LETTER. Synaptic potentiation onto habenula neurons in the learned helplessness model of depression

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1 doi:.38/nature974 Synapti potentiation onto habenula neurons in the learned helplessness model of depression Bo Li,, Joaquin Piriz, Martine Mirrione,3, ChiHye Chung, Christophe D. Proulx, Daniela Shulz 3, Fritz Henn,3 & Roberto Malinow The ellular basis of depressive disorders is poorly understood. Reent studies in monkeys indiate that neurons in the lateral habenula (LHb), a nuleus that mediates ommuniation between forebrain and midbrain strutures, an inrease their ativity when an animal fails to reeive an expeted positive reward or reeives a stimulus that predits aversive onditions (that is, disappointment or antiipation of a negative outome) 4. LHb neurons projet to, and modulate, dopamine-rih regions, suh as the ventral tegmental area (VTA),5, that ontrol reward-seeking behaviour 6 and partiipate in depressive disorders 7. Here we show that in two learned helplessness models of depression, exitatory synapses onto LHb neurons projeting to the VTA are potentiated. Synapti potentiation orrelates with an animal s helplessness behaviour and is due to an enhaned presynapti release probability. Depleting transmitter release by repeated eletrial stimulation of LHb afferents, using a protool that an be effetive for patients who are depressed 8,9, markedly suppresses synapti drive onto VTA-projeting LHb neurons in brain slies and an signifiantly redue learned helplessness behaviour in rats. Our results indiate that inreased presynapti ation onto LHb neurons ontributes to the rodent learned helplessness model of depression. To study the ellular basis of behavioural depression, we examined the synapti iruitry in the LHb of rats showing learned helplessness (Fig. ), a model of depression whereby animals show redued esape from esapable foot shok. We used two well-established animal models: aute learned helplessness (), whih is indued by subjeting rats to periods of inesapable and unpreditable shok ; and ongenital learned helplessness (), whih is present in a strain of rats produed by seletive breeding of animals that show the greatest amount of,. In addition to showing redued esape from esapable foot shok, animals with and also showed greater immobility in the fored swim test, another widely used animal model for depression 3, than ontrol animals (Fig. a, b). We examined transmission onto LHb neurons. These neurons reeive major inputs from numerous brain regions involved in stress response (suh as the entopedunular nuleus, lateral hypothalamus, lateral preopti area, medial prefrontal ortex and the bed nuleus of the stria terminalis) 4 (Supplementary Fig. ) and an ontrol dopaminergi funtion in the midbrain 4. We wished to determine whether synapti transmission onto LHb neurons is different in animals with learned helplessness and normal animals. To reord seletively from LHb neurons that an regulate the ativity of dopamine-produing neurons, we injeted a retrograde traer, holera toxin onjugated to the dye Alexa Fluor 488, into the VTA in vivo. Two to three days later, we prepared brain slies that ontained the LHb. A minority of neurons in the LHb were fluoresent, whih indiated their projetion to the VTA (Supplementary Fig. a, b). Notably, the LHb neurons that projet to the VTA and to the rostromedial tegmental nuleus a newly identified GABA (-aminobutyri aid)-produing, inhibitory relay station a VTA-p VTA-p b 4 mepsc frequeny (Hz) d 3 Cumulative probability ms 4 3 ms mepsc frequeny (Hz) pa 5 ms e Cumulative probability VTA-p ms 4 Number of ells ms VTA-p 3 4 mepsc frequeny (Hz) Amplitude (pa) 3 4 mepsc amplitude (pa) ms Figure Inreased exitatory synapti transmission onto VTA-projeting LHb neurons in the learned helplessness models of depression. a, Examples of mepscs reorded from VTA-projeting LHb neurons (VTA-p) from wildtype ontrol () animals and animals with, ms or. b, Means (histogram bars) and individual reordings (open irles) of mepsc frequeny from VTA-projeting LHb neurons in various groups of animals. Left:,.4 6.3, n 5 65 (6 animals);, , n 5 85 (8 animals), P,.5 bootstrap method; ms, , n 5 7 (8 animals), P,. bootstrap. Right:,.8 6., n 5 9 (4 animals);, , n 5 3 (4 animals), P,.5 bootstrap. Results are presented as mean 6 s.e.m. P values are ompared with group. Shaded region beyond dashed lines indiates highfrequeny mepscs. n, number of ells., Frequeny distribution of mepsc frequenies of all ells reorded (n 5 63 from 34 animals) showed bimodal distribution. d, The umulative probability of mepsc frequeny of VTAprojeting LHb neurons in different groups of animals (P,.5, Kolmogorov Smirnov (K S) test omparing with any other group). e, The amplitude of mepscs did not differ among the four animal groups (P..3, bootstrap). Inset shows mean 6 s.e.m. n, number of ells; NS, not signifiant. NS Center for Neural Ciruits and Behavior, Departments of Neurosiene and Biologial Sienes, 95 Gilman Drive # 634, University of California at San Diego, La Jolla, California 993, USA. Cold Spring Harbor Laboratory, Bungtown Road, Cold Spring Harbor, New York 74, USA. 3 Brookhaven National Laboratory, Upton, New York 973, USA. These authors ontributed equally to this work. 4 FEBRUARY VOL 47 NATURE 535 Mamillan Publishers Limited. All rights reserved

