons opposing of the activation overall the antagonize drive only behaviors, not valence-specific antagonize They also but behaviors, opposing levels. electrophysiological and population, and (green) Rspo2-Cre or R t in characterized values ( resistance ( Ppp1r1b *** test, exact Fisher excitation; than rather inhibition mutual by predominately interact groups * test, signed-rank ( magnocellular both (for application GBZ (GBZ+) after and (GBZ−) before amplitude IPSC ( Ppp1r1b-ChR2 of train) (10-Hz stimulation in recorded (IPSCs) currents ( ChR2 in recorded potentials postsynaptic ( mice Rspo2-ChR2 ( respectively. neurons, mice) ( magnocellular 6 Figure nature nature ( assays (22) = 2.472; ( = 2.472; (22) m m Previous inactivation studies have implicated a greater contribu greater a implicated have studies inactivation Previous , e c a

n P ( Magnocellular Rspo2 473 nm ) Recorded magnocellular (green) and parvocellular (red) neurons were confirmed using soma diameter and anatomical position ( position anatomical and diameter soma using confirmed were neurons (red) parvocellular and (green) magnocellular ) Recorded = 0.0003, = 0.0003, f ) fibers. Green ( Green ) fibers.

Fig. Fig. + ) + NEUR Rspo2 (red) neurons were similar. ( similar. were neurons (red) neurons are the principal neurons that represent and elicit and elicit represent that neurons principal are the neurons Rspo2 R m 4 ) and membrane capacitance ( capacitance membrane ) and ), we found no evidence suggesting that that suggesting evidence no found we ), OSCI Rspo2 + m t and and Rspo2 (22) = 4.341. Results show mean mean show Results = 4.341. (22) + ) AP distance from bregma bregma from distance ) AP P and and d = 0.0313. ( = 0.0313. ). Main scale bar, 200 scale Main ). EN Ppp1r1b + Ppp1r1b ) ) ( 2 e Figure Figure + 7 C Ppp1r1b ) and red ( red ) and . Here we dissociated, using specific genetic genetic specific using dissociated, we Here . Ppp1r1b-ChR a and and E Ppp1r1b ) and parvocellular ( parvocellular ) and advance online publication online advance Rspo2 c , 2 Ppp1r1b 0.5 + + 473 nm d BLA neurons establish reciprocal inhibitory connections. ( connections. inhibitory reciprocal establish neurons BLA ; unpaired ; unpaired positive neurons. However, from the the from However, neurons. positive i ) Sagittal view of biocytin-filled biocytin-filled of view ) Sagittal , + s j ) Probability of connection, connection, of ) Probability + neurons constitute virtually all BLA BLA all virtually constitute neurons f + 2 neurons or Cartpt-Cre (red) ( (red) Cartpt-Cre or neurons Rspo2 ) traces represent average trace of 20 sweeps recorded during periods without spikes. ( spikes. without periods during recorded sweeps 20 of trace average represent ) traces (magnocellular; (magnocellular; 20 mV l b f ) IPSC amplitude was greater in in greater was amplitude ) IPSC d + 473 nm neurons in a set of behavioral behavioral of set a in neurons + 473 nm t ( -test * -test µ Rspo2-ChR e g C m; inset, 50 50 inset, m; P ) and ) and ) and Rspo2-ChR2 ( Rspo2-ChR2 ) and Ppp1r1b m < 0.0001, < 0.0001, ) ) ( P n ). ). < 0.05, ** < 0.05, Ppp1r1b ± 2 g s.e.m. ( s.e.m. Rspo2 1 ) and ) and 2 + and the pBLA in in pBLA the and ) ) ( Rspo2 µ b t (22) = 6.681; soma soma = 6.681; (22) m. Asterisks in in Asterisks m. ) neurons, while stimulating stimulating while ) neurons, + Ppp1r1b + and and ( g Rspo2 Rspo2 ( Parvocellula Ppp1r1b P Ppp1r1 f , + h ) BLA neurons by 10-Hz optogenetic stimulation of Ppp1r1b-ChR2 ( Ppp1r1b-ChR2 of stimulation optogenetic 10-Hz by neurons ) BLA

< 0.01, **** < 0.01, negative negative Rspo2 a , k – Ppp1r1b h , 0.5 n ) fibers. Currents are blocked by bath application of gabazine (GBZ, 10 10 (GBZ, gabazine of application bath by blocked are Currents ) fibers. l ). Colors: neurons recorded or virus-infected transgenic mouse, mouse, transgenic virus-infected or recorded neurons Colors: ). ). b + + + ) s and and and and (parvocellular; (parvocellular; + r + Ppp1r1b BLA neurons in Ppp1r1b-ChR2 mice ( mice Ppp1r1b-ChR2 in neurons BLA to to 20 mV - -

+ c Rspo2 cells were statistically distinct in all four parameters and consistent with with consistent and parameters four all in distinct statistically were cells and and l k i g P the antagonistic control of affective emotional states and emotional emotional and states emotional affective of control antagonistic the for site key a is BLA the that suggests neurons positive and negative respectively neurons, parvocellular and to magnocellular correspond and BLA of the subfields posterior and anterior the as defined been previously has what define and BLA the entire BLA showed that of types two neurons can these be considered intermingled; however,pBLA, examination ofand the aBLA the between transition the At respectively. pBLA, and aBLA the into segregated spatially are rons gled intermin be may neurons BLA positive and negative that suggested species. vertebrate and invertebrate across and CNS the throughout be motif may common a information positive and negative of representation gated in neurons body mushroom amygdala amygdala medial in the as demonstrated brain, in the tion informa and of positive negative representation segregated spatially the of evidence growing the to add findings Together, these valence. Ppp1r1b Excitatory Inhibitory < 0.0001 ( < 0.0001 473 nm Onset (ms) GBZ+ GBZ– 10 15 P + The identification of a mutually inhibitory microcircuit between between microcircuit inhibitory mutually a of identification The Previous 0 5 d < 0.0001, < 0.0001, (red) than in in than (red) + denote the cells recorded in in recorded cells the denote 15 connection ( connection , 1 h 6 0 . . However, our results suggest that negative and neu positive ) BLA neurons (clamped at 0 mV) in response to optogenetic optogenetic to response in 0 mV) at (clamped neurons ) BLA + Probabillity ofconnection(% 3/12 Ppp1r1b neurons participate in behaviors or associations across across associations or behaviors in participate neurons 2 9 n a in vivo and gustatory cortex gustatory and m 25 , = 6) ( = 6) b , t n ) Scheme for the experimental setup for recording in recording for setup experimental the for ) Scheme (22) = 5.857; ( = 5.857; (22) ). ( ). Rspo2 P i IPSP (mV) 12/1 ) and ) and 0 2 4 6 8 < 0.001. ( < 0.001. + 50 k electrophysiology and studies stimulus-dependent electrophysiology g (Ppp1r1b-ChR2 mice) and and mice) (Ppp1r1b-ChR2 GBZ GBZ– ) ) ) and parvocellular ( parvocellular ) and 2 P = 0.7441, = 0.7441, + + Rspo2 (green) neurons; unpaired unpaired neurons; (green) 75

