Ventral tegmental area GABA neurons mediate stress-induced … · GABA neurons (Fig. 3b) 39. Restraint stress reduced the firing rates of both VTA DA and . 137. GABA neurons (Extended

Ventral tegmental area GABA neurons mediate stress-induced anhedonia. 1

Daniel C. Lowes1, Linda A. Chamberlin1, Lisa N. Kretsge1, Emma S. Holt1, Atheir I. 2 Abbas3, Alan J. Park1,2,4, Lyubov Yusufova1, Zachary H. Bretton1, Ayesha Firdous1, 3 Joshua A. Gordon5, and Alexander Z. Harris 1,2 4

Affiliations: 5 1. Department of Psychiatry, Columbia University, College of Physicians and Surgeons, 6 New York, New York 10032; 7 8 2. Division of Systems Neuroscience, New York State Psychiatric Institute, New York, 9 New York 10032 10 11 3. VA Portland Health Care System, Department of Behavioral Neuroscience and 12 Department of Psychiatry, Oregon Health & Science University, Portland, OR, 97239 13 14 4. The Mortimer B. Zuckerman Mind Brain Behavior Institute at Columbia University, 15 New York, New York 10032 16 17 5. National Institute of Mental Health, Bethesda, MD 20892 18 19 Correspondence to: 20 21 Alexander Z. Harris 22 Department of Psychiatry 23 Columbia University 24 1051 Riverside Drive, Unit 87 25 Kolb Annex Room 140 26 New York, NY 27 10032 28 Phone: (646) 774-7116, E-mail: [email protected] 29

30

.CC-BY-NC-ND 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (whichthis version posted May 31, 2020. . https://doi.org/10.1101/2020.05.28.121905doi: bioRxiv preprint

mailto:[email protected]

mailto:[email protected]

https://doi.org/10.1101/2020.05.28.121905

http://creativecommons.org/licenses/by-nc-nd/4.0/

Abstract 31

Stressful experiences frequently precede depressive episodes1. Depression results in 32

anhedonia, or disrupted reward-seeking, in most patients2. In humans3,4 and rodents5,6, 33

stress can disrupt reward-seeking, providing a potential mechanism by which stress can 34

precipitate depression7-9. Yet despite decades investigating how stress modulates 35

dopamine neuron transmission between the ventral tegmental area (VTA) and nucleus 36

accumbens (NAc), the underpinnings of the stress-anhedonia transition remain elusive10-37

13. Here we show that during restraint stress, VTA GABA neurons drive low frequency 38

NAc LFP oscillations, rhythmically modulating NAc firing rates. The strength of these 39

stress-induced NAc oscillations predict the degree of impaired reward-seeking upon 40

release from restraint. Inhibiting VTA GABA neurons disrupts stress-induced NAc 41

oscillations and reverses the effect of stress on reward-seeking. By contrast, mimicking 42

these oscillations with rhythmic VTA GABA stimulation in the absence of stress blunts 43

subsequent reward-seeking. These experiments demonstrate that VTA GABA inputs to 44

the NAc are both necessary and sufficient for stress-induced decreases in reward seeking 45

behavior, elucidating a key circuit-level mechanism underlying stress-induced anhedonia. 46

Main 47

Most patients with major depressive disorder have substantial reward processing 48

impairments14-16. Existing therapies incompletely treat anhedonia17-23 and its presence 49

strongly predicts clinical outcome24,25. Neural activity in the ventral tegmental area (VTA) 50

and nucleus accumbens (NAc) are key to reward processing26-29. Yet despite 40 years of 51

research into the dopamine (DA) anhedonia hypothesis10, we do not understand how 52

stress disrupts the different aspects of reward processing and its underlying neural 53