2 RESEARCH LETTER a Fration of immobility Failure rate d 5 mepsc frequeny (Hz).4.3. s between the LHb and the VTA 5,6 are largely non-overlapping populations (Supplementary Fig. ), indiating that we would be able to seletively target LHb neurons that diretly projet to the VTA. LHb neurons projeting to the VTA were glutamatergi, as indiated by their o-loalization with the glutamate transporter EAAC (also known as SLCA) and lak of GABAergi marker expression (Supplementary Fig. 3). We performed whole-ell path-lamp reordings on VTA-projeting neurons in aute parasagittal brain slies from rats that were wild-type ontrol, had or (naive), or had and had been exposed to mild stress (ms; see Behavioural paradigms in the Methods setion). We examined miniature exitatory postsynapti urrents (mepscs) (in the presene of tetrodotoxin to blok ation potentials and pirotoxin to blok GABA A -mediated synapti urrents), whih were mediated by AMPA (a-amino-3-hydroxy-5-methyl-4-isoxazole propioni aid)-type glutamate reeptors (Supplementary Fig. 4a) and represent responses from individual synapses onto the ells studied. bfailure rate Esape/avoidane.5 pa.4.3. Spontaneous spiking frequeny (Hz) Figure Enhaned synapti transmission onto VTA-projeting LHb neurons orrelates with helpless behaviour of individual animals. a, b, Animals with or show behavioural defiits in the fored swim test and in the esape/avoidane test. a, Fration of immobile time over the 5 min of the fored swim test:,. 6.5;, ; and, Results are mean 6 s.e.m; n (number of animals) indiated in histogram bars;, P,.5, Kruskal Wallis test. b, Rate of failure to esape during 3 trials of esapable foot shok:,. 6.3;,.3 6.3; and, Results are mean 6 s.e.m; n (number of animals) is indiated in the histogram bars;, P,., F (,36) 5.57, one-way ANOVA., The mean frequeny of mepscs onto VTA-projeting LHb neurons orrelates with an animal s helpless behaviour, measured as the fration of sessions in whih animals failed to esape (R 5.69, grey line; P,. by a linear regression; n 5 3 animals; n $ 5 ells for eah animal). Error bars, s.e.m. d, The spontaneous spiking rate measured in a ell-attahed onfiguration was higher in animals with than in ontrol animals: left, an example; right, histogram shows mean 6 s.e.m., and open irles are data from individual ells (,.9 6.3, n 5 7;, ; n 5 5,, P,., bootstrap). a b Hz 5 Hz Normalized peak amplitude ms 5 pa ms Normalized peak amplitude The mean frequeny of mepscs reorded from VTA-projeting LHb neurons of rats with learned helplessness ( Hz (mean 6 s.e.m.), n 5 3), ( Hz, n 5 85) and ms ( Hz, n 5 7) was higher than that of wild-type ontrols (.3 6. Hz, n 5 84; F (3,5) 5 3., P,.3 omparing the wild-type ontrol group with any other group, analysis of variane (ANOVA)) (Fig. a, b). In general, the distribution of mepsc frequenies reorded aross different ells in all groups had a bimodal distribution (Fig. ). Notably, the prevalene of neurons with high-frequeny mepscs (.8 Hz; Fig. b d, shaded region) was signifiantly higher 5 pa ms Number of trains Number of trains NS.6 3 ms pa 5 pa Failure rate Spine density (number μm ) Amplitude of non-failures (pa) NS ms 7 9 Figure 3 Presynapti mehanism underlying the inrease in exitatory synapti transmission onto VTA-projeting LHb neurons in helpless animals. a, Two-photon laser sanning images of a VTA-projeting LHb neuron. The neuron was labelled by in vivo injetion into the VTA of a herpes simplex virus that expresses green fluoresent protein and is transported in a retrograde manner. The neuron is shown at low magnifiation (left) and high magnifiation (entre, image of area demarated by white retangle in left panel). Sale bars, 5 mm (left) and mm (entre). Right, dendriti spine density on VTA-projeting LHb neurons of animals or animals with :,.5 6., 3 ells,,5.3 mm total dendriti length; and,.8 6., 7 ells,,.4 mm total dendriti length; P.., Student s t-test. b, Top, evoked EPSCs onto VTA-projeting LHb neurons of animals or animals with in response to stimulus trains ( Hz or 5 Hz). Bottom, plot of peak EPSCs normalized to first EPSC (, Hz n 5, 5 Hz n 5 ; and, Hz n 5 4, 5 Hz n 5 3). Compared with animals, animals with showed a faster synapti depression ( Hz, F (9,98) 5.3, P 5.; 5 Hz, F (9,98) , P,.; one-way ANOVA with repeated measures) and more extensive synapti depression ( Hz, F (,) 5 6.6, P 5.; 5 Hz, F (,) 5 7.5, P 5.; one-way ANOVA with repeated measures)., Minimally evoked EPSCs onto VTA-projeting LHb neurons show more failures in ontrol animals than in animals with (,.5 6., n 5 7;,. 6., n 5 9;, P,., Student s t-test). Mean amplitude of suessful trials (right) (, 3. 6, n 5 7; : , n 5 9; P..6, Student s t-test). a, Error bars, s.e.m. 5 pa 536 NATURE VOL 47 4 FEBRUARY Mamillan Publishers Limited. All rights reserved

