Synaptic potentiation ontohabenula neurons in the learned helplessness model of depression

LETTER

doi:10.1038/nature09742

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

Bo Li 1,2*,Joaquin Piriz 1*,Martine Mirrione 2,3*,ChiHye Chung 1*,Christophe D.Proulx 1,Daniela Schulz 3,Fritz Henn 2,3&Roberto Malinow 1

The cellular basis of depressive disorders is poorly understood 1.Recent studies in monkeys indicate that neurons in the lateral habenula (LHb),a nucleus that mediates communication between forebrain and midbrain structures,can increase their activity when an animal fails to receive an expected positive reward or receives a stimulus that predicts aversive conditions (that is,disappointment or anticipation of a negative outcome)2–4.LHb neurons project to,and modulate,dopamine-rich regions,such as the ventral tegmental area (VTA)2,5,that control reward-seeking behaviour 6and partici-pate in depressive disorders 7.Here we show that in two learned helplessness models of depression,excitatory synapses onto LHb neurons projecting to the VTA are potentiated.Synaptic potentia-tion correlates with an animal’s helplessness behaviour and is due to an enhanced presynaptic release probability.Depleting transmitter release by repeated electrical stimulation of LHb afferents,using a protocol that can be effective for patients who are depressed 8,9,markedly suppresses synaptic drive onto VTA-projecting LHb neurons in brain slices and can significantly reduce learned help-lessness behaviour in rats.Our results indicate that increased pre-synaptic action onto LHb neurons contributes to the rodent learned helplessness model of depression.

To study the cellular basis of behavioural depression,we examined the synaptic circuitry in the LHb of rats showing learned helplessness (Fig.1),a model of depression whereby animals show reduced escape from escapable foot shock 10.We used two well-established animal models:acute learned helplessness (aLH),which is induced by subjecting rats to periods of inescapable and unpredictable shock 10;and congenital learned helplessness (cLH),which is present in a strain of rats produced by selective breeding of animals that show the greatest amount of aLH 11,12.In addition to showing reduced escape from escapable foot shock,animals with cLH and aLH also showed greater immobility in the forced swim test,another widely used animal model for depression 13,than control animals (Fig.2a,b).

We examined transmission onto LHb neurons.These neurons receive major inputs from numerous brain regions involved in stress response (such as the entopeduncular nucleus,lateral hypothalamus,lateral preoptic area,medial prefrontal cortex and the bed nucleus of the stria terminalis)14(Supplementary Fig.1)and can control dopaminergic function in the midbrain 4.We wished to determine whether synaptic transmission onto LHb neurons is different in animals with learned helplessness and normal animals.To record selectively from LHb neu-rons that can regulate the activity of dopamine-producing neurons,we injected a retrograde tracer,cholera toxin conjugated to the dye Alexa Fluor 488,into the VTA in vivo .Two to three days later,we prepared brain slices that contained the LHb.A minority of neurons in the LHb were fluorescent,which indicated their projection to the VTA (Sup-plementary Fig.2a,b).Notably,the LHb neurons that project to the VTA and to the rostromedial tegmental nucleus—a newly iden-tified GABA (c -aminobutyric acid)-producing,inhibitory relay station

*These authors contributed equally to this work.

1

Center for Neural Circuits and Behavior,Departments of Neuroscience and Biological Sciences,9500Gilman Drive #0634,University of California at San Diego,La Jolla,California 92093,USA.2Cold Spring Harbor Laboratory,1Bungtown Road,Cold Spring Harbor,New York 11724,USA.3Brookhaven National Laboratory,Upton,New York 11973,

USA.a

VTA-p WT VTA-p cLH VTA-p cLHms VTA-p aLH

mEPSC frequency (Hz)

d

C u m u l a t i v e p r o b a b i l i t y

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mEPSC frequency (Hz)mEPSC amplitude (pA)

e

Figure 1|Increased excitatory synaptic transmission onto VTA-projecting LHb neurons in the learned helplessness models of depression.a ,Examples of mEPSCs recorded from VTA-projecting LHb neurons (VTA-p)from wild-type control (WT)animals and animals with cLH,cLHms or aLH.b ,Means (histogram bars)and individual recordings (open circles)of mEPSC frequency from VTA-projecting LHb neurons in various groups of animals.Left:WT,2.460.3,n 565(6animals);cLH,4.060.7,n 585(8animals),P ,0.05bootstrap method;cLHms,4.760.7,n 570(8animals),P ,0.001bootstrap.Right:WT,1.860.2,n 519(4animals);aLH,3.760.8,n 523(4animals),P ,0.05bootstrap.Results are presented as mean 6s.e.m.P values are