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300 100 pA * s  - - - - © 2016 Nature America, Inc. All rights reserved. the identification of genetic markers for distinct populations of BLA of BLA populations for distinct markers of genetic identification the neurons effector CeM putative the and neurons BLA tive Rspo2 to similarly behaviors negative supports which CeL, and CeC the in of population a identified study recent A CeC. the that may this route suggest be an through our indirect findings CeM, from BLA negative neurons neurons to to in connections the effector role of the amygdala central in appetitive behaviors the for support further provide and CeM and CeL the to projections are (which neurons BLA parvocellular that demonstrating studies anatomical with consistent are findings our projections, CeL and CeM to regards In findings. our by supported to directly the drive CeM negative projecting behavior neurons the that neurons—namely, BLA of definition jection-based not BLA neurons to project Here, positive the CeC. prothe previous negative BLA neurons project to the CeM and CeL, while not negative but but positive that suggest data our hypotheses, previous these to CeM the in neurons effector and/or neurons CeL neurons to from BLA principal of information negative transmission are that of a BLA neurons NAc-projecting proportion larger observation to-NAc projections mediate positive behaviors negative and positive BLA neurons distinguishing for insufficient is definition projection-based a such ( behaviors of tion NAcwere to projected showed grade projection experiments that ~30% of BLA neurons that defining a be may neurons BLA positive of feature projections NAc that suggested studies These criteria target-based projection using behaviors positive and tive memories. and behavior emotional underlying mechanisms addition to genetic models for elucidation of further and the circuitry cellular and targeting for pharmacological the treatment of in disease, ticipate in an antagonistic circuit provides an avenue for more precise markers for neurons that molecular of par distinct the identification Therefore, disorders. in emotional may mechanism be an underlying neurons BLA positive and negative between excitation of imbalance BLA the in excitability elevated with these two populations. Studies have correlated negative affective states information positive on the basis of the of balance excitation between and negative of range continuous a associating and representing for memories. The antagonistic BLA circuit provides a circuit mechanism mean ( (red) mice Cartpt-Cre and (green) mice Rspo2-Cre Colors: IL. and NAc, CeM, ( PL. and NAc, CeC, the in in micrographs ( CeL/CeM the into injected CTB ( CeC ( ( CTB of site Injections group). mice, individual represent traces mean, represent ( ( areas amygdala supplemental and amygdala the to targeted CTB from BLA the of mm) −2.8 to mm −0.8 bregma from distance (coronal axis AP the across neurons ( areas. prefrontal and nuclei amygdaloid 7 Figure t r a 1 dissociation anatomical and functional the permitted has neurons k g 0 , , It is widely thought that the amygdala fear circuit involves direct direct involves circuit fear amygdala the that thought widely is It of nega study for the neurons BLA have targeted studies Previous m h

)—or dual CTB targeted to prefrontal cortex ( cortex prefrontal to targeted CTB dual )—or Ppp1r1b ) Colabeling of of ) Colabeling k ± o + ) and NAc ( NAc ) and , s.e.m. BLA neurons and thus may be nega an between intermediate p Rspo2

C I ). Scale bars: 250 250 bars: Scale ). Rspo2 Supplementary Fig. 9 Fig. Supplementary + e l ( + Supplementary Figure 7 Figure Supplementary somata or their NAc projections resulted in negative negative in resulted NAcprojections their or somata + Table and and m s Rspo2 ). ( ). Ppp1r1b 1 l , ). n ) Co-labeling of of ) Co-labeling Rspo2 mRNA in the BLA with CTB targeted to the the to targeted CTB with BLA the in mRNA p ) ) Ppp1r1b-ChR2 µ m m ( + c a BLA neurons project to distinct distinct to project neurons BLA + , )—CeC ( )—CeC l e c ) and NAc ( NAc ) and BLA neurons. Furthermore, stimula Furthermore, neurons. BLA , – g j 1 , , 8 i o ). These findings demonstrate that that demonstrate findings These ). 3 ) and CTB ) and . However, retrograde and antero and However,retrograde . , 4 p . Previous observations that BLA- ), 25 25 ), . . ( c a Ppp1r1b o , – 33 d ) ) Rspo2-ChR2 j ), CeL/CeM ( CeL/CeM ), ) Quantification of CTB of ) Quantification , n + 3 µ fibers are found in the CeL, CeL, the in found are fibers ); quantification in in quantification ); 4 m m ( , which suggest that the the that suggest which , + BLA neurons ( neurons BLA b Ppp1r1b k 8 )—PL and IL ( IL and )—PL mRNA in the BLA with with BLA the in mRNA n , – 1 = 3 mice per per = 3 mice n 6 are likely due to the ). Results show show Results ). 26 18 e , Calcrl + , , 39– 35– f fibers are found found are fibers + 18 ), NAc NAc ), ) send strong strong send ) 4 , 3 35– 3 8 4 . . In regards d . Contrary Contrary . 4 + 3 , i . Overall, Overall, . neurons neurons , f Table 8 j ,

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j i –2.8 –2.8 online version of version online NEUR NAc CTB CeL/CeM CTB CeC CTB OSCI IL CTB PL CTB EN C E © 2016 Nature America, Inc. All rights reserved. 12. 11. 10. 9. 8. 7. 6. 5. 4. 3. 2. 1. reprints/index.htm at online available is information permissions and Reprints The authors declare no competing interests.financial and S.T. and discussed commented on the manuscript. electrophysiological data. J.K. and S.T. wrote the manuscript. J.K., M.P., S.X., S.I. J.K. and S.X., and collected analyzed behavioral data. M.P. and collected analyzed the transgenic Rspo2-Cre mouse. J.K. and collected analyzed histological data. J.K. and S.T. conceived the study. J.K. geneidentified markers. S.I. generated Hughes Medical Institute and the JPB Foundation (to S.T.). (to T32GM007287 J.K.) and by the RIKEN Brain Institute,Science the Howard support. This work is supported in part by NIH Pre-Doctoral Training Grant on the manuscript, and all the members of the Tonegawa laboratory for their X. Liu for cloning the apparatuses, the MIT BioMicroCenter for support the collecting RNA array data, We acknowledge A. Wagatsuma and Redondo R.L. for help designing behavioral on Note: Any Supplementary Information and Source Data files are available in the codes. Accession nature nature 15. 14. 13. COM AUTH Acknowledgmen

