LHb VTA. VTA-projecting RMTg-projecting overlay. Supplemental Figure 2. Retrograde labeling of LHb neurons. a. VTA-projecting LHb

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Harold Joseph
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SUPPLEMENTARY INFORMATION Supplementl Figure 1 doi:10.1038/nture09742 Lterl 1.0 mm from midline mpfc BNST mpfc BNST Lterl 2.1 mm from midline LHA LHA Lterl 2.

The niml ws trnsrdilly perfused with 4% PFA 8 dys lter nd the retrogrde leling y holer toxin ws exmined in 200 µm sgittl rin setions.

1 SUPPLEMENTARY INFORMATION Supplementl Figure 1 doi: /nture09742 Lterl 1.0 mm from midline mpfc BNST mpfc BNST Lterl 2.1 mm from midline LHA LHA Lterl 2.7 mm from midline SUPPLEMENTAL INFORMATION EP EP Supplementl Figure 1. Mjor rin res tht send projetions to the. Alex 488-ojugted Choler toxin ws injeted into the of mle, 55-dy-old WT rt (; red rrow). The niml ws trnsrdilly perfused with 4% PFA 8 dys lter nd the retrogrde leling y holer toxin ws exmined in 200 µm sgittl rin setions. Prominent leling (see rrowheds) ws found in the medil prefrontl ortex (mpfc; green oxes in ), ed nuleus of the stri terminlis (BNST; red oxes in ), lterl hypothlmus re (LHA; green oxes in ), nd the entopedunulr nuleus (EP; green oxes in ). Imges on the right side re higher mgnifition pitures of the oxed res on the left. Sle r: 1 mm. W W W. N A T U R E. C O M / N A T U R E 1

RESEARCH SUPPLEMENTARY INFORMATION VTA VTA D3V M & L ntomy ChTx nti-neun overly VTA-projeting RMTg-projeting

. VTA-projeting neurons leled y in vivo injetion of retrogrde trer in VTA.

fixed with 4% PFA. Right: higher mgnifition of the region to visulize the GFP-leled, VTA-projeting neurons.

2 RESEARCH SUPPLEMENTARY INFORMATION VTA VTA D3V M & L ntomy ChTx nti-neun overly VTA-projeting RMTg-projeting overly Supplementl Figure 2. Retrogrde leling of neurons.. VTA-projeting neurons leled y in vivo injetion of retrogrde trer in VTA. Left: prsgittl setion of rt rin tht ontins oth (red ox) nd VTA (rrow); middle: the sme rin setion viewed under fluoresene mode. A retrogrdely-trnsported HSV virus expressing GFP ws injeted into VTA (rrow) 2 dys efore the niml ws perfused nd fixed with 4% PFA. Right: higher mgnifition of the region to visulize the GFP-leled, VTA-projeting neurons. Sle r: 1 mm.. Left: digrm of henul in oronl setion (MH: medil henul; M: the medil setion of lterl henul; L: the lterl setion of lterl henul). Right pnels: holer toxin (ChTx; onjugted with Alex 488) ws injeted into VTA 2 to 3 dys efore the niml ws perfused nd oronl rin setions ontining henul were prepred. 2

3 SUPPLEMENTARY INFORMATION RESEARCH Setions were stined with n nti-neun ntiody to visulize ll the neurons. Typilly there were only smll frtion of ChTx positive, VTA-projeting neurons in. Sle r: 200 µm.. An imge of the with neurons leled y ChTx injeted into oth the VTA (Alex488 onjugted; green) nd RMTg (Alex594 onjugted; red). The green nd red ells, representing VTA-projeting nd RMTg-projeting neurons, respetively, re lrgely non-overlpping. The oordintes (mm) for the injetions re: VTA -5.3 (AP), 0.9 (ML), nd -8 to -8.2 (DV); RMTg -6.8 (AP), 0.4 (ML), nd -7.7 to -8 (DV). Injetions were lso mde in different lotions within RMTg (from 0 to 1 ML), whih resulted in similr non-overlpping leling of neurons (not shown). Sle r: 50 µm. 3