compared with WT group.Shaded region beyond dashed lines indicates high-frequency mEPSCs.n ,number of cells.c ,Frequency distribution of mEPSC frequencies of all cells recorded (n 5263from 34animals)showed bimodal distribution.d ,The cumulative probability of mEPSC frequency of VTA-projecting LHb neurons in different groups of animals (P ,0.05,Kolmogorov–Smirnov (K–S)test comparing WT with any other group).e ,The amplitude of mEPSCs did not differ among the four animal groups (P .0.3,bootstrap).Inset shows mean 6s.e.m.n,number of cells;NS,not significant.

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between the LHb and the VTA 15,16—are largely non-overlapping populations (Supplementary Fig.2c),indicating that we would be able to selectively target LHb neurons that directly project to the VTA.LHb neurons projecting to the VTA were glutamatergic,as indicated by their co-localization with the glutamate transporter EAAC1(also known as SLC1A1)and lack of GABAergic marker expression (Sup-plementary Fig.3).

We performed whole-cellpatch-clamp recordings on VTA-projecting neurons in acute parasagittal brain slices from rats that were wild-type control,had aLH or cLH (naive),or had cLH and had been exposed to mild stress (cLHms;see ‘Behavioural paradigms’in the Methods section).We examined miniature excitatory postsynaptic currents (mEPSCs)(in the presence of tetrodotoxin to block action potentials and picrotoxin to block GABA A -mediated synaptic currents),which were mediated by AMPA (a -amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)-type glutamate receptors (Supplementary Fig.4a)and represent responses from individual synapses onto the cells studied.

The mean frequency of mEPSCs recorded from VTA-projecting LHb neurons of rats with learned helplessness—aLH (3.760.8Hz (mean 6s.e.m.),n 523),cLH (4.060.7Hz,n 585)and cLHms (4.760.7Hz,n 570)—was higher than that of wild-type controls (2.360.2Hz,n 584;F (3,251)53.1,P ,0.03comparing the wild-type control group with any other group,analysis of variance (ANOVA))(Fig.1a,b).In general,the distribution of mEPSC frequencies recorded across different cells in all groups had a bimodal distribution (Fig.1c).Notably,the prevalence of neurons with high-frequency mEPSCs (.8Hz;Fig.1b–d,shaded region)was significantly

higher

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Figure 2|Enhanced synaptic transmission onto VTA-projecting LHb neurons correlates with helpless behaviour of individual animals.

a ,

b ,Animals with aLH or cLH show behavioural deficits in the forced swim test and in the escape/avoidance test.a ,Fraction of immobile time over the 5min of the forced swim test:WT,0.1260.05;aLH,0.3360.08;and cLH,0.3160.05.Results are mean 6s.e.m;n (number of animals)indicated in histogram bars;*,P ,0.05,Kruskal–Wallis test.b ,Rate of failure to escape during 30trials of escapable foot shock:WT,0.1260.03;aLH,0.3160.03;and cLH,0.3860.05.Results are mean 6s.e.m;n (number of animals)is indicated in the histogram bars;***,P ,0.001,F (2,136)511.57,one-way ANOVA.

c ,The mean frequency of mEPSCs onto VTA-projecting LHb neurons correlates with an animal’s helpless behaviour,measure

d as th

e fraction o

f sessions in which animals failed to escape (R 250.69,grey line;P ,0.001by a linear regression;n 513animals;n $5cells for each animal).Error bars,s.e.m.d ,The spontaneous spikin

g rate measured in a cell-attached configuration was higher in animals wit

h cLH than in WT control animals:left,an example;right,histogram shows mean 6s.e.m.,and open circles are data from individual cells (WT,0.9260.32,n 517;cLH,3.0360.82;n 525,**,P ,0.01,

bootstrap).