3 RESEARCH in rats with (7%), (4%) and ms (%) than in wildtype ontrol rats (%; P,. omparing the wild-type ontrol group with any other group, x test). To determine whether the observed exitatory synapti potentiation was quantitatively orrelated to an animal s helpless behaviour, we first tested animals (either wild type or with ) by using an esape avoidane task and subsequently prepared brain slies and made reordings from the VTA-projeting neurons. For eah animal, we reorded from at least five ells and plotted the mean mepsc frequeny against the animal s helpless behaviour (as measured by the fration of trials in whih the animal failed to esape from an esapable -s foot shok; see also Fig. b). The signifiant orrelation (R 5.69, F (,) , P,., n 5 3 for all animals; and R 5.64, F (,6) 5.7, P,.5, n 5 8 for animals with ) (Fig. ) indiates that the potentiation of exitatory transmission onto VTA-projeting LHb neurons is linked with an individual animal s helpless behaviour. To examine the output of VTA-projeting neurons, we measured their spontaneous ation potentials, whih were more frequent in animals with than in wild-type ontrols (Fig. d). We observed no differenes among the various groups with respet to the amplitude of mepscs (Fig. e) or the frequeny or amplitude of miniature inhibitory postsynapti urrents (Supplementary Fig. 4b, ). These results indiate that the exitatory synapti input onto LHb neurons that projet to the VTA is potentiated in the learned helplessness model. a b Number of lever presses d Baseline Fration of immobility 3 Lever-press test (Baseline) (Day ). Lever-press test 5 μa Sham DBS. NS 5 ms 5 mv 9 Day Day Normalized slope DBS st pulse nd pulse 5 5 Time (min) DBS DBS DBS ( h) 4 h 4 h Test I Test II (5 μa) (3 μa) (Day ) LHb sham (n = 4) LHb DBS (n = 9) LPLR DBS (n = 7) 3 μa Baseline 5 μa 3 μa e 3 Test ompletion time (min) LHb DBS LPLR DBS The enhaned mepsc frequeny ould result from an inrease in either the number of synapses or the probability of presynapti neurotransmitter release. To distinguish between these possibilities, we first measured the density of dendriti spines, whih are the sites of exitatory synapses, on the dendrites of VTA-projeting LHb neurons. There was no signifiant differene in dendriti spine density between wild-type ontrols and animals with (Fig. 3a), and there was no obvious differene in the patterns of dendriti branhing between the two groups (data not shown), suggesting that there was no major differene in the number of synapses between wild-type ontrol animals and those with. To determine whether there is a hange in the effiay of presynapti neurotransmitter release, we examined evoked transmission. Synapti transmission onto LHb neurons (eliited by plaing a stimulating eletrode in the LHb) showed distint properties: the evoked exitatory synapti response had a very small NMDA (N-methyl-D-aspartate) reeptor omponent (Supplementary Fig. 4d), and the AMPA reeptor omponent showed strong inward retifiation (Supplementary Fig. 4e). To probe presynapti funtion, we evoked transmission with highfrequeny stimulation trains (ten stimuli delivered at Hz or 5 Hz). The derease in the amplitude of EPSCs in response to suessive pulses during a train of stimuli reflets presynapti vesile depletion; more depletion orrelates with a higher release probability 7. VTA-projeting LHb neurons of animals with showed a faster synapti depression (at Hz F (9,98) 5.3, P,.5; at 5 Hz F (9,98) , P,., one-way ANOVA with repeated measures) and a more extensive synapti depression (at Hz F (,) 5 6.6, P,.5; at 5 Hz F (,) 5 7.5, P,.5, one-way ANOVA with repeated measures) than those in wild-type ontrol animals (Fig. 3b). Furthermore, with minimal stimulation, whih is designed to ativate few synapses (as indiated by the amplitude of non-failure responses, whih is similar to the mepsc amplitude; Fig. 3), we measured synapti transmission failure rate. Exitatory synapti transmission onto VTA-projeting LHb neurons of animals with had a signifiantly lower failure rate than that of wild-type ontrol animals (Fig. 3; Figure 4 DBS in the LHb suppresses exitatory synapti transmission and reverses learned helplessness. a, Left, example exitatory postsynapti potentials (EPSPs) (paired pulses) reorded from a VTA-projeting LHb neuron before (), during () and after (3) stimulation mimiking DBS. Arrows indiate when paired pulses were given. Right, mean EPSP slope at indiated time points before, during and after DBS: () before (first pulse. 6., seond pulse.9 6., n 5 6 (4 animals)); () during (first pulse.5 6.7, seond pulse.3 6.3, P,. for both pulses ompared with those in, Student s t-test); and (3) after (first pulse. 6.3, seond pulse.9 6.). b,a shemati diagram showing the experimental proedures., Number of lever presses (left) and test ompletion time (right) for animals that reeived DBS or sham stimulation in the LHb or DBS in the LPLR (lateral post-thalami nulei, laterorostral), before (baseline) or after DBS of different intensities. For DBS in the LHb (n 5 9), lever press baseline. 6.4; 5 ma session ; and 3 ma session (n 5 8) For DBS in the LHb (n 5 9), test ompletion time baseline ; 5 ma session 7. 