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circuitry. By contrast, VTA GABA neurons are well situated to mediate the impact of stress 54

on reward-seeking7. These neurons fire in response to aversive stimuli26 and play a 55

crucial role in calculating reward prediction27. They are increasingly recognized to play 56

important roles in stress-mediated addiction behavior30, yet previous studies investigating 57

the role of VTA-NAc on stress-induced reward-seeking deficits focused on dopaminergic 58

signaling12,13,31. Here, we tested whether VTA GABA projections to the NAc mediate 59

stress-induced anhedonia. 60

To determine how stress impacts reward processing, we trained mice to collect 61

rewards after hearing a reward-predicting cue (CS+). After a 1.5 second delay from CS+ 62

onset, reward was available for 5 seconds. After reaching stable performance, we 63

recorded VTA-NAc reward circuit activity in mice over two days as they either explored a 64

familiar environment or underwent acute restraint stress in a counterbalanced order (Fig. 65

1a). A prominent low frequency (2-7 Hz) oscillation of local field potential (LFP) activity 66

emerged in the NAc (Fig. 1b,c; Extended Fig. 1a) during the restraint stress. A restraint-67

induced oscillation also appeared in other brain regions, including the prefrontal cortex, 68

where stress has previously been reported to induce low-frequency oscillations32,33. 69

However, simultaneous recordings revealed that the restraint-induced oscillation was 70

largest in the NAc (Extended Fig. 1b,c). The magnitude of the restraint-induced 71

oscillation did not differ between the core and shell of the NAc (Extended Fig. 1d). This 72

restraint-induced oscillation straddles the hippocampal theta frequency range (4-12 Hz), 73

yet simultaneous recordings in the hippocampus and NAc revealed that the restraint-74

induced oscillation was distinct from hippocampal theta (Extended Fig. 1e). We did not 75

observe similar oscillations during periods of voluntary immobility in the familiar 76


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environment, suggesting that this neural activity reflects restraint, rather than decreased 77

movement (Extended Fig. 1f). As further evidence that stress induces this oscillation, it 78

persisted at the same magnitude throughout the restraint session and only abated when 79

the mice were released from restraint (Extended Fig. 1g). Moreover, exposure to another 80

stressor (a mouse aggressor in socially defeated mice) induced a similar low-frequency 81

oscillation in the NAc (Extended Fig. 1h). These data support rhythmic low-frequency 82

LFP activity in the NAc as a novel electrophysiological biomarker of acute stress. 83

Immediately after undergoing 30 minutes of restraint stress, freely moving mice 84

showed increased latency to retrieve rewards and decreased anticipatory (delay period) 85

licking relative to mice exposed to a neutral environment for an equivalent period (Fig. 86

1d,e). This decreased reward anticipation returned to control levels when measured the 87

following day (Extended Fig. 1i). Stress did not significantly alter lick rates during reward 88

consumption (Fig. 1f), nor did it decrease general mouse movement (Extended Fig. 1j) 89

or non-reward predicting cue (CS-) associated behavior (Extended Fig. 1k), suggesting 90

a selective deficit in reward anticipation. Notably, the magnitude of stress-induced 91

oscillations correlated with the degree of reward-seeking impairment (Fig. 1g,h), 92

suggesting that this biomarker reflects the circuit activity by which stress disrupts reward-93

seeking. 94

Low frequency oscillations typically represent the collective synchronous activity 95

of subthreshold synaptic inputs to a structure34. Such input would be expected to 96

modulate NAc neural firing rates. Indeed, restraint stress also robustly affected NAc single 97

unit activity. The firing rates of approximately 75% of NAc single units were significantly 98

modulated by restraint. Most significantly modulated neurons had reduced activity, while 99


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a minority had increased firing rates (Fig. 2a,b). On average, single unit firing rates 100

became rhythmically entrained to the restraint-induced oscillation (Extended Fig. 2a,b). 101

By contrast, prefrontal cortex single units did not phase-lock to the oscillation recorded in 102

the prefrontal cortex (Extended Fig. 2c), suggesting that the oscillation originates in the 103