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Goosens, K.A. & Maren, S. Contextual and auditory fear conditioning are mediated are conditioning fear auditory and Contextual S. Maren, & K.A. Goosens, Ambroggi, F., Ishikawa, A., Fields, H.L. & Nicola, S.M. Basolateral amygdala neurons Stuber, G.D. R.L. Redondo, alpha of innervation GABAergic F. Mascagni, & J.F. Muller, A.J., McDonald, receptor 3A type 5-HT of subpopulation novel A A.J. McDonald, & F. Mascagni, cortical-like a as complex amygdaloid basolateral The L. Heimer, & J. Carlsen, Golgi a nuclei: amygdaloid basolateral and lateral the of Neurons A.J. McDonald, study.Golgi a cat: the of amygdala The E. Hall, amygdala? the is What G.D. Petrovich, & L.W. Swanson, amygdaloid basolateral and lateral the of organization Neuronal A.J. McDonald, Pitkänen, A., Savander, V. & LeDoux, J.E. Organization of intra-amygdaloid circuitries oe F. Gore, fear-conditioned of types Different B.J. Everitt, & T.W. Robbins, S., Killcross, stimulus- in amygdala the of Involvement B.J. T.W.Everitt, Robbins, & M., Cador, 323–331 (1998). 323–331 amygdala mediate innate and learned responses. learned and innate mediate amygdala (1997). amygdala. within nuclei separate by mediated behaviour (1989). striatum. ventral the with interaction associations: reward rats. in nuclei amygdaloid central (2001). and 148–155 basal, lateral, the by 59 neurons. accumbens nucleus exciting by behavior reward-seeking facilitate seeking. reward facilitates engram. memory contextual hippocampal amygdala. basolateral rat (2002). the in pyramidal immunoreactive neurons kinase protein calcium/calmodulin-dependent II type 144 amygdala. basolateral rat the in interneurons immunoreactive subunit structure. rat. the in study (1972). 439–458 rat. the in nuclei amygdala. the of functions TrendsNeurosci. understanding for framework emerging an rat: the in P , 648–661 (2008). 648–661 , O E , 1015–1024 (2007). 1015–1024 , TI R R NEUR CON NG t al. et Brain Res. Brain FI et al. TRIBUTI OSCI N l . erl ersnain o ucniind tml i basolateral in stimuli unconditioned of representations Neural

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© 2016 Nature America, Inc. All rights reserved. fear conditioning protocol (three shocks, 0.75 mA, 2 s duration) (shock week aftergroup) operation, mice were ortaken off the Dox diet for 2 d and underwent a bilaterally injected with AAV9-Fos-tTA and AAV9-TRE-Pabpc1-Flag virus. OneR before behavioral experiments. weekpost-operation for recovery. Mice were handled by the investigator 2–3 d Eppendorf tube, was fixed ontoblack themL implant1.5 a usingusing dentalmade implant,cement. the surroundingMice cap spentprotective a 3–4 cured, tained two screws that lay medially to the implant site. Once the dental cement con which skull, (Teetsthe cementA-MCure;Systems)dentalto using Cold cemented was cannula optic fiber mono the BLA, the above positioned Once and above the NAc of Rspo2-Cre (AP +1.3 mm, ML +0.75 mm, DV −4.0 mm). BLA of Rspo2-Cre and Cartpt-Cre (AP −2.0 mm, ML +3.3 mm, DV −4.3 mm), implanted(unilaterallybilaterally,or experiment)abovethethe ondepending implantation. Fiber incubated 4 d before electrophysiological experiments. was injected into the BLA of Rspo2-Cre and Cartpt-Cre mice 4–5 weeks old and slice electrophysiological experiments, 200 nL of AAV9-Ef1a-DIO-ChR2-eYFP brainFor sacrifice. before d 7 forincubated and mm) −2.6 DV mm, +0.3 ML intothePL (AP−1.75 mm,ML+0.3 mm,DV −2.3 mm)and IL(AP−1.6 mm, injected was 647) CTB-Alexaand 555 (CTB-Alexa CTB Dual beforesacrifice. or CeC (100 nL, AP −1.0 mm, ML +2.9 mm, DV 4.3 mm) and incubated for 7 d DV −4.8 mm), (100 CeM/CeL nL, AP −1.35 mm, ML +2.9 mm, DV −4.7 mm) mm, +0.75 ML mm, +1.0 AP nL, NAc(300 the into injected unilaterally was Fluor555–conjugatedAlexa tracing, (1cholera toxin(CTB)subunit B retrograde For experiments. behavioral before weeks 3–4 for incubated and DV −4.85 mm) and Cartpt-Cre mice (AP −2.0 mm, ML ent viruses was injected into the BLA of Rspo2-Cre (AP −1.6 mm, ML ments. For behavioral experiments, 200 of doxycycline (Dox)-fed mice and incubated for 7 d before downstream experi into the BLA (distance from bregma, AP −1.4 mm, ML AAV9-cfos-tTA and AAV9-TRE-Pabpc1-Flagringe. For(1:1 activity-dependent mixture) transcriptional were profiling,bilaterally 200 nL injectedofinjected ~2.0using a × mineral10 oil–filled glass micropipette attached to a 1- tized under isoflurane. Standard stereotactic procedures were used. Viruses were Stereotactic injections. Medicine Vector Core. of School PennsylvaniaUniversityof from obtained was (CS0633-3CS) eYFP from University of North Carolina at Chapel Hill VectorChR2-eYFP (AV5226B), Core. and AAV9-Ef1a-ChR2-AAV5-Ef1a-DIO-eYFP (AV4310D) were obtained Medical School. AAV5-Ef1a-DIO-eArch3.0-eYFP (AV5257), AAV5-Ef1a-DIO- UniversityMassachusettsVectorofthe andat TherapyCenterCore Gene the TRE-PABP-Flagplasmid.AAV wereplasmidsintopackagedAAV9 vectorsby into the tetracycline responsive element ( subcloned was #MR209653), Cat. (Origene, Myc-DDKtag C-terminal a with Fos-tTA vector. A cDNA clone of mouse polyadenylate-bindingclonedinto anadeno-associated (AAV)virus backbone togenerate pAAV-the protein 1, site)tionalstartfollowed advancedbythetetracycline transactivator, tTA, was Viruses. National Institutes of Health (NIH). of Technology (MIT) Committee on Animal Care (CAC) and guidelines by the maintained in accordance with protocols approved by the Massachusettsof 3–5 or,Institute for postoperative animals, individually. All subjects were cared for and of age. All subjects were male mice housed in a 12:12 h regulatory lightelements of cycle either in cohorts the by driven construct Cre a with (RP32-39M21) clone(BAC) chromosome fortwo generations. Rspo2-Cre mice were generated using artificial bacterial a C57BL/6J to backcrossed were mice Cartpt-Cre (MMRRC). Center Research and Resource Mouse Mutant the from obtained were project, GENSAT the Jackson Laboratory. Cartpt-Cre mice (stock #036659-UCD), produced through Subjects. ONLINE METHODS nature nature exposure to a female mouse in the home cage for 2 h (female group). Immediately N A immunoprecipitation. The mouse minimal idtp C7L6 (tc #064, ie ee band from obtained were mice #000664), (stock C57BL/6J Wild-type NEUR OSCI Mono fiber optic cannulas (5.0 mm, Doric Lens) were Lens) Doric mm, (5.0 cannulas optic fiber Mono EN Rspo2 Subjects undergoing stereotactic injections were anesthe C E Twelve wild-type male mice kept on Dox diets were . Experiments were performed in mice 8–16 weeks Fos promoter (−623 to +1050 from the transcrip µ TRE L of viral stocks of AAV5 Cre-depend ) backbone to generate the pAAV- ± ± 3.3 mm, DV −4.85 mm) 3.4 mm, DV −4.9 mm) µ L microsy ± 3.3 mm, Pabpc1 µ 9 GC of g/ µ L) ------,