6 ; 3 ma session (n 5 8) For sham stimulation in the LHb (n 5 4), lever press baseline.8 6.5; 5 ma session.4 6.9; and 3 ma session.4 6. For sham stimulation in the LHb, test ompletion time baseline ; 5 ma session ; and 3 ma session For DBS in the LPLR (n 5 7), lever press baseline.4 6.; 5 ma session 6 ; and 3 ma session For DBS in the LPLR, test ompletion time baseline.6 6.; 5 ma session 9 6.; and 3 ma session For DBS in the LHb group,, P,.5 ompared with baseline (bootstrap). For the sham and DBS in LPLR groups, P..5 for both measurements at both sessions ompared with baseline (bootstrap). d, Immobility during the fored swim test. For DBS, day,.6 6.; day,.6 6.; n 5 9. For sham, day,.5 6.; day,.7 6.5; n 5. DBS versus sham on day,, P,. (bootstrap). e, Representative resyl violet staining of oronal brain setions after DBS in the LHb or LPLR. Arrows indiate the eletrode trak in the LHb (top) or LPLR (bottom). Dashed lines indiate the border of the habenula. Sale bars, mm. a,, d, Error bars, s.e.m. 4 FEBRUARY VOL 47 NATURE 537 Mamillan Publishers Limited. All rights reserved

4 RESEARCH LETTER n 5 7 9, P,., bootstrap method). These results indiate that the exitatory synapti inputs onto VTA-projeting LHb neurons of helpless animals have a higher synapti release probability; therefore, repeated stimulation an deplete synapti vesiles faster and more effiiently in helpless animals. One treatment for linial depression that is urrently under evaluation is deep brain stimulation (DBS). This therapy onsists of ontinuously delivered high-frequeny eletrial stimulation to various brain regions 8,8. In a reent linial ase, DBS of the LHb produed a marked remission of treatment-resistant depression 9. Notably, depression reurred when DBS was stopped (in two aidental episodes 9 ). To examine the ellular effets of DBS, we used brain slies and reorded synapti transmission onto VTA-projeting LHb neurons, whih was evoked by plaing a stimulating eletrode in the LHb. After a baseline period of evoked transmission, a DBS protool used in patients (trains onsisting of seven stimuli at 3 Hz followed by a 4 ms interval with no stimuli) was ontinuously delivered through the same stimulation eletrode; stimuli were interleaved with the DBS trains, allowing us to monitor evoked synapti transmission (see Methods for details). The DBS protool produed a marked depression of exitatory synapti transmission, whih persisted for the DBS protool period and was reversed on essation of the DBS protool (Fig. 4a). Thus, a DBS protool an effetively redue exitatory synapti transmission onto VTA-projeting LHb neurons. We wished to test whether reduing synapti drive onto LHb neurons an modulate helpless behaviour. Remarkably, when the DBS protool that was used in brain slies was delivered to the LHb in animals with, it markedly ameliorated their helpless behaviour, as indiated by an inrease in esape behaviour (Fig. 4b, and Supplementary Fig. 5a). This effet was dependent on both the intensity of stimulation and the plaement of the stimulation eletrode: stimulating at 3 ma had a stronger behavioural effet and affeted a larger volume within the LHb than stimulating at 5 ma (Fig. 4b, and Supplementary Fig. 5b, ). In addition, only if the eletrode was plaed in the LHb, but not in the nearby thalamus, did DBS reverse the helplessness (Fig. 4b,, e). Furthermore, DBS in the LHb, but not sham stimulation in the LHb, prevented the inrease in immobility in the fored swim test (Fig. 4d). Thus, suppression of synapti transmission at the LHb through DBS an autely reverse helpless behaviour in rats. Several hanges in neural funtion have been identified in depressed humans and rodent models of depression, probably owing to the multifaeted nature of depressive disorders 9 3. The reent identifiation of the LHb as a brain region in monkeys that an enode disappointment and expetation of negative onditions,3 led us to investigate its role in the learned helplessness rodent model of depression. Our findings indiate that exitatory synapti ativity onto VTA-projeting neurons in the LHb may be a key modulator of learned helplessness. The two learned helplessness models examined showed potentiated exitatory synapti ativity onto these neurons. Interestingly, the major modifiation was an inrease in the proportion of ells showing high-frequeny mepscs (from % to 4 %) in animals with learned helplessness. This finding suggests that large hanges in a small proportion of ells in the LHb may be apable of modifying an animal s behaviour. A ruial role for transmission onto LHb neurons is further supported by the strong orrelation between the potentiation of synapti transmission onto VTA-projeting LHb neurons and an individual animal s helpless behaviour. Given the presynapti nature of synapti potentiation, we examined the effets of synapti depression by repeated afferent stimulation, a protool that mimis linially used DBS. Reduing synapti transmission onto LHb neurons through a DBS protool led to aute reversal of learned helplessness. Suppression of transmission onto