NAc and may be volume conducted to other brain regions. Interestingly, while NAc single 104

units with significantly decreased firing rates showed an increase in phase locking, those 105

with significantly increased firing rates did not, suggesting that the stress-induced 106

oscillation reflects a net inhibitory input to the NAc (Fig. 2c,d). Since the magnitude of the 107

oscillation correlates with the degree of impaired reward seeking (Fig. 1g,h), these results 108

raise the possibility that an inhibitory input to the NAc may mediate stress-induced reward 109

impairments. 110

We hypothesized that since the VTA projects densely to the NAc and provides 111

GABAergic in addition to dopaminergic input, it could be the source of stress-induced 112

oscillations. Consistent with this hypothesis, coherence between VTA and NAc LFPs 113

increased sharply during restraint stress (Fig. 2e) in the same frequency range as the 114

restraint-induced oscillation. This increase in VTA-NAc coherence was significantly 115

greater than any changes in NAc coherence with the prefrontal cortex, amygdala, or 116

hippocampus (Extended Fig. 2d). Since the VTA and NAc reciprocally communicate, we 117

inferred directionality by examining the lag at which the phase locking of VTA multiunit 118

activity (MUA) to the NAc restraint-induced oscillation peaks35,36. In the familiar 119

environment, VTA MUA activity of the future was best phase-locked to the NAc LFP. By 120

contrast, during restraint stress, VTA MUA activity of the past was best phase-locked to 121


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the NAc LFP. These results suggest that during restraint stress the predominant 122

directionality of reward circuit activity is from the VTA to the NAc (Fig. 2f). 123

We next examined the role of VTA inputs in driving the restraint-induced NAc 124

oscillation. We infused the GABAA agonist muscimol (0.5 µg/side) or saline control into 125

the VTA while recording NAc LFP activity in restrained mice. Muscimol treatment 126

significantly reduced the restraint-induced oscillation, suggesting that VTA activity is 127

necessary for NAc restraint-induced activity (Fig. 3a). While recent work has proposed 128

that respiration-driven oscillations originating in the olfactory bulbs and piriform cortex 129

represent the true source of NAc oscillations37,38, these results and our data showing that 130

the oscillation modulates NAc single unit activity (Fig. 2c,d), indicate that restraint-131

induced oscillations reflect a true NAc oscillation. 132

To assess which VTA cell types provide input to the NAc during restraint stress, 133

we injected Cre-dependent adeno-associated virus (AAV) carrying archaerhodopsin 134

(Arch) into the VTA of Dat-ires-Cre and Vgat-ires-Cre mice to respectively tag DA and 135

GABA neurons (Fig. 3b)39. Restraint stress reduced the firing rates of both VTA DA and 136

GABA neurons (Extended Fig. 3a-c). However, the lag of peak phase-locking of VTA 137

GABA neural firing was shifted to the past during restraint, indicating that VTA GABA 138

neuron activity precedes the NAc restraint-induced oscillation (Fig. 3c). In contrast, the 139

lag of peak phase-locking of VTA DA neural firing did not suggest a particular directionality 140

and did not change with restraint (Fig. 3d). Based on these findings, we hypothesized 141

that VTA GABA, but not DA, neurons induce rhythmic NAc activity during restraint stress. 142

To test this hypothesis, we inhibited VTA DA or GABA neurons with archaerhodopsin and 143

compared the power of NAc restraint-induced oscillations. Only inhibiting VTA GABA 144


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neurons reduced NAc restraint-induced oscillations (Extended Fig. 3d-f). We observed 145

that Arch inhibition of VTA GABA neurons waned over the course of 1 minute, while 146

halorhodopsin (eNpHR) robustly inhibited VTA GABA neurons for 30 minutes (Extended 147

Fig. 3g,h). Thus, to determine the role VTA GABA mediated NAc oscillations play in 148

stress-induced anhedonia, we inhibited VTA GABA neurons throughout restraint stress 149

using eNpHR. As expected, prolonged inhibition of VTA GABA neurons during restraint 150

reduced low frequency NAc oscillations (Fig. 3e), but inhibiting VTA GABA neurons in the 151

familiar environment or with control virus had no impact on NAc oscillations (Extended 152

Fig. 3f,4a). Critically, inhibiting VTA GABA neurons during restraint reversed subsequent 153

reward-seeking deficits, decreasing latency to lick for reward and increasing anticipatory 154

licking (Fig. 3f; Extended Fig. 4b), suggesting that the activity of VTA GABA neurons 155

during stress is necessary for subsequent disrupted reward-seeking. 156

To assess if VTA GABA neural activity is sufficient to induce a low frequency NAc 157

oscillation, we injected Cre-dependent AAV carrying channelrhodopsin-2 (ChR2) into the 158