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Figure 3|Presynaptic mechanism underlying the increase in excitatory synaptic transmission onto VTA-projecting LHb neurons in helpless animals.a ,Two-photon laser scanning images of a VTA-projecting LHb neuron.The neuron was labelled by in vivo injection into the VTA of a herpes simplex virus that expresses green fluorescent protein and is transported in a retrograde manner.The neuron is shown at low magnification (left)and high magnification (centre,image of area demarcated by white rectangle in left panel).Scale bars,50m m (left)and 10m m (centre).Right,dendritic spine density on VTA-projecting LHb neurons of WT animals or animals with cLH:WT,0.1560.02,3cells,1,125.3m m total dendritic length;and cLH,

0.1860.02,7cells,1,002.4m m total dendritic length;P .0.1,Student’s t -test.b ,Top,evoked EPSCs onto VTA-projecting LHb neurons of WT animals or animals with cLH in response to stimulus trains (20Hz or 50Hz).Bottom,plot of peak EPSCs normalized to first EPSC (WT,20Hz n 510,50Hz n 511;and cLH,20Hz n 514,50Hz n 513).Compared with WT animals,animals with cLH showed a faster synaptic depression (20Hz,F (9,198)52.32,P 50.02;50Hz,F (9,198)53.83,P ,0.001;one-way ANOVA with repeated measures)and more extensive synaptic depression (20Hz,F (1,22)56.62,P 50.02;50Hz,F (1,22)57.15,P 50.01;one-way ANOVA with repeated measures).

c ,Minimally evoke

d EPSCs onto VTA-projecting LHb neurons show mor

e failures in WT control animals than in animals with cLH (WT,0.560.1,n 57;cLH,0.260.1,n 59;***,P ,0.001,Student’s t -test).Mean amplitude o

f successful trials (right)(WT,23.262,n 57;cLH:24.862.5,n 59;P .0.6,Student’s t -test).a –c ,Error bars,s.e.m.

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in rats with aLH (17%),cLH (14%)and cLHms (20%)than in wild-type control rats (2%;P ,0.01comparing the wild-type control group with any other group,x 2test).

To determine whether the observed excitatory synaptic potentiation was quantitatively correlated to an animal’s helpless behaviour,we first tested animals (either wild type or with cLH)by using an escape avoidance task and subsequently prepared brain slices and made recordings from the VTA-projecting neurons.For each animal,we recorded from at least five cells and plotted the mean mEPSC fre-quency against the animal’s helpless behaviour (as measured by the fraction of trials in which the animal failed to escape from an escapable 10-s foot shock;see also Fig.2b).The significant correlation (R 250.69,F (1,11)524.85,P ,0.001,n 513for all animals;and R 250.64,F (1,6)510.7,P ,0.05,n 58for animals with cLH)(Fig.2c)indicates that the potentiation of excitatory transmission onto VTA-projecting LHb neurons is linked with an individual animal’s helpless behaviour.To examine the output of VTA-projecting neurons,we measured their spontaneous action potentials,which were more frequent in animals with cLH than in wild-type controls (Fig.2d).We observed no differences among the various groups with respect to the amplitude of mEPSCs (Fig.1e)or the frequency or amplitude of miniature inhibi-tory postsynaptic currents (Supplementary Fig.4b,c).These results indicate that the excitatory synaptic input onto LHb neurons that project to the VTA is potentiated in the learned helplessness model.

The enhanced mEPSC frequency could result from an increase in either the number of synapses or the probability of presynaptic neuro-transmitter release.To distinguish between these possibilities,we first measured the density of dendritic spines,which are the sites of excitatory synapses,on the dendrites of VTA-projecting LHb neurons.There was no significant difference in dendritic spine density between wild-type controls and animals with cLH (Fig.3a),and there was no obvious difference in the patterns of dendritic branching between the two groups (data not shown),suggesting that there was no major difference in the number of synapses between wild-type control animals and those with cLH.

To determine whether there is a change in the efficacy of presynaptic neurotransmitter release,we examined evoked transmission.Synaptic transmission onto LHb neurons (elicited by placing a stimulating electrode in the LHb)showed distinct properties:the evoked excitatory synaptic response had a very small NMDA (N -methyl-D -aspartate)receptor component (Supplementary Fig.4d),and the AMPA receptor component showed strong inward rectification (Supplementary Fig.4e).To probe presynaptic function,we evoked transmission with high-frequency stimulation trains (ten stimuli delivered at 20Hz or 50Hz).The decrease in the amplitude of EPSCs in response to suc-cessive pulses during a train of stimuli reflects presynaptic vesicle depletion;more depletion correlates with a higher release probability 17.VTA-projecting LHb neurons of animals with cLH showed a faster synaptic depression (at 20Hz F (9,198)52.32,P ,0.05;at 50Hz F (9,198)53.83,P ,0.001,one-way ANOVA with repeated measures)and a more extensive synaptic depression (at 20Hz F (1,22)56.62,P ,0.05;at 50Hz F (1,22)57.15,P ,0.05,one-way ANOVA with repeated measures)than those in wild-type control animals (Fig.3b).Furthermore,with minimal stimulation,which is designed to activate few synapses (as indicated by the amplitude of non-failure responses,which is similar to the mEPSC amplitude;Fig.3c),we measured syn-aptic transmission failure rate.Excitatory synaptic transmission onto VTA-projecting LHb neurons of animals with cLH had a significantly lower failure rate than that of wild-type control animals (Fig.