VTA-projeting LHb neurons probably had a role in mediating this benefiial effet, although modulation of LHb neurons, or axons of passage, projeting to other targets may also be involved. Our study provides ellular mehanisms that may explain previously reported phenomena: the inrease in LHb metaboli ativity observed in humans who are depressed 4,5 and in animal models of depression 6,7 ; and that lesion 8,9 or pharmaologial silening 3 of the LHb an modulate depression-like symptoms in animal models. Our findings suggest an aberrant ellular proess that has not previously been examined in the ontext of mood disorders and that may be ruial in the aetiology of depression. Future studies aimed at determining the hanges in moleular signalling that underlie the synapti hyperativity onto LHb neurons may lead to novel and effetive treatments able to reverse some forms of depressive disorders. METHODS SUMMARY Standard surgial proedures were followed for the in vivo injetion of retrograde traers. Rats with were bred as desribed previously,. To prepare animals with, rats were exposed to a learned helplessness training session after in vivo injetion of retrograde traers into the VTA. This session onsisted of inesapable, unontrollable eletri foot shoks, with random shok duration and unpreditable inter-shok intervals. Control animals were plaed in the shoking hamber in parallel, without being shoked. To prepare the ms group, rats with were treated with brief, esapable foot shok. Aute brain slies from the various groups were prepared for eletrophysiologial reordings. To evaluate learned helplessness behaviour, we used both a lever-pressing task and an ative avoidane task. In the lever-pressing task, a lever was added to the shoking hamber in the testing session. Foot shook was terminated if the animal pressed the lever. The ative avoidane task was performed in a shuttle box equipped with an eletrial grid floor and a door separating the two halves. Foot shok was terminated if the animal rossed to the other side of the age. For the fored swim test, the animal was fored to swim in a ylinder of water at 5 6 uc, and the animal s immobility in the water was measured. To test the effet of DBS on learned helplessness behaviour, rats were first trained and tested, and those that met the learned helplessness riteria were hosen for eletrode implantation in the LHb. After reovery from surgery, rats underwent a training session followed by a baseline learned helplessness test. DBS at different intensities was subsequently applied, and animals were tested again for the learned helplessness behaviour. To test the effets of DBS on the fored swimtest, DBS in the LHb was applied before the seond day of swimming to determine its effet on the animal s immobility. Full Methods and any assoiated referenes are available in the online version of the paper at Reeived 4 Otober 9; aepted 7 Deember.. Krishnan, V. & Nestler, E. J. The moleular neurobiology of depression. Nature 455, (8).. Matsumoto, M. & Hikosaka, O. Lateral habenula as a soure of negative reward signals in dopamine neurons. Nature 447, 5 (7). 3. Matsumoto, M. & Hikosaka, O. Representation of negative motivational value in the primate lateral habenula. Nature Neurosi., (9). 4. Hikosaka, O. The habenula: from stress evasion to value-based deision-making. Nature Rev. Neurosi., (). 5. Ji, H. & Shepard, P. D. 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5 RESEARCH 7. Zuker, R. S. & Regehr, W. G. Short-term synapti plastiity. Annu. Rev. Physiol. 64, (). 8. Sartorius, A. & Henn, F. A. Deep brain stimulation of the lateral habenula in treatment resistant major depression. Med. Hypotheses 69, (7). 9. Airan, R. D. et al. High-speed imaging reveals neurophysiologial links to behavior in an animal model of depression. Siene 37, (7).. Berton, O. & Nestler, E. J. New approahes to antidepressant drug disovery: beyond monoamines. Nature Rev. Neurosi. 7, 37 5 (6).. Pittenger, C. & Duman, R. S. Stress, depression, andneuroplastiity: a onvergene of mehanisms. Neuropsyhopharmaology 33, 88 9 (8).. Sahay, A. & Hen, R. Adult hippoampal neurogenesis in depression. Nature Neurosi., 5 (7). 3. Mill, J. & Petronis, A. Moleularstudiesofmajor depressive disorder: the epigeneti perspetive. Mol. Psyhiatry, (7). 4. Morris, J. S. et al. Covariation of ativity in habenula and dorsal raphe nulei following tryptophan depletion. Neuroimage, 63 7 (999). 5. Roiser, J. P. et al. The effets of tryptophan depletion on neural responses to emotional words in remitted depression. Biol. Psyhiatry 66, (9). 6. Shumake, J. & Gonzalez-Lima, F. Brain systems underlying suseptibility to helplessness and depression. Behav. Cogn. Neurosi. Rev., 98 (3). 7. Caldeott-Hazard, S., Mazziotta, J. & Phelps, M. Cerebral orrelates of depressed behavior in rats, visualized using 4 C -deoxygluose autoradiography. J. Neurosi. 8, (988). 8. Amat, J. et al. The role of the habenular omplex in the elevation of dorsal raphe nuleus serotonin and the hanges in the behavioral responses produed by unontrollable stress. Brain Res. 97, 8 6 (). 