of a single brain. of the BLA, we allowed the loss of no more than two sections during sectioning for temperature 30room min at before dried storagewere at Brains −80 mm. °C. In −2.0 order AP to obtainand a mm homogenous −0.8 representation AP slide resulted in 12 coronal brain sections representing 0.1 mm intervals betweenin a staggered fashion (begun on the 6th, 11th, 16th, etc. slide). Therefore, each −2.0 mm were taken from each brain. SectionsAP to mm −0.8 AP fromslides—sectionsfrom 60 of each on section eachoneproduced subsequent brain started sectioned coronally at 20 sectioning, brains were equilibrated to −16 °C in a cryostat. Brains were serially and 60 Superfrost Plus slides (25 × 75 mm, Fisherbrand). Thirty minutes before stored at −80 °C. A single session of sectioning consisted of 12 wild-type brains quickly dissected and immediately flash frozen on aluminum foil on dry icedwere and anesthetized with isoflurane and were sacrificed by decapitation.Tissue. Brains were positive controls. Sst Ppp1r1b Gpr137 femalegroupthe enrichedin were selected: were thatcandidates gene 21 BLA, the in patternsexpression of basis the On (RLT lysis buffer from Qiagen RNeasy kits with 10 plexes were dissociated from magnetic beads by vortexing samples in lysis buffer MgCl mM 12 KCl,buffersaltM a (0.3washed inthree times NP-40, 1% TrismM 50 7.4, pH overnight at 4 °C. Magnetic beads were separated using a magnetic tube rack andMagnetic Beads were washed in HB, added to the homogenates, and incubated Flag (F7425, Sigma) was added and incubated for 6 h at 4 °C. 200 5 and tube microcentrifuge new a intoseparated Supernatantwas 10,000 at centrifuged were and tubes microcentrifuge 1.5-mL to transferred and,subsequently,A pestle usingHomogenizedenized B. pestle samples were Samplesadded. weretransferred Douncehomogenizer2-mL a to andhomog protease1%heparin, cycloheximide,inhibitorsmg/mL 1 wasSigma)) (P8340, MgCl mM 12 7.4, pH milliliterofhomogenization bufferNP-40,(HB,1% KCl, TrismM mM100 50 singlea 1.5-mL microcentrifuge tube. Thisyielded ~30 the BLAs were crudely dissected from two mouse brains and were collected into 300- cryostat; ratory (University of Washington) tation was performed in a similar fashion to that described by cage,the beforeMcKnight paraformaldehyde labo fixation (described below). RNA immunoprecipiabove), in addition to kainic acid–induced seizure (20 mg/kgunderwent kainic either acid)fear orconditioning home or reward conditioning off Dox (as described−80 °C until RNA immunoprecipitation. Mice undergoing histological analysis kept on a Dox diet. Brains were dissected,were flash frozen on ice, dry and stored mice at control Two decapitation. by sacrificed were and isoflurane with after,mice were returned to Doxa diet. Two days later, mice were anesthetized Gria4 enriched in the shock group were selected:were that candidates gene 16 BLA, the in patternsexpression of basis the On ( Atlas Expression Brain Mouse Allen the on icance, enriched in either the RMA or MAS5 normalized data set were screened obtained from the array, the top gene candidates, independent Screening ofand statisticalselecting B signif accession code (GEO), OmnibusExpression Gene NCBI the to deposited been haveanalysis Affymetrix through three samples from analyzed the female-exposed mice were grouped. The data from this were files Transcriptome Analysis Console 2.0; CHP three samples from the shocked mice and Subsequently, Console Software. Expression Affymetrix the through MAS5 or RMA by normalized BioMicroCenter.chipbyMIT 2.0 chipMousewere2.0from the 430 files CEL R Micro Kit. RNA samples were stored at −80 °C until further experiments. Magnetic beads were drawn off and RNA was isolated using the Qiagen RNeasy N , A analysis. A Vip , Htr2c , For screening candidate gene markers, wild-type mice 12–16 weeks old , Gpr165 , Pvalb Slc24a4 , Htr3a 2 µ —and pyramidal cell markers— , 100 , RNA samples were analyzed using the Affymetrix Mouse 430 Mouse Affymetrix the using analyzed were samples RNA G m sections across the BLA were collected. Using a razor blade, razorUsing a collected. were BLA the across sections m , Gria1 S , E Slc30a3 , 7 Nptx2 µ 8 g/mL cycloheximide, 0.5 mM DTT). Protein–RNA cycloheximide,DTT). g/mL mM 0.5 com 1 2 , , 200 U/mL Promega RNasin, 1 mM DTT,100 mM 1 RNasin,Promega U/mL 200 , 3 Grin1 7 µ , . , L Nrxn3 m and thaw-mounted onto slides. Each mouse brain Stx1a A gene marker candidates. , Oprl1 , , Pthlh 2 Synpo 0 . Brains were thawed for 30 min in a −16 °C , Neurl1a , Pcdh18 . Interneuronalmarkers— . Acvr1c Adrbk1 , Nos1 Camk2a http://m , Rspo2 , Cdh9 µ , Nos1ap , L/mL Aig1 , , , Sema5a ouse.brain-map.org On the basis of the data Thy1 Crhbp µ g ofg brain tissue. One , β doi: Esrra , -mercaptoethanol). Ntrk3 —were selected as , 10.1038/nn.4414 , Gabra1 Slc30a1 , µ Gipc1 , L Pierce A/G Ntng2 Calb1 µ L of anti- of L , , , Gabra2 Zfpm2 µ Gpr39 , , g/mL Penk Npy / ) 2 g 4 - - - - - , , , . , . .