3c;

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Figure 4|DBS in the LHb suppresses excitatory synaptic transmission and reverses learned helplessness.a ,Left,example excitatory postsynaptic potentials (EPSPs)(paired pulses)recorded from a VTA-projecting LHb neuron before (1),during (2)and after (3)stimulation mimicking DBS.Arrows indicate when paired pulses were given.Right,mean EPSP slope at indicated time points before,during and after DBS:(1)before (first pulse 1.160.1,second pulse 0.960.1,n 56(4animals));(2)during (first pulse 0.1560.07,second pulse 0.0360.03,P ,0.001for both pulses compared with those in 1,Student’s t -test);and (3)after (first pulse 1.260.3,second pulse 0.960.2).b ,A schematic diagram showing the experimental procedures.c ,Number of lever presses (left)and test completion time (right)for animals that received DBS or sham stimulation in the LHb or DBS in the LPLR (lateral post-thalamic nuclei,laterorostral),before (baseline)or after DBS of different intensities.For DBS in the LHb (n 59),lever press baseline 1.260.4;150m A session 3.961;and 300m A session (n 58)5.862.For DBS in the LHb (n 59),test completion time baseline 19.960.4;150m A session 17.161;300m A session (n 58)15.462.For sham stimulation in the LHb (n 514),lever press baseline 1.860.5;150m A session 2.460.9;and 300m A session 2.461.For sham stimulation in the LHb,test completion time baseline 19.260.5;150m A session 18.760.9;and 300m A session 18.561.1.For DBS in the LPLR (n 57),lever press baseline 0.460.2;150m A session 261;and 300m A session 1.160.3.For DBS in the LPLR,test completion time baseline 20.660.2;150m A session 1961.2;and 300m A session 19.960.3.For DBS in the LHb group,*,P ,0.05compared with baseline (bootstrap).For the sham and DBS in LPLR groups,P .0.05for both measurements at both sessions compared with baseline (bootstrap).d ,Immobility during the forced swim test.For DBS,day 1,0.0660.01;day 2,0.0660.01;n 59.For sham,day 1,0.0560.01;day 2,0.1760.05;n 510.DBS versus sham on day 2,***,P ,0.001(bootstrap).e ,Representative cresyl violet staining of coronal brain sections after DBS in the LHb or LPLR.Arrows indicate the electrode track in the LHb (top)or LPLR (bottom).Dashed lines indicate the border of the habenula.Scale bars,1mm.a ,c ,d ,Error bars,s.e.m.

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n57–9,P,0.001,bootstrap method).These results indicate that the excitatory synaptic inputs onto VTA-projecting LHb neurons of help-less animals have a higher synaptic release probability;therefore, repeated stimulation can deplete synaptic vesicles faster and more efficiently in helpless animals.

One treatment for clinical depression that is currently under evalu-ation is deep brain stimulation(DBS).This therapy consists of con-tinuously delivered high-frequency electrical stimulation to various brain regions8,18.In a recent clinical case,DBS of the LHb produced a marked remission of treatment-resistant depression9.Notably,depres-sion recurred when DBS was stopped(in two accidental episodes9).To examine the cellular effects of DBS,we used brain slices and recorded synaptic transmission onto VTA-projecting LHb neurons,which was evoked by placing a stimulating electrode in the LHb.After a baseline period of evoked transmission,a DBS protocol used in patients(trains consisting of seven stimuli at130Hz followed by a40ms interval with no stimuli)was continuously delivered through the same stimulation electrode;stimuli were interleaved with the DBS trains,allowing us to monitor evoked synaptic transmission(see Methods for details).The DBS protocol produced a marked depression of excitatory synaptic transmission,which persisted for the DBS protocol period and was reversed on cessation of the DBS protocol(Fig.4a).Thus,a DBS pro-tocol can effectively reduce excitatory synaptic transmission onto VTA-projecting LHb neurons.