4 Supplementl Figure 3 RESEARCH SUPPLEMENTARY INFORMATION ChTx dorsl nti-eaac1 overly L Setions were stined with n nti-neun ntiody to visulize ll the neurons. Typilly there were only smll frtion of ChTx positive, VTA-projeting neurons in. Sle r: 200 µm.. An imge of the with neurons leled y ChTx injeted into oth the ventrl ntomy HSV-GFP overly VTA (Alex488 onjugted; green) nd RMTgnti-GABA (Alex594 onjugted; red). The green nd red ells, representing VTA-projeting nd RMTg-projeting neurons, respetively, re lrgely non-overlpping. The oordintes (mm) for the injetions re: VTA -5.3 (AP), 0.9 (ML), nd -8 to -8.2 (DV); RMTg -6.8 (AP), 0.4 (ML), nd -7.7 to -8 (DV). Injetions were lso mde in different lotions within RMTg (from 0 to 1 ML), whih resulted in similr non-overlpping leling of neurons (notdg shown). Sle r: 50 µm. nti-gad67 nti-gad67 nti-gad67 Supplementl Figure 3. Glutmtergi neurons re dominnt in the.. Injetion ws done s desried in supplementl figure 2. Imges were tken using twophoton lser snning mirosope. Setions were stined with n ntiody ginst the glutmte trnsporter EAAC1 (red). On the right re higher mgnifition pitures of the oxed region in the left. Aout 90% of the ChTx positive, VTA-projeting neurons (green) o-lolized with EAAC1 (rrowheds). Sle r: 50 µm.. Left: digrm of henul in prsgittl setion. Right pnels: Two dys fter in vivo injetion of the HSV-GFP virus into VTA, prsgittl rin setions ontining (left) were prepred nd stined with n nti-gaba ntiody. The o-loliztion of VTAprojeting neurons (GFP) nd GABA-positive neurons (rrow hed) ws exmined. Among the 391 GFP positive-neurons exmined from 9 setions, whih ontined 58 GABA-positive neurons, no o-loliztion of GFP nd GABA expression in the sme 4 W W W. N A T U R E. C O M / N A T U R E neuron ws found. Sle r: 100 µm. Coronl rin setions ontining nd

5 SUPPLEMENTARY INFORMATION RESEARCH 58 GABA-positive neurons, no o-loliztion of GFP nd GABA expression in the sme neuron ws found. Sle r: 100 µm. Coronl rin setions ontining nd dentte gyrus (DG) were stined with n nit-gad67 ntiody. On the sme rin setions, mny GAD67 positive ells were found in the DG, wheres very few GAD67 positive neurons ould e found in the. On the right re higher mgnifition pitures of the oxed regions in the left. Sle r: 200 µm. 5

RESEARCH SUPPLEMENTARY INFORMATION efore CNQX fter CNQX efore pirotoxin fter pirotoxin ontrol LH d NMDA/AMPA rtio VTAprojeting rndom mipsc frequeny (Hz)

. Smple mepsc tres reorded from neurons efore (left) nd fter (right) pplition of CNQX. Similr results were otined from 4 ells.

7, n=12; LH: 1.9±0.7, n=10; p>0.05) nd mplitude (ontrol: 25.5±1.6 pa, n=11; LH: 27.9±2.8 pa, n=10; p>0.05).. Smple mipsc tres reorded from neurons efore (left) nd fter (right) pplition of pirotoxin.

Evoked EPSCs from oth VTA-projeting nd rndomly reorded neurons ontined very little NMDAR-medited omponent (NMDA/AMPA rtio: VTA-projeting: 0.05±0.

6 RESEARCH SUPPLEMENTARY INFORMATION efore CNQX fter CNQX efore pirotoxin fter pirotoxin ontrol LH d NMDA/AMPA rtio VTAprojeting rndom mipsc frequeny (Hz) mipsc mplitude (pa) ontrol LH ontrol LH frequeny mplitude e retifition index VTAprojeting rndom Supplementl Figure 4. Synpti properties of neurons.. Smple mepsc tres reorded from neurons efore (left) nd fter (right) pplition of CNQX. Similr results were otined from 4 ells.. Left: exmples of the mipscs reorded from VTA-projeting neurons of ontrol or LH nimls. Right: Quntifition of mipsc frequeny (ontrol: 2.3±0.7, n=12; LH: 1.9±0.7, n=10; p>0.05) nd mplitude (ontrol: 25.5±1.6 pa, n=11; LH: 27.9±2.8 pa, n=10; p>0.05).. Smple mipsc tres reorded from neurons efore (left) nd fter (right) pplition of pirotoxin. Similr results were otined from 3 ells. d. Evoked EPSCs from oth VTA-projeting nd rndomly reorded neurons ontined very little NMDAR-medited omponent (NMDA/AMPA rtio: VTA-projeting: 0.05±0.01, n=8; rndom: 0.06±0.03, n=9; p>0.05). e. Evoked EPSCs from oth VTA-projeting nd rndomly reorded neurons showed similrly strong inwrd retifition (retifition index: VTAprojeting: 5.6±1, n=5; rndom: 5±0.8, n=5; p>0.05). All error rs represent s.e.m. 6