© 2016 Nature America, Inc. All rights reserved. in 50%, 70%, 100%, and 100% ethanol for 5 min each. Slides were Brain sectionsdried were fixed in for4% paraformaldehyde 5 for min.15 min and then washed and 5 Atlas Expression Brain Mouse Allen on reported ers Lipid RNeasy (Qiagen Tissue brains Mini Kit). PCR primers were mouse the same as the forward and from reverse prim extracted RNA on (Qiagen) kit RT Omniscript the using transcription reverse via obtained was cDNA brain Mouse markers. gene candidate of cDNA cloning for was backbone the EcoRI, as used with cut vector, pCRII-TOPO (ThermoFisher). Vector TOPO pCRII- the from generated probes RNA using performed was (FISH) zation Fluorescence sections spaced 0.2 mm apart. on a vibratome and serially collected into four wells. Each well contained coronal12 h, before storage in PBS at 4 °C. Coronally cut brains were sectioned at 50 were dissected and stored in 4% paraformaldehyde at room temperature for 8 to perfused with phosphate-buffered saline (PBS) and 4% paraformaldehyde. Brains temperature for 60 min before storage at −80 °C. 0.16 mm apart spanning ML 3.8 mm to ML 2.8 mm. Slides were dried at room 20 at sectioned serially was slides brain cut Sagittally mm. −2.8 AP to mm −0.8 AP spanning apart mm 0.2 spaced sections, brain 12 or 11 containing slide each slides. Coronally cut brain slides were serially sectioned at 20 an individual brain was serially sectioned and thaw-mountedlected through ontothe flash freezing Superfrostmethod (as described above). UsingPlus a cryostat, doi: for 3) solution (pretreatment solution protease using digested were Proteins (NEB, HiScribe T7 High Yield RNA Synthesis Kit) with digoxigenin-labeled digoxigenin-labeled with Kit) Synthesis RNA Yield High T7 HiScribe (NEB, with HindIII and transcribing the antisense strand using a T7 RNA polymerase Cells (Clontech). RNA probes were generated by cutting pCRII-TOPO plasmids TOPO cloning (Clontech In-Fusion HD) and maintained in Stellar Competent candidate gene was isolated and cloned into the backbone pCRII-TOPO using of the and primer forward primer,reverse respectively. PCR for products each DNA oligonucleotide probes were designed for designed were probes oligonucleotideDNA C2 and C1 Custom Diagnostics). Cell (Advanced Kit Multiplex Fluorescent rescence fluorescence Single-molecule of BLA principal cells as shown from quantification of centage of the total large (<10 taining DAPI (Vector Laboratories). Slides were coverslipped and mounted using VectaShield mountingtheslides for 30 min and solutionthen washed away with of a series three TNT washes. con 594–conjugated tyramide signal amplification (TSA) solution was applied over Alexa washes. TNT three of series a byremoved was perature. Anti-dig-POD reagent tem room blocking at h 2 for with Roche) (anti-dig-POD, fragmentFAB anti-digoxigenin buffer TNT in blocked (PerkinElmer) (TNB) before were being incubated with 1:100 peroxidase-conjugated Slides buffer. TNT hydrogen peroxide in Tris-NaCl-Tween (TNT) buffer,in 0.1× SSPE at 60 °C. followedEndogenous peroxidase activity was removed with 0.3% by three washes in phosphate-EDTA buffer (SSPE), 50% formamide in 2× SPPE at 60 °C, and saline-sodium twice washes—2× stringency post-hybridization underwent slides 2, day On °C. 60 at incubationovernight forcoverslipped andtRNA and buffer hybridization of solution a in slides to applied were then ice, on cooled and werefor dried ath2 room temperature. RNA probes were denatured at 100 °C and washed with decreasing concentrations of ethanol (100%, 95%). The probes concentrationsing delipidated100%),ethanol95%, of (70%,chloroform, with increas in washed (SSC), solution citrate saline-sodium 2× in washed dride, water(DEPC)triethanolamineandbuffer, (TEA) pretreatedanhy acetic with at 4 °C, washed twice in phosphate buffer pH 7.4, rinsed in diethylpyrocarbonate mouse brain FISH protocols. On day 1, slides were fixed in 4% paraformaldehyde isolated were probes RNA Kit) and Mini stored RNeasy at °C. −80 (Qiagen, antisense Digoxigenin-labeled (Roche). UTP ′ -CAGTGTGCTGGAATT-3 Forimmunohistochemistry, weremice euthanized byAvertin overdose and For single-molecule fluorescence Percent labeling was calculated as the number of RNA-positive cells as a per Tissue preparation for single-label FISH was performed similarly to standard 10.1038/nn.4414 Gad1 µ m onto 8 slides, each slide containing at least 12 brain sections, spaced sections, brain 12 containingleast atslide each slides, onto8 m n situ in probes were purchased from the Advanced Diagnostics Cell catalog. in situ in yrdzto (mIH ws efre uig RNAscope a using performed was (smFISH) hybridization hybridization. µ m) DAPI ′ and 5 n situ in in situ ′ Single-label fluorescence fluorescence Single-label -GATATCTGCAGAATT-3 + hybridization. BLA cells, which is an indirect indicator hybridization, mouse brains were col Rspo2 Camk2a 2 and ige oeue fluo molecule Single 4 with the addition of addition the with Ppp1r1b µ m onto 10 slides, + in situin cells ( ′ to the 5 . hybridi Fig. 1 Camk2a ′ end µ l ). m ------