9. Yang, L. M. et al. Lateral habenula lesions improve the behavioral response in depressed rats via inreasing the serotonin level in dorsal raphe nuleus. Behav. Brain Res. 88, 84 9 (8). 3. Winter, C. et al. Pharmaologial inhibition of the lateral habenula improves depressive-like behavior in an animal model of treatment resistant depression. Behav. Brain Res. 6, (). Supplementary Information is linked to the online version of the paper at Aknowledgements We thank K. Deisseroth for help and suggestions, A. Gifford and A. Biegon for sharing equipment and laboratory spae, and members of the Malinow Lab and LiLab for disussions. Thisstudywassupported bythe DanaFoundation (B.L.), the Biobehavioral Researh Awards for Innovative New Sientists (BRAINS) from the National Institute of Mental Health, National Institutes of Health (RMH993-) (B.L.) and the Shiley-Maros Endowment (R.M.). Author Contributions B.L., J.P., M.M. and C.C ontributed equally to the study. B.L., J.P., M.M., C.C., C.D.P. and D.S. performed and analysed the experiments. C.C. and B.L. made the figures. B.L., F.H. and R.M. designed the study. B.L. and R.M. wrote the manusript. Author Information Reprints and permissions information is available at The authors delare no ompeting finanial interests. Readers are welome to omment on the online version of this artile at Correspondene and requests for materials should be addressed to B.L. (bli@shl.edu) or R.M. (rmalinow@usd.edu). 4 F E B R U A RY VO L 4 7 N AT U R E Mamillan Publishers Limited. All rights reserved

6 RESEARCH LETTER METHODS Animals. Wild-type, male Sprague Dawley rats were purhased from Taoni Farms and allowed to alimatize to the animal faility for weeks before experiments were arried out. The rats were bred as desribed,. The rats were housed under a -h light dark yle (7 a.m. to 7 p.m. light), with food and water freely available. All proedures involving animals were approved by the Institute Animal Care and Use Committees of Cold Spring Harbor Laboratory, University of California, San Diego, and Brookhaven National Laboratory. Retrograde labelling of VTA-projeting LHb neurons in vivo. Standard surgial proedures were followed for in vivo injetion 3. To label the VTA-projeting LHb neurons, we injeted in vivo Alexa-Fluor-488-onjugated holera toxin ( mg ml ; Moleular Probes) or a herpes simplex virus expressing enhaned GFP (HSV GFP, NeuroVex), both of whih are retrograde traers, into the VTA. Animals were anaesthetized with isoflurane (Baxter) using an isoflurane vaporizer (Paragon Medial) and positioned in a stereotaxi apparatus that was onneted to a omputer system with a digital rat brain atlas (Angle Two Stereotaxi System, myneurolab.om). Injetions of traer solutions (3 5 injetion sites along the vertial axis, nl per injetion) were delivered with a glass miropipette through a skull window ( 3 mm ) by pressure appliation (5 psi, ontrolled by a Pirospritzer II; General Valve). The injetions were performed within the following stereotaxi oordinates: 5.3 mm from bregma;.96 mm lateral from the midline; and mm vertial from the ortial surfae. Rats were injeted subutaneously with 5 mg kg arprofen (an NSAID) after surgery. During proedures, animals were kept on a heating pad and were brought bak to their home ages after regaining movement. We waited 3 days to allow the retrograde labelling of neurons in the LHb before we killed the animals for experiments. Preparation of aute brain slies and eletrophysiology. Male rats of 4 5 days of age were used for all of the eletrophysiology experiments. Animals were anaesthetized with isoflurane, deapitated and their brains quikly removed and hilled in ie-old dissetion buffer (. mm holine hloride, 5. mm NaHCO 3,.5 mm NaH PO 4,.5 mm KCl,.5 mm CaCl, 7. mm MgCl, 5. mm gluose,.6 mm asorbi aid and 3. mm pyruvi aid, gassed with 95% O and 5% CO ). Sagittal slies (4 mm) aross the LHb were ut in dissetion buffer, by using a VT S vibratome (Leia), and subsequently transferred to a storage hamber ontaining artifiial erebrospinal fluid (ACSF) (8 mm NaCl,.5 mm KCl, 6. mm NaHCO 3, mm NaH PO 4, mm gluose, 4 mm MgCl and 4 mm CaCl, at 5 uc, ph 7.4, gassed with 95% O and 5% CO ). After at least h reovery time, slies were transferred to the reording hamber and were onstantly perfused with ACSF maintained at 7 uc. Experiments were always performed on interleaved wild-type ontrol and or animals. About three-quarters of the experiments were arried out blinded to the experimental group. These showed the same results as the non-blinded experiments, and the data were ombined. Whole-ell path-lamp reordings were obtained with Axopath-D amplifiers (Axon Instruments) onto neurons in the LHb under visual guidane using transmitted light illumination. For evoked EPSCs, synapti transmission was evoked with a bipolar stimulating eletrode plaed lose to the stria medullaris, typially.. mm away from ell bodies. Responses were reorded at holding potentials of 6 mv (for AMPA-reeptor-mediated responses) and 4 mv (for detetion of any NMDA-reeptor-mediated responses and measurement of retifiation). NMDA-reeptor-mediated