VTA of Vgat-ires-Cre mice. Stimulating these VTA neurons at a low frequency (4 Hz) 159

drove the emergence of a low frequency oscillation in the NAc (Fig. 4a, Extended Fig. 160

5a). Next, we tested the impact of stimulating VTA GABA neurons on reward anticipation. 161

Thirty minutes of low frequency VTA GABA neuron stimulation in a familiar environment 162

increased reward latency and decreased anticipatory licking during the subsequent 163

reward seeking session (Fig. 4b, Extended Fig. 5b). By contrast, stimulating VTA GABA 164

neurons at 20 Hz did not produce an oscillation in the NAc, nor did it significantly blunt 165

reward-seeking (Extended Fig. 5c,d). These results indicate that VTA GABA neurons 166


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suffice to induce low frequency rhythmic NAc activity and to reduce subsequent reward-167

seeking, mimicking the effect of stress on NAc physiology and reward behavior. 168

However, it remained unclear whether direct GABAergic projections from VTA to 169

NAc sufficed to induce low frequency oscillations and blunt reward-seeking. Previous 170

studies reported that stimulating the terminals of NAc-projecting VTA GABA neurons does 171

not impact reward-seeking40-42. However, we speculated that robust, rhythmic stimulation 172

mimicking the activity seen during stress might be necessary to reduce reward 173

anticipation. We therefore injected the Cre-dependent retrogradely transported virus 174

rAAV2 carrying channelrhodopsin into the NAc of Vgat-ires-Cre mice. Stimulating the VTA 175

cell bodies of NAc-projecting GABA neurons at low frequencies for thirty minutes in a 176

familiar environment induced rhythmic NAc activity and blunted subsequent reward-177

seeking, with increased reward latency and decreased anticipatory licking (Fig. 4c,d), 178

indicating that this small VTA GABAergic sub-population can recapitulate both the 179

physiologic signature of restraint stress and its impact on reward-seeking. 180

The emergence of VTA GABA neurons as key mediators of stress-induced reward 181

circuit activity suggests a mechanism by which stress disrupts reward-seeking. The 182

orchestrated activity of VTA DA and GABA neurons underlies reward processing. Brief 183

phasic activity of VTA DA neurons in response to reward predicting cues encodes reward 184

anticipation, while prolonged cue evoked firing of VTA GABA neurons sets the level of 185

expectation, resulting in characteristic firing patterns for each cell type27 (Fig. 4e,f). We 186

reasoned that restraint stress would alter these firing patterns, causing decreased reward 187

anticipation. Indeed, after undergoing restraint mice showed enhanced GABA-like cue-188

evoked activity, while DA-like cue evoked activity remained intact (Fig. 4e,f). These data 189


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indicate that stress-induced low-frequency activity of VTA GABA neurons alters their 190

normal function even after stress has abated. Collectively, these data reveal a VTA-NAc 191

circuit mechanism by which stress can cause anhedonia. 192

Discussion 193

Previous efforts to link stress to anhedonia have yielded mixed results10-13. Here 194

we show that during acute restraint stress, VTA GABA neurons entrain rhythmic activity 195

in the NAc, producing a newly described 2-7 Hz NAc oscillation. Remarkably, the strength 196

of this oscillation predicts subsequent deficits in reward anticipation. Furthermore, we 197

establish that VTA GABA activity is both necessary and sufficient for stress-induced 198

reward-seeking deficits, as inhibiting VTA GABA neurons during stress reverses the 199

impact on reward-seeking, and mimicking stress-evoked activity in NAc-projecting VTA 200

GABA neurons depresses reward pursuit in unstressed mice. 201

Our study reveals the cellular mediators of a novel VTA-NAc restraint-induced 202

oscillation. Long-range oscillations form an important motif in neural circuit 203

communication, yet with the exception of theta generation in the medial septo-204

hippocampal circuit43, the cellular mediators of these oscillations remain obscure in most 205

circuits. We show that long-range GABA projections mediate restraint-induced 206

oscillations. NAc-projecting VTA GABA neurons may preferentially inhibit NAc cholinergic 207

interneurons44. These cholinergic interneurons represent less than 1% of NAc neurons45, 208

yet ramify broadly within the striatum46, inhibiting ~80% of NAc neurons47. As a result, 209