1 h, and then incubated overnight at 4 °C with chicken anti-GFP (Invitrogen, anti-GFP chicken with °C 4 at overnight incubated then and h, 1 for blocked min, 10 for times three PBST in washed were slices brain iments, DAPI (Vector Laboratories). were coverslipped and mounted using VectaShield mounting solution555 containing(Invitrogen, A21428, 1:1,000). After three more 10-min PBST washes, slides Alexa Fluor 488 (Invitrogen, A11039, 1:1,000) and goat anti-rabbit Alexa Fluor (Santa Cruz, sc-52, 1:2,000). Secondary antibodies used were goat anti-chicken anti-Fosrabbit and 1:1,000) A10262, (Invitrogen,anti-GFP chicken 1:1,000), peratureantibodieswereforPrimary h.used rabbit2 anti-Flag (F7425,Sigma, ofPBST and incubated antibody secondary in blockingin buffer at room tem overnight at 4 °C. On the next day, brains were washed with three 10-min5% normal goat and serum), incubatedwashes in primary antibody in blocking buffer (PBST,bufferblockingin TritonX)h 3% for1blocked min, for10 times three Immunohistochemistry. mounting medium with DAPI (ThermoFisher). buffer, slides were immediately coverslipped using ProLong Diamond Antifade green)C2,depending oncolorthe combination ofchoice. wash final After the red; (C1, AMPB or red) C2, green; (C1, AMP4A using protocol kit standard to accordingsteps reaction colorimetric the underwent then and buffer wash humidified°Cincubator 40 RNAscopea inSlides inweretwice forh. rinsed 3 Probesmin. 10were applied wereslides,whichthe to coverslipped for andplaced bath water °C 40 a in heated were probes C2 or and C1 parallel, In PBS. in twice washed were slides afterward, Immediately eYFP-expressingtissues. on s 5–10 and tissues, CTB-expressing on s 30 tissues, wild-type on s 60–90 pBLA was calculated by (number of aBLA)/(totalthenumber incells of in ZEN and were exported into image files for quantification in a blind fashion. Background signals levels of c-Fos and of water exposure, 15 min after satiety, which took <5 min after water exposure. above) (described afterminendof15stimulus the the exposure or, case the in stimulus exposure protocol but were sacrificed using the flash freezing method nohistochemical analysis of c-Fos. For smFISH of exposure, mice were sacrificed using Avertin overdose and perfused for immu bottle without a spout was placed into the home cage. Ninety minutes quinineafter initialhydrochloride dihydrate), sucrose water (5% sucrose), or an emptyand habituatedwater for 4 h before hydration. A bottle of water, quinine water (0.05% mice were transported in home cages to an experimental room, food removed, pipetted into the center of the home cage. Water-deprived (overnight) wild-type dH in TMT (10% TMT of milliliter One exposure. odorbefore h habituated4 for cages to an experimental room; water and food were removed, homeand in transportedwere micethe removed.Odor-exposed were food miceandwater were the fear conditioning chamber for 500 s then returned to the home cage, where to exposed were mice Context-exposedmouse. female wild-type a to exposed exposed mice were transported in home cages to an experimental room and thenreturned tohomethewere cage, where water and were food removed. Female- (Med Associates) for 500 s and received three footshocks (0.75 mA, 2 1 s h duration),of the dark cycle. Shocked mice were exposed to a fear conditioning chamber for 3 d before stimulus exposure. Stimulus exposure experimentsc-Fos experiment.occurred within fluorescent labeling. for green colored were representations figure Main software. edition) (black ZEN Zeiss using microscope confocal M2 AxioImager Zeiss or microscope fluorescent Zeiss a M containing DAPI (Vector Laboratories). solution mounting VectaShield using mounting and coverslipping before h 6 least attemperaturefor room at dried were slides washes, PBST more10-min anti-GFP (Invitrogen, A10262, 1:1,000) for 2 chicken h in at incubated room then temperature. and min, After10 for three times three PBST in washed were streptavidin CF555 and fibers day, next the slices On neurons. recorded visualize to 1:100) 29038, (Biotium, ChR2-eYFP visualize to 1:1,000) A10262, icroscopy and histological representation. For immunohistochemistry of brains subjected to electrophysiological exper The relativeThe 2 O), 1 mL of peanut oil, or 1 mL of benzaldehyde (0.25% in 70% ethanol) were Fos Rspo2 expressionbycalculated(number was of aBLA for the Wild-type mice were handled by investigator once each day + Free floating brain sections were washed in PBST (PBS, neurons and red for red and neurons Fos Fos + Fos + cells in the pBLA)/(total number of cells in the BLA); likewise, thatthelikewise, inBLA); the incells plus Micrographs were obtained using Rspo2 Ppp1r1b Fos nature nature , mice underwent the same or + Ppp1r1b irrespective of native of irrespective NEUR were adjusted OSCI EN Fos Fos C E + + - - -

© 2016 Nature America, Inc. All rights reserved. 180 s without light stimulation. Freezing behavior was scored manually usingmanually scored was behaviorstimulation.Freezing lightwithout s 180 for chamber conditioning fear the to returned and cords patch optic fiber to the optic cannula at the 180-s time point for 180 s. On berday for2, mice360 weres. 20-Hz,connected 473-nm light (10–15 mW) was unilaterallyO delivered through the 5 s baseline period before cue onset. µ ( tion were calculated in 100-ms time bins and quantified with a pokes was counted for the period. Percent in-port time during the cue presenta constructed from the first rewarded trial to 150 trials thereafter. Total number of andreward deliveries were andloggedanalyzed withMatlab. Dataarrays were pokes nose light, rewards.Timestamps watercue forelicit not did pokes nose s, 15 and 10 between distributed randomly interval, inter-trial following the 20-Hz pulse train of 15-ms pulses of 473-nm light (10–15 mW) was used.532-nm light (10–15 mW) wasDuring used; for optical activation experiments, a 10-s, of pulse constant10-sdelivery. rewarda inactivationexperiments, Foroptical ally delivered to the implanted optic cannula, was issued at the same time as the the port and the cue light was turned off. A TTL triggering a laser pulse, bilater time during the 5 s, a reward was immediately delivered through a water spoutreward contingentin on a nose poke, lasting 5 s. Upon a successful nose-poke any At the start of each trial, the onset of the cue light signaled tioningthe availabilitychamber (Islandof waterMotion) with one reward port equipped with Rewarda conditioning. cue light. three bins (1 min). binnedintotest,wasfreezing min 3 theinter-shock day2, On s). (80 intervals fear conditioning trial to the first shock (198 s), then subsequently between the trial. Freezing scored in bins ( the of end to beginning the from 2 day and trial the of end the to shock first in a blind fashion. Freezing was scored ( laser was delivered. Freezing behavior was scored manually using JWatchers1.0 cords and placed in the fear conditioning chamber for 180 s, where no shock or (10–15 mW) light was used. On day 2, mice were connected to optic fiber patch 473-nm of pulses 15-ms of train 20-Hz 10-s, a experiments,activation optical (10–15 mW) was delivered through the optical cannulas for duration of 20 s; for shocks at the 198-s, 278-s and 358-s time points, a constant pulse of 532-nm time points.light For optical inactivation 358-s experiments, and simultaneously278-s 198-s, with the the foot at footshocks received and s 500 cords for Lens) patch (Doric fiber optic to connected bilaterally while Associates) (Med ber Fear conditioning. quantified in a blind fashion. of sections Three fashion. adjusted in ZEN and were exported into image files for quantification in a blind of levels signals Background above). water exposure, mice were sacrificed using the flash freezing method (described satiety, after or, min ofwater30 <5 exposure, took case which the in contingent on drinking water. Thirty minutes after end of the stimulus exposure menter) received a 20-Hz train of 15-ms pulses at 473 nm bilaterally to the BLA butexperiments, in above manually wild-type in the (bydescribed experi the Rspo2-eYFP mice ( and Water-deprivedRspo2-ChR2 (overnight) BLA. the to bilaterally nm 473 simultaneously with footshocks received a 10-s, 20-Hz train of 15-ms pulses at ( mice mined by a mouse brain atlas and was accurate within unilateral BLAs on three 20- for unilateral BLAs on 50- aBLA are graphically represented ( parisonof thepBLAwere redundant. ofBecause this, only thestatistics for the differentconditions, significance values for comparison ofand aBLAthe com pBLA are mutually exclusive. Thus, when statistically comparing values between boundaries ( cells in the BLA). The aBLA and pBLA were determined using mouse brain atlas nature nature JWatchers1.0 in a blind fashion. z and = ( ptogenetic freeze test. For the the For x − σ Fig. 5g Fig. are the mean and s.d. of percent time spent in the rewardduringthepercent portof spentands.d. meanin aretime the µ NEUR )/ Fos σ Supplementary Fig. 4 ), where – experiments in in experiments i ) were exposed to three footshocks in a similar manner, but manner, similar a in footshocks three to exposed were ) OSCI On day 1, mice were placed in to a fear conditioning cham Fig. 5j EN x Water-restricted mice were placed in an operant condi is the average percent time spent in the reward port and On day 1, mice were placed in a fear conditioning cham C µ – E m sections. smFISH l Fos µ ) ) were exposed to water in a similar manner to that m sections per mouse. The AP position was deter Fig. 5 plus Figure Figure Fig. 3d ). The relative b ) was binned on day 1 from the start of the Rspo2 Fos 6 Fig. 4 – , Ppp1r1b-ChR2 and Ppp1r1b-eYFP Ppp1r1b-eYFP and Ppp1r1b-ChR2 , and f ). IHC c-Fos counting was performed or c Fos Ppp1r1b ) on day 1 from the onset of the Fos Fos plus counting was performed for expression of the aBLA and ± per mouse brain were brain mouse per Rspo2 0.2 mm. z or -score procedure Ppp1r1b were ------