We wished to test whether reducing synaptic drive onto LHb neu-rons can modulate helpless behaviour.Remarkably,when the DBS protocol that was used in brain slices was delivered to the LHb in animals with aLH,it markedly ameliorated their helpless behaviour, as indicated by an increase in escape behaviour(Fig.4b,c and Supplementary Fig.5a).This effect was dependent on both the intensity of stimulation and the placement of the stimulation electrode:stimu-lating at300m A had a stronger behavioural effect and affected a larger volume within the LHb than stimulating at150m A(Fig.4b,c and Supplementary Fig.5b,c).In addition,only if the electrode was placed in the LHb,but not in the nearby thalamus,did DBS reverse the help-lessness(Fig.4b,c,e).Furthermore,DBS in the LHb,but not sham stimulation in the LHb,prevented the increase in immobility in the forced swim test(Fig.4d).Thus,suppression of synaptic transmission at the LHb through DBS can acutely reverse helpless behaviour in rats. Several changes in neural function have been identified in depressed humans and rodent models of depression,probably owing to the multi-faceted nature of depressive disorders19–23.The recent identification of the LHb as a brain region in monkeys that can encode disappointment and expectation of negative conditions2,3led us to investigate its role in the learned helplessness rodent model of depression.Our findings indicate that excitatory synaptic activity onto VTA-projecting neurons in the LHb may be a key modulator of learned helplessness.The two learned helplessness models examined showed potentiated excitatory synaptic activity onto these neurons.Interestingly,the major modifica-tion was an increase in the proportion of cells showing high-frequency mEPSCs(from2%to14–20%)in animals with learned helplessness. This finding suggests that large changes in a small proportion of cells in the LHb may be capable of modifying an animal’s behaviour.A crucial role for transmission onto LHb neurons is further supported by the strong correlation between the potentiation of synaptic transmission onto VTA-projecting LHb neurons and an individual animal’s helpless behaviour.Given the presynaptic nature of synaptic potentiation,we examined the effects of synaptic depression by repeated afferent stimu-lation,a protocol that mimics clinically used DBS.Reducing synaptic transmission onto LHb neurons through a DBS protocol led to acute reversal of learned helplessness.Suppression of transmission onto VTA-projecting LHb neurons probably had a role in mediating this beneficial effect,although modulation of LHb neurons,or axons of passage,projecting to other targets may also be involved.

Our study provides cellular mechanisms that may explain previ-ously reported phenomena:the increase in LHb metabolic activity observed in humans who are depressed24,25and in animal models of depression26,27;and that lesion28,29or pharmacological silencing30of the LHb can modulate depression-like symptoms in animal models.Our findings suggest an aberrant cellular process that has not previously been examined in the context of mood disorders and that may be crucial in the aetiology of depression.Future studies aimed at deter-mining the changes in molecular signalling that underlie the synaptic hyperactivity onto LHb neurons may lead to novel and effective treat-ments able to reverse some forms of depressive disorders. METHODS SUMMARY

Standard surgical procedures were followed for the in vivo injection of retrograde tracers.Rats with cLH were bred as described previously11,12.To prepare animals with aLH,rats were exposed to a learned helplessness‘training session’after in vivo injection of retrograde tracers into the VTA.This session consisted of inescapable, uncontrollable electric foot shocks,with random shock duration and unpredictable inter-shock intervals.Control animals were placed in the shocking chamber in parallel,without being shocked.To prepare the cLHms group,rats with cLH were treated with brief,escapable foot shock.Acute brain slices from the various groups were prepared for electrophysiological recordings.To evaluate learned helplessness behaviour,we used both a lever-pressing task and an active avoidance task.In the lever-pressing task,a lever was added to the shocking chamber in the testing session. Foot shook was terminated if the animal pressed the lever.The active avoidance task was performed in a shuttle box equipped with an electrical grid floor and a door separating the two halves.Foot shock was terminated if the animal crossed to the other side of the cage.For the forced swim test,the animal was forced to swim in a cylinder of water at25–26u C,and the animal’s immobility in the water was mea-sured.To test the effect of DBS on learned helplessness behaviour,rats were first trained and tested,and those that met the learned helplessness criteria were chosen for electrode implantation in the LHb.After recovery 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 effects of DBS on the forced swim test,DBS in the LHb was applied before the second day of swimming to determine its effect on the animal’s immobility.

Full Methods and any associated references are available in the online version of the paper at https://www.sodocs.net/doc/d613156991.html,/nature.

Received4October2009;accepted7December2010.

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Supplementary Information is linked to the online version of the paper at

https://www.sodocs.net/doc/d613156991.html,/nature.