responses were quantified as the mean urrent between ms and 6 ms after stimulation. Bathing solution (ACSF) ontained 9 mm NaCl,.5 mm KCl, mm CaCl, mm MgCl,6.mMNaHCO 3, mm NaH PO 4, mm gluose, and.mm pirotoxin, gassed with 5% CO and 95% O,at7uC (unless otherwise noted). Internal solution for voltage-lamp experiments ontained 5 mm aesium methanesulphonate, mm CsCl, mm HEPES,.5 mm MgCl, 4 mm Na - ATP,.4 mm Na-GTP, mm Na-phosphoreatine and.6 mm EGTA (ph 7.). Spermine ( mm) was inluded in the internal solution for measurement of retifiation. mepscs were reorded at 7 uc in the presene of mm tetrodotoxin (TTX) and mm pirotoxin in sagittal slies and analysed using Mini Analysis Program (Synaptosoft). To isolate miniature inhibitory spontaneous responses (mipscs), mm TTX, mm APV(D-()--amino-5-phosphonopentanoi aid) and 3 mm NBQX were added. For the experiments in whih high-frequeny stimulation trains were used to determine presynapti release probability, QX34 (5 mm) was inluded in the internal solution to prevent the generation of sodium spikes. To reruit the maximal number of axon terminals that an be stimulated by the high-frequeny trains, thereby minimizing the effets of axonal failures and reduing the variability in responses, a low onentration ( nm) of NBQX was inluded in the bath ACSF. This allowed stimulation at a higher intensity without evoking large EPSCs that ould ativate voltage-dependent ondutanes. For experiments testing the effets of DBS on synapti transmission onto VTA-projeting LHb neurons, evoked EPSCs were monitored before, during and after a stimulation protool mimiking linial DBS. Stimulation onsisted of episodes of 44 trains of stimuli separated by 4 ms. During eah train, seven stimuli were applied at a frequeny of about 3 Hz. The inter-episode interval was ms, during whih two stimuli separated by 5 ms were applied to monitor the amplitude and slope of the EPSP. The DBS protool and the paired-pulse stimulation were delivered using the same eletrode. Two-photon imaging of dendriti spines. Image aquisition and analysis were desribed previously 3,33. Images were aquired on a ustom built dual hannel two-photon laser sanning mirosope (based on the Olympus FluoView laser sanning mirosope) using a Ti:Sapphire Chameleon laser (Coherent), whih was mode loked to 9 nm. Full three-dimensional (3D) image staks were aquired using a 36.9 NA objetive lens at 35 digital zoom (FluoView software, Olympus), 7 nm per pixel. Eah image plane was resampled three times and spaed.5 mminthez dimension. Behavioural paradigms. Methods for the learned helplessness paradigm have been optimized previously 34. To prepare animals with learned helplessness (the group) for behavioural testing and eletrophysiologial reording, rats were exposed to a learned helplessness training session 5 days after in vivo injetion of retrograde traers into the VTA. This session onsisted of inesapable, unontrollable eletri foot shoks at.8 ma over 4 min in the shoking hambers (Coulbourn Instruments; hambers were inhes wide 3 inhes deep 3 inhes high and were ontrolled by preision adjustable shokers), with random shok duration ranging from 5 s to 5 s and unpreditable inter-shok intervals (ITIs). Experiments were performed on pairs of littermates housed in the same age. Control animals were plaed in the shoking hamber in parallel for 4 min, without being shoked. Eletrophysiologial reordings on aute brain slies were performed 4 48 h after shoking. Animal identity was oded for blinding the researher with respet to treatment. To prepare animals exposed to mild stress (the ms group), rats were treated with a proedure essentially the same as the ative avoidane task (see below), during whih animals reeived an average of s of esapable foot shok (n 5 8), and aute brain slies were prepared after h. To evaluate learned helplessness behaviour, we used both a lever-pressing task and an ative avoidane task. The lever-pressing task was desribed previously 34. Briefly, an illuminated lever was added to the shoking hamber in the testing session, whih omprised 5 esapable foot shoks lasting up to 6 s (shorter if terminated by a lever press) over min, and with fixed ITIs of 4 s. The ative avoidane task was performed in a shuttle box ( inhes wide 3 inhes deep 3 inhes high; Coulbourn Instruments) equipped with an eletrial grid floor, a door separating the two halves, and photoell detetors. The shuttle box was plaed in a sound-attenuating hamber to minimize external stimuli. Testing was fully automated using Graphi State software (Coulbourn Instruments). Animals were allowed to explore the shuttle box for 5 min, and helpless behaviour was evaluated over 3 trials of unexpeted and esapable foot shok (. ma intensity, s duration, with random ITIs of 4 6 s) following a 5-s ue tone. Foot shok was terminated if the animal ompletely rossed to the other side of the age. When an animal rossed the age during the 5-s ue tone presentation, avoidane was sored. If an animal rossed during the -s shoks, the mean esape lateny was measured. Failure was reorded if no rossing was made during the -s shok. For the fored swim test, animals were fored to swim for 5 min in a ylinder of water (water temperature was 5 6 uc; the ylinder was 3 m in diameter and 4 m high; the depth of the water was set to prevent animals from touhing the bottom with their hind limbs). Animal behaviour was videotaped using a PC6EX3 infrared amera (SuperCiruits). The immobile time eah animal spent during the test was manually ounted offline, with the evaluator being blind to the treatment of the animals. Deep brain stimulation (DBS). To prepare animals with learned helplessness for the DBS experiments, animals were first treated with a training session and then 4 h later, a testing session, as desribed above. On the basis of the test results, animals that met the riteria (those that pressed the lever only 5 times, and took between 6 and min to finish the test) were used to test the effets of DBS. For inreased stringeny, only lever presses ourring within the first s of shok onset were ounted. Fifty two male Sprague Dawley rats were trained and tested, and 6 of these animals met the riteria. Three days later, standard surgial proedures were used to implant bipolar onentri eletrodes (8 mm long,.8 mm tip diameter; Plastis One) unilaterally into the LHb (oordinates 3.7 mm AP, 6.7 mm ML and 5.4 mm DV) in rats that met the riteria. After 3 5 days reovery from surgery, rats underwent a training session followed by a baseline learned helplessness test. Three animals, in whih the eletrodes were implanted into the LHb, were exluded from further study beause their performane did not meet the riteria during the baseline test. Immediately following the baseline test, DBS (seven stimulus trains of 3 Hz, separated by 4 ms intervals; 5 ma intensity) in the LHb or the thalamus, or no stimulation (sham), was applied for h. Twenty-four hours later, another -h session of DBS Mamillan Publishers Limited. All rights reserved

7 RESEARCH or sham stimulation was given immediately before and during the learned helplessness test. DBS intensity was 5 ma. Another 4 h later, the final -h session of DBS (or sham stimulation) was given at a higher intensity (3 ma), immediately before and during the final learned helplessness test (see Fig. 4b for a shemati diagram showing the experimental proedures). Only animals with the eletrode orretly plaed in the LHb or thalamus (LPLR) were inluded for the respetive behavioural analysis. To test the effets of DBS on the fored swim test, eletrodes were implanted in the same way as desribed above, exept that in the DBS group two rats had bilateral implants and that in the sham group three rats had bilateral implants. The rest of the animals had unilateral implants (total animals used for the fored swim test: DBS n 5 9 and sham n 5 ). Immobility time was reorded during the first 5 min of a 5-min swimming session on day. DBS (5 ma) or sham stimulation was applied for h following the fored swim test on day. Twenty-four hours later (day ), another h of DBS (5 ma) or sham stimulation was applied, and immobility time was reorded during the 5-min swimming session. To determine the volume of tissue affeted by DBS in the LHb, in a separate set of experiments, animals were perfused with 4% PFA h after the onset of DBS, and brains were proessed for immunohistohemistry to examine Fos expression. Immunohistohemistry. Immunohistohemistry experiments were performed following standard proedures on 5-mm brain setions fixed with 4% PFA. The antibodies used were anti-neun antibody (Chemion), anti-eaac antibody (Chemion), anti-gaba antibody (Sigma), anti-gad67 antibody (Chemion) and anti-fos antibody (Santa Cruz Biotehnology). After finishing the immunohistohemistry proess, images were taken using either an LSM 5 onfoal mirosope (Zeiss; for double labelling with two olours) or a BX4 histology mirosope (Olympus; for single labelling with one olour), using 3 objetives. Statistis and data presentation. To ompare the means of non-normally distributed data sets, we used a bootstrap proedure. Two data sets (N and M of size n and m) were randomly sampled n and m times, respetively, allowing resampling, and means (N i and M i ) were generated. This proedure was repeated, times. If N j was more than M j fewer than 5% of the times, then the probability that N is more than M was estimated to be less than.5. Similar alulations established probabilities less than.. All other statistial tests are indiated when used. All data are presented as mean 6 s.e.m. 3. Rumpel, S., LeDoux, J., Zador, A. & Malinow, R. Postsynapti reeptor traffiking underlying a form of assoiative learning. Siene 38, (5). 3. Kope, C. D. et al. Glutamate reeptor exoytosis and spine enlargement during hemially indued long-term potentiation. J. Neurosi. 6, 9 (6). 33. Kope, C. D., Real, E., Kessels, H. W. & Malinow, R. GluR links strutural and funtional plastiity at exitatory synapses. J. Neurosi. 7, (7). 34. Vollmayr, B. & Henn, F. A. Learnedhelplessness inthe rat: improvements in validity and reliability. Brain Res. Brain Res. Proto. 8, 7 (). Mamillan Publishers Limited. All rights reserved