manner in the left or right hemisphere. For all behavioral experiments, two- experiments, behavioral all For hemisphere. right or left the in manner unilateral implants, mice received implants randomly and in a counterbalanced control groups. Groups of mice did not undergo multiple behavioral assays. For possible,divided equally on the basis of age and parents into experimental and surgery and behavior in a pseudorandom fashion in that mice were, as much as mice and initiated within 2 h of the onset of dark cycle. Animals were selected for tion in the stimulated side) − (duration in the non-stimulated side). video tracking software (Noldus). The difference score was calculated by (dura take from both trials. The position of the mice was tracked using EthoVision XT stimulationofcounterbalanced.side wasthe average The difference scorewas preselected half of the boxrandomly for a 5into min. entry Each on mousecontingent mW) ran two (10–15 stimulationtrial light1 h 473-nm apart, in which continuous20-Hz, received mice box, the into entry uponImmediately cues. whereboxcm)ofcontainedendeachthe 31 × 25 × gularbox(70distinct wall O quantified by MED-PC (Med Associates) software on day 1 and day 2. no food pellet) for 15 min without light stimulation. Total number of pokes was fiber optic patch cords and returned to the reward conditioning chamber (with operantthe totalin spendaofh 1 chamber. dayOnweremice 2, connected to Mice port. nose the in break beam contingentcannulaa opticon the through 20-Hz,port. 473-nmlight (10–15mW, duration)s 5 unilaterallywas delivered nose port. The nose port contained a single food pellet to attract the mouseinto an operantto conditioningthe chamber (Med Associates) equipped with a single O repeated 20 times every 4 s or train of 15 light pulses at 10 Hz repeated 20 times maximum power was employed. Slices were stimulated by single 2-ms light(subtracted pulseoffline). Light power on the sample was 33 mW mm 25 of onset delay a with input TTL by driven Dynamics) Lumen (XLED1, obtained from Tocris. Pro (Wavemetrics). Software and code are available upon request. Gabazine was 2 kHz, digitized (20 kHz) and acquired using custom software running on Igor dual-channeltwo amplifiers (Multiclamp 700B, Molecular filteredDevices), at the access resistance was beyond 20 M or mV −50 above depolarized was potential membrane resting the whenever throughout the duration of the experiment and data acquisition was suspended 0.5% biocytin (pH 7.3, osmolarity 290 mOsm). Access resistance was monitored 2.8 NaCl, 5 TEA chloride, 4 Mg-ATP, 0.3 Na-GTP, 10 QX314, 0.1 spermine and cellularsolution cesium117mM): (inmethansulfonate, HEPES,EGTA,0.420 Recordings in voltage clamp mode were performed by using the following intra mOsm).osmolarity 2907.25, (pH phosphocreatine biocytin mM 0.5%10and ATP,GTP,mM mM 4 0.3 HEPES, mM 10 KCl, mM gluconate,10 potassium M 3–5 of ances Borosilicate glass pipettes were fabricated (P97, Sutter Instrument) with a resist manipulators(Luigs Neumann)& cameraandautomatic CCD (Orca a Hamamatsu). R2, four 0.8), (N.A. objective 40× immersion water a with Olympus) formed by using an infrared differential interference contrast microscope (BX51, 36 °C ACSF at a rate of 3 ml min transferred to a submerged experimental chamber and perfused with oxygenated cose, saturated with 95% O/5% CO (pH 7.3, osmolarity 300 mOsm). Slices were CaCl osmolarity 340 mOsm). The ACSF contained 124 mM NaCl, 3 mM KCl, 2 mM NaHPO contained 3 mM KCl, 0.5 mM CaCl ~23 °C in oxygenated artificial cerebrospinal fluid (ACSF). The cutting solution amygdala in oxygenated cutting solution at ~4 °C. Slices were then incubated at Leica), we prepared 300- tized by isoflurane and their brains were dissected. Using a vibratome (VT1000S, O sible during the experimentation. from analysis. Experimenters were blind during data analysis and whenever pos and control groups. Mice lacking expression or misplaced fibers were excluded unpairedtailed Student’s ptogenetic slice electrophysiology. ptogenetic place preference test. ptogeneticself-stimulation test. All behavioral experiments were performed by a set of mice in cohorts of 4–16 Optogenetic stimulation was achieved through a 460-nm LED light source light LED 460-nm a through achieved wasstimulation Optogenetic per were mode voltage-clamp or current-clamp in recordings Whole-cell 2 , 1.3 mM MgSO mM 1.3 , 4 , 10 mM Ω d and filled with the following intracellular solution: 110 mM 110followingsolution:intracellular the with filled and -glucose, 230 mM sucrose, saturated with 95% O/5% CO (pH 7.3, 4 , 25 mM NaHCO mM 25 , µ t m-thick parasagittal slices containing the basolateral -testswereexperimentalgroupsperformedbetween −1 . Mice were placed into the center of a rectan On day 1, food-deprivedday1,were Onmice placed 2 , 10 mM MgCl Male mice (mean-PND 45 d) were anesthe Ω . Recordings were amplified using up to 3 , 1.2 mM NaHPO mM 1.2 , 2 , 25 mM NaHCO doi: 4 10.1038/nn.4414 , 10 mM mM 10 , −2 , and only the 3 , 1.2 mM d -glu µ s ------