Acknowledgements We thank K.Deisseroth for help and suggestions,A.Gifford and A.Biegon for sharing equipment and laboratory space,and members of the Malinow Lab and Li Lab for discussions.This study was supported by the Dana Foundation(B.L.), the Biobehavioral Research Awards for Innovative New Scientists(BRAINS)from the National Institute of Mental Health,National Institutes of Health(1R01MH091903-01) (B.L.)and the Shiley-Marcos Endowment(R.M.).

Author Contributions B.L.,J.P.,M.M.and C.C contributed 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 manuscript. Author Information Reprints and permissions information is available at

https://www.sodocs.net/doc/d613156991.html,/reprints.The authors declare no competing financial interests. Readers are welcome to comment on the online version of this article at

https://www.sodocs.net/doc/d613156991.html,/nature.Correspondence and requests for materials should be addressed to B.L.(bli@https://www.sodocs.net/doc/d613156991.html,)or R.M.(rmalinow@https://www.sodocs.net/doc/d613156991.html,).

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24F E B R U A R Y2011|V O L470|N A T U R E|539

METHODS

Animals.Wild-type,male Sprague Dawley rats were purchased from Taconic Farms and allowed to acclimatize to the animal facility for1–2weeks before experiments were carried out.The cLH rats were bred as described11,12.The rats were housed under a12-h light–dark cycle(7a.m.to7p.m.light),with food and water freely available.All procedures 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-projecting LHb neurons in vivo.Standard surgical procedures were followed for in vivo injection31.To label the VTA-projecting LHb neurons,we injected in vivo Alexa-Fluor-488-conjugated cholera toxin(2m g m l21; Molecular Probes)or a herpes simplex virus expressing enhanced GFP(HSV–GFP,NeuroVex),both of which are retrograde tracers,into the VTA. Animals were anaesthetized with isoflurane(Baxter)using an isoflurane vaporizer (Paragon Medical)and positioned in a stereotaxic apparatus that was connected to a computer system with a digital rat brain atlas(Angle Two Stereotaxic System, https://www.sodocs.net/doc/d613156991.html,).Injections of tracer solutions(3–5injection sites along the vertical axis,100–200nl per injection)were delivered with a glass micropipette through a skull window(2–3mm2)by pressure application(5–12psi,controlled by a Picrospritzer II;General Valve).The injections were performed within the following stereotaxic coordinates:25.3mm from bregma;0.96mm lateral from the midline;and8–8.4mm vertical from the cortical surface.Rats were injected subcutaneously with5mg kg21carprofen(an NSAID)after surgery.During pro-cedures,animals were kept on a heating pad and were brought back to their home cages after regaining movement.We waited2–3days to allow the retrograde labelling of neurons in the LHb before we killed the animals for experiments. Preparation of acute brain slices and electrophysiology.Male rats of40–50days of age were used for all of the electrophysiology experiments.Animals were anaes-thetized with isoflurane,decapitated and their brains quickly removed and chilled in ice-cold dissection buffer(110.0mM choline chloride,25.0mM NaHCO3, 1.25mM NaH2PO4,2.5mM KCl,0.5mM CaCl2,7.0mM MgCl2,25.0mM glu-cose,11.6mM ascorbic acid and3.1mM pyruvic acid,gassed with95%O2and5% CO2).Sagittal slices(400m m)across the LHb were cut in dissection buffer,by using a VT1000S vibratome(Leica),and subsequently transferred to a storage chamber containing artificial cerebrospinal fluid(ACSF)(118mM NaCl,2.5mM KCl,26.2mM NaHCO3,1mM NaH2PO4,20mM glucose,4mM MgCl2and 4mM CaCl2,at22–25u C,pH7.4,gassed with95%O2and5%CO2).After at least 1h recovery time,slices were transferred to the recording chamber and were constantly perfused with ACSF maintained at27u C.