7 SUPPLEMENTARY INFORMATION RESEARCH Supplementl Figure time (min) lever press (#) dentte gyrus (DG) were stined with n nit-gad67 ntiody. On the sme rin individul rts individul rts setions, mny GAD67 positive ells were found in the DG, wheres very few GAD67 Shm 150 ua 300 ua positive neurons ould e found in the. On the right re higher mgnifition pitures of the oxed regions in the left. Sle r: 200 µm. Supplementl Figure 4. Synpti properties of neurons.. Smple mepsc tres reorded from neurons efore (left) nd fter (right) pplition of CNQX. Similr results were otined from 4 ells.. Left: exmples of the mipscs reorded from VTA-projeting neurons of ontrol or LH nimls. Right: Quntifition of mipsc frequeny (ontrol: 2.3±0.7, n=12; LH: 1.9±0.7, n=10; p>0.05) nd mplitude (ontrol: 25.5±1.6 pa, n=11; LH: 27.9±2.8 pa, n=10; p>0.05).. Smple mipsc tres reorded from neurons efore (left) nd fter (right) pplition of pirotoxin. Similr results were otined from 3 ells. d. Evoked EPSCs from oth VTA-projeting nd rndomly reorded neurons ontined very little NMDAR-medited omponent (NMDA/AMPA rtio: VTA-projeting: 0.05±0.01, n=8; rndom: 0.06±0.03, n=9; p>0.05). e. Evoked EPSCs from oth VTA-projeting nd rndomly reorded neurons showed similrly strong inwrd retifition (retifition index: VTAprojeting: 5.6±1, n=5; rndom: 5±0.8, n=5; p>0.05). All error rs represent s.e.m. Supplementl Figure 5. The volume of tissue ffeted y DBS in the.. Behvior of 52 nimls fter lerned helplessness indution (see methods). Red symols represent 26 nimls displying most severe lerned helplessness tht were hosen for eletrode implnttion (see methods for seletion riteri). Behviorl mesurements 4 W W W. N A T U R E. C O M / N A T U R E 7

8 RESEARCH SUPPLEMENTARY INFORMATION inluded the numer of suessful lever presses (left) within 20 seonds of shok onset, nd the test ompletion time (right). As lever presses led to termintion of the shok, low lever presses nd long test ompletion times indite lerned helplessness ehvior.. Representtive imges of -Fos expression in the oronl rin setions of nimls reeived Shm stimultion or DBS t 150 or 300 µa urrent intensities in the. The is outlined y dotted lines. Upper: rin setions 200 µm nterior to where the stimulting eletrodes were implnted. Both left nd right were shown. Stimulting eletrodes were implnted in the left. Lower: rin setions whih ontined the stimulting eletrodes (rrows). Only the left were shown. Note the prominent -Fos expression in the 300 µa group, whih ws onfined to the nd did not spred to the djent medil henul nd thlmi regions. The 150 µa group hd less -Fos expression, wheres the shm group hd no -Fos expression.. Quntifition of -Fos expression. Left: the numer of -Fos positive ells s funtion of distne from the tip of stimulting eletrode in rin setions tht ontined the stimulting eletrodes (p = , F = y One-wy ANOVA; Shm vs. 300 µa: p<0.05). Right: the numer of -Fos positive ells within the s funtion of distne from the stimulting eletrode long the nterior / posterior xis (p = , F = y One-wy ANOVA; Shm vs. 300 µa: p<0.01; 150 µa vs. 300 µa: p<0.05). DBS t oth 150 nd 300 µa signifintly indued -Fos expression ompred to the shm stimultion. DBS t 300 µa ws most effetive nd ffeted volume of out 1 mm 3, whih is out the size of. All error rs represent s.e.m. Sle r: 200 µm. 8

Supplementary Figure 1. Scheme of unilateral pyramidotomy used for detecting compensatory sprouting of intact CST axons.

Supplementary Figure 1. Scheme of unilateral pyramidotomy used for detecting compensatory sprouting of intact CST axons. () BDA 2 weeks fter Py () AAVs Cre or GFP t P1 BDA 2 weeks fter Py CSMN CST () Py t P7 or 2 months () Py t 2 months Supplementry Figure 1. Sheme of unilterl pyrmidotomy used for deteting ompenstory sprouting