© 2016 Nature America, Inc. All rights reserved. strand synthesis, reverse transcriptase was inactivated by 5 min incubation on an Superscript III RT) and incubated on a 50 °C heat block for 50 samplemin. the toof (6 RT added was mix After the first 8 synthesis, strand first For ice. on chilled then and min, 5 for block heat CellsDirect cDNA Synthesis Kit (ThermoFisher). Samples were placed on 2 a 70 °C 10 with filled tube PCR 0.2-mL a to transferred quickly was pipette glass the suctioned, were contents cytoplasmic the Once pipette. the recordingof the applyingpressurebynegativeto collected was neuronrecorded cytoplasm the recordings, clamp patch the of end the at slices, brain reaction. chain polymerase quantitative Single-cell in 4% paraformaldehyde for morphological identification. are presented as mean test was employed to verify the significance of the connection probability. Data Wilcoxon signed-rank or rank-sum test wasunpaired with or employedpaired tested two-tailed a accordingly.and was Kolmogorov-Smirnovtest the data A the Fisher of exact distribution The (Microsoft). Excel or (Prism), cell or interneuron). expressioncharacterized atbyleastone responsive postsynaptic (principalcell ability of connection ( baseline and a parabolic fit of the rising phase of the EPSC. To computeto the thestarting probpoint of the response estimated through the intercept between the light pulse starts. EPSC onset was measured from the beginning of the light averagepulse maximum peak response by subtracting a baseline obtained 5 ms before were determined by averaging 20 trials. EPSC amplitude was measured from the step injection of 1 s duration. Synaptic connections, in voltage or current mode, estimated by single exponential fit of the recovery time from a −100 pA current threshold was tested with a current ramp injection. Membrane time constantrelationship (injectionwas of 10–12 current steps of 1-s duration). Action potential with the cell held at −70 mV. Input resistance was estimated by linear fit of the and action potentials were measured at resting potentials. ment and at 0 mV for IPSC measurements, whereas in 6every s. Incurrent voltage-clamp mode cells modewere held at EPSPs,−70 mV for EPSC measure IPSPs doi: µ As the recorded cells were filled with biocytin, slices were recovered and fixed GraphPad (Wavemetrics), Igor using performed was analysis Statistical mode current measured were properties electrophysiological intrinsic The L oligo(dT), 1 1 oligo(dT), L 10.1038/nn.4414 µ L dNTP, L 1 n ± successes/ s.e.m. µ L RNaseOUT provided by the SuperScript III III SuperScript the by provided RNaseOUT L n tests) we used only slices with reliable ChR2 µ L 5× RT 5× L Buffer, 1 In wild-type mouse mouse wild-type In µ µ L RNase-free water, RNase-free L L 0.1 M DTT, M 0.1 L 1 t -test or a or -test I µ µ - V L L - -

neurons, respectively. Therefore, were not statistical different from unconfirmed, magnocellular and parvocellular threshold cycles (CT) <50 cycle for most cells ( positiveterionamplification.of Therefore, The majority of cells did not result in amplification of either cein (FAM) channel with the standard qPCR reaction protocol for 60–80 cycles. formed in an Applied Biosystems 7500 Real-Time PCR System using the fluores7 Expression Assay for 25 of the ratio between Biosystems). The genetic identity of BLA neuron using qPCR was determined by chain reaction (qPCR). Samples heatblock. werequantitative °C storeduntil 85 °C at −20 polymerase and histology analyses basis of and were similar to those in previous studies the on examiningdeterminedwere tests statistical and samplesizes ourbut sample sizes, similar behaviors predetermine to used were methods statisticalNo 6.0. GraphPadPrismusing analysis. Statistical from bregma, resistance, and capacitance ( in the statistically compared ( and found on NCBI Gene Expression Omnibus (GEO), accession code D analyses were two-sided. A stimulation test. All data are represented as meanself- optogenetic the in Ppp1r1b-eYFP vs. Ppp1r1b-ChR2 or test freeze netic optoge the in Rspo2-eYFP vs. Rspo2-ChR2 of case the in as such dramatic, the exception from groups where the effects of experimental manipulations were groups that were compared and met the assumptions of the statistical tests with tested for all statistical tests basis of similar measurements from previous literature but this was not formally µ ata availability.ata qPCR was performed using the TaqMan Gene Expression Assays (AppliedTaqManAssaysExpression the Gene using performed was qPCR L of cDNA template, and 17.5 Ppp1r1b µ Figure L 2× TaqMan2.5 Mix,Expression2× Master Gene L Rspo2 + ) and unconfirmed (magnocellular and parvocellular) neurons were 6 experiment using a combination of soma diameter, AP distance + and Data available upon request. RNA microarray data may be be may data microarrayRNA request. upon available Data Rspo2 Statistical analysis and statistical graphics was generated was graphics statistical and analysis Statistical Rspo2 10 Table Ppp1r1b and , 2 9 (Mm00555790_m1) or . Data distribution was assumed to be normal on the 10 Supplementary , 1 2 Ppp1r1b 9 ). qPCR-confirmed . Variance was not significantly different between + neurons were identified on the basis of the cri µ Rspo2 Rspo2 L of RNase free water. qPCR reaction was per expression. The qPCR reaction consisted + + or and Fig. 6k Ppp1r1b M Ppp1r1b Fig. 1 ethods ± Rspo2 – Ppp1r1b s.e.m. All nature nature n f + ). qPCR-confirmed ( ) amplification appeared at + C neurons were identified + hecklist and µ L 20× TaqMan20× L Gene (Mm00454892_m1), t NEUR Rspo2 Ppp1r1b -test and ANOVA is available. or GSE7813 OSCI + Ppp1r1b neurons Rspo2 EN 7 C . E + - - - - .