Experiments were always performed on interleaved wild-type control and cLH or aLH animals.About three-quarters of the experiments were carried out blinded to the experimental group.These showed the same results as the non-blinded experi-ments,and the data were combined.Whole-cell patch-clamp recordings were obtained with Axopatch-1D amplifiers(Axon Instruments)onto neurons in the LHb under visual guidance using transmitted light illumination.For evoked EPSCs, synaptic transmission was evoked with a bipolar stimulating electrode placed close to the stria medullaris,typically.0.2mm away from cell bodies.Responses were recorded at holding potentials of260mV(for AMPA-receptor-mediated res-ponses)and140mV(for detection of any NMDA-receptor-mediated responses and measurement of rectification).NMDA-receptor-mediated responses were quantified as the mean current between110ms and160ms after stimulation. Bathing solution(ACSF)contained119mM NaCl,2.5mM KCl,2mM CaCl2, 1mM MgCl2,26.2mM NaHCO3,1mM NaH2PO4,11mM glucose,and0.1mM picrotoxin,gassed with5%CO2and95%O2,at27u C(unless otherwise noted). Internal solution for voltage-clamp experiments contained115mM caesium methanesulphonate,20mM CsCl,10mM HEPES,2.5mM MgCl2,4mM Na2-ATP,0.4mM Na-GTP,10mM Na-phosphocreatine and0.6mM EGTA(pH7.2). Spermine(100m M)was included in the internal solution for measurement of rec-tification.mEPSCs were recorded at27u C in the presence of1m M tetrodotoxin (TTX)and100m M picrotoxin in sagittal slices and analysed using Mini Analysis Program(Synaptosoft).To isolate miniature inhibitory spontaneous responses (mIPSCs),1m M TTX,100m M APV(D-(2)-2-amino-5-phosphonopentanoic acid) and3m M NBQX were added.

For the experiments in which high-frequency stimulation trains were used to determine presynaptic release probability,QX314(5mM)was included in the internal solution to prevent the generation of sodium spikes.To recruit the maximal number of axon terminals that can be stimulated by the high-frequency trains, thereby minimizing the effects of axonal failures and reducing the variability in responses,a low concentration(100nM)of NBQX was included in the bath ACSF. This allowed stimulation at a higher intensity without evoking large EPSCs that could activate voltage-dependent conductances.For experiments testing the effects of DBS on synaptic transmission onto VTA-projecting LHb neurons,evoked EPSCs were monitored before,during and after a stimulation protocol mimicking clinical DBS.Stimulation consisted of episodes of44trains of stimuli separated by 40ms.During each train,seven stimuli were applied at a frequency of about130Hz. The inter-episode interval was200ms,during which two stimuli separated by50ms were applied to monitor the amplitude and slope of the EPSP.The DBS protocol and the paired-pulse stimulation were delivered using the same electrode.

Two-photon imaging of dendritic spines.Image acquisition and analysis were described previously32,33.Images were acquired on a custom built dual channel two-photon laser scanning microscope(based on the Olympus FluoView laser scanning microscope)using a Ti:Sapphire Chameleon laser(Coherent),which was mode locked to910nm.Full three-dimensional(3D)image stacks were acquired using a3600.9NA objective lens at35digital zoom(FluoView software, Olympus),70nm per pixel.Each image plane was resampled three times and spaced0.5m m in the z dimension.

Behavioural paradigms.Methods for the learned helplessness paradigm have been optimized previously34.To prepare animals with learned helplessness(the aLH group)for behavioural testing and electrophysiological recording,rats were exposed to a learned helplessness‘training session’5days after in vivo injection of retrograde tracers into the VTA.This session consisted of120inescapable,uncon-trollable electric foot shocks at0.8mA over40min in the shocking chambers (Coulbourn Instruments;chambers were12inches wide310inches deep312 inches high and were controlled by precision adjustable shockers),with random shock duration ranging from5s to15s and unpredictable inter-shock intervals (ITIs).Experiments were performed on pairs of littermates housed in the same cage.Control animals were placed in the shocking chamber in parallel for40min, without being shocked.Electrophysiological recordings on acute brain slices were performed24–48h after shocking.Animal identity was coded for blinding the researcher with respect to treatment.To prepare cLH animals exposed to mild stress(the cLHms group),cLH rats were treated with a procedure essentially the same as the active avoidance task(see below),during which animals received an average of152627s of escapable foot shock(n58),and acute brain slices were prepared after2h.

To evaluate learned helplessness behaviour,we used both a lever-pressing task and an active avoidance task.The lever-pressing task was described previously34. Briefly,an illuminated lever was added to the shocking chamber in the testing session,which comprised15escapable foot shocks lasting up to60s(shorter if terminated by a lever press)over21min,and with fixed ITIs of24s.The active avoidance task was performed in a shuttle box(20inches wide310inches deep3 12inches high;Coulbourn Instruments)equipped with an electrical grid floor,a door separating the two halves,and photocell detectors.The shuttle box was placed in a sound-attenuating chamber to minimize external stimuli.Testing was fully automated using Graphic State software(Coulbourn Instruments).Animals were allowed to explore the shuttle box for5min,and helpless behaviour was evaluated over30trials of unexpected and escapable foot shock(1.2mA intensity,10s duration,with random ITIs of24612s)following a5-s cue tone.Foot shock was terminated if the animal completely crossed to the other side of the cage. When an animal crossed the cage during the5-s cue tone presentation,avoidance was scored.If an animal crossed during the10-s shocks,the mean escape latency was measured.Failure was recorded if no crossing was made during the10-s shock. For the forced swim test,animals were forced to swim for5min in a cylinder of water(water temperature was25–26u C;the cylinder was30cm in diameter and 40cm high;the depth of the water was set to prevent animals from touching the bottom with their hind limbs).Animal behaviour was videotaped using a PC6EX3 infrared camera(SuperCircuits).The immobile time each animal spent during the test was manually counted 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 24h later,a‘testing’session,as described above.On the basis of the test results, animals that met the criteria(those that pressed the lever only0–5times,and took between16and21min to finish the test)were used to test the effects of DBS.For increased stringency,only lever presses occurring within the first20s of shock onset were counted.Fifty two male Sprague Dawley rats were trained and tested, and26of these animals met the criteria.Three days later,standard surgical procedures were used to implant bipolar concentric electrodes(8mm long, 0.8mm tip diameter;Plastics One)unilaterally into the LHb(coordinates 23.7mm AP,60.7mm ML and25.4mm DV)in rats that met the criteria. After3–5days recovery from surgery,rats underwent a training session fol-lowed by a‘baseline’learned helplessness test.Three animals,in which the elec-trodes were implanted into the LHb,were excluded from further study because their performance did not meet the criteria during the baseline test.Immediately following the baseline test,DBS(seven stimulus trains of130Hz,separated by 40ms intervals;150m A intensity)in the LHb or the thalamus,or no stimulation (sham),was applied for1h.Twenty-four hours later,another1-h session of DBS

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or sham stimulation was given immediately before and during the learned help-lessness test.DBS intensity was150m A.Another24h later,the final1-h session of DBS(or sham stimulation)was given at a higher intensity(300m A),immediately before and during the final learned helplessness test(see Fig.4b for a schematic diagram showing the experimental procedures).Only animals with the electrode correctly placed in the LHb or thalamus(LPLR)were included for the respective behavioural analysis.

To test the effects of DBS on the forced swim test,electrodes were implanted in the same way as described above,except 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 forced swim test:DBS n59and sham n510).Immobility time was recorded during the first5min of a15-min swimming session on day1.DBS(150m A)or sham stimulation was applied for1h following the forced swim test on day1. Twenty-four hours later(day2),another1h of DBS(150m A)or sham stimulation was applied,and immobility time was recorded during the5-min swimming session.

To determine the volume of tissue affected by DBS in the LHb,in a separate set of experiments,animals were perfused with4%PFA2h after the onset of DBS,and brains were processed for immunohistochemistry to examine Fos expression. Immunohistochemistry.Immunohistochemistry experiments were performed following standard procedures on50-m m brain sections fixed with4%PFA.The antibodies used were anti-NeuN antibody(Chemicon),anti-EAAC1antibody (Chemicon),anti-GABA antibody(Sigma),anti-GAD67antibody(Chemicon) and anti-Fos antibody(Santa Cruz Biotechnology).After finishing the immuno-histochemistry process,images were taken using either an LSM510confocal microscope(Zeiss;for double labelling with two colours)or a BX41histology microscope(Olympus;for single labelling with one colour),using320objectives. Statistics and data presentation.To compare the means of non-normally dis-tributed data sets,we used a bootstrap procedure.Two data sets(N and M of size n and m)were randomly sampled n and m times,respectively,allowing resampling, and means(N i and M i)were generated.This procedure was repeated10,000times. If N j was more than M j fewer than5%of the times,then the probability that N is more than M was estimated to be less than0.05.Similar calculations established probabilities less than0.01.All other statistical tests are indicated when used.All data are presented as mean6s.e.m.

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underlying a form of associative learning.Science308,83–88(2005).

32.Kopec,C.D.et al.Glutamate receptor exocytosis and spine enlargement during

chemically induced long-term potentiation.J.Neurosci.26,2000–2009(2006).

33.Kopec,C.D.,Real,E.,Kessels,H.W.&Malinow,R.GluR1links structural and

functional plasticity at excitatory synapses.J.Neurosci.27,13706–13718(2007).

34.Vollmayr,B.&Henn,F.A.Learned helplessness in the rat:improvements in validity

and reliability.Brain Res.Brain Res.Protoc.8,1–7(2001).

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