VTA GABA neuron rhythmic pacing of NAc cholinergic neurons could underlie the 210

widespread restraint-induced NAc LFP oscillation and single unit firing modulation. 211


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Lending further support to this hypothesis, selectively silencing NAc cholinergic 212

interneurons leads to decreased sucrose preference48. 213

While long-range oscillations are typically used to assay the activity of long-range 214

circuits49, restraint-induced oscillations seem to reflect the degree to which stress-induced 215

VTA GABA activity induces plastic changes in VTA-NAc circuitry that outlast the stressor 216

and result in impaired reward anticipation. Plasticity affecting VTA GABA neurons has 217

been implicated in the response to cocaine50 and stress7 and represents an attractive 218

therapeutic target. Our findings thus reveal a VTA GABA-NAc neural circuit which 219

underlies the transition from stress to anhedonia, a critical discovery for advancing 220

depression treatment. 221

222

Figure Legends 223

Figure 1. Restraint stress re-organizes the NAc LFP and impairs subsequent reward-224

seeking. 225

a. Experimental design. 226

b. Representative traces of the NAc LFP of a mouse freely exploring a familiar 227

environment (grey) or during restraint (red). Black traces are the 2-7 Hz filtered signal. 228

c. (Left) Average NAc LFP power spectra of mice exploring a familiar environment (black) 229

or during restraint stress (red). (Right) Peak power in the 2-7 Hz range was higher in 230

restrained mice compared to mice freely exploring a familiar environment (***P<0.001 231

Wilcoxon signed-rank test W=91, n=13 mice). Data are plotted as mean ± s.e.m. 232


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d. Latency to reward retrieval in the cued-reward task for mice exploring a familiar 233

environment (black) or experiencing restraint stress (red). Restraint stress increases the 234

time to first lick during the reward availability period (***P<0.001 Wilcoxon signed-rank 235

test, W=89, n=13 mice). Data are plotted as mean ± s.e.m. 236

e. Average anticipatory lick rate for mice exploring a familiar environment (black) or 237

experiencing restraint stress (red). Restraint stress decreases average anticipatory lick 238

rate between CS+ onset and reward availability (*P<0.05 paired t-test, t(12)=3.01, n=13 239

mice). Data are plotted as mean ± s.e.m. 240

f. Average consummatory lick rate for mice exploring a familiar environment (black) or 241

experiencing restraint stress (red). Restraint stress does not affect average lick rate 242

during reward consumption (P>0.05 paired t-test t(12)=0.935, n=13 mice). Data are 243

plotted as mean ± s.e.m. 244

g. Relationship between peak 2-7 Hz NAc power during restraint and the change in 245

latency to reward retrieval from after familiar environment exploration to after restraint 246

(*P<0.01, r(11)=0.73, n=13 mice) 247

h. Relationship between peak 2-7 Hz NAc power during restraint and the change in 248

anticipatory lick rate from after familiar environment exploration to after restraint (*P<0.05, 249

r(11)=-0.60, n=13 mice). 250

251

Figure 2. VTA neural activity leads NAc activity during restraint. 252


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a. (Left) Relationship between the firing rate of NAc single units during familiar 253

environment exploration and restraint stress, with line of equality for reference. Areas 254

above and below the line of equality are shaded green or purple to clarify whether firing 255

rate values increased or decreased (respectively) during restraint. (Right) Restraint 256

reduced the firing rate of NAc single units (**P<0.001 Wilcoxon signed-rank test W=-4537, 257

n=126 neurons). Data are plotted as mean ± s.e.m. 258

b. Distribution of NAc neurons excited, inhibited, and unmodulated by restraint (total 259

neurons n=126). 260

c. (Left) An example of the spike-phase relationship during familiar environment 261

exploration and restraint for a restraint-inhibited NAc neuron. Activity was uniformly 262

distributed across phase angles in the 2-7 Hz filtered NAc LFP during familiar 263

environment exploration (left), but phase-locked during restraint (right). (Centre) 264

Relationship between the pairwise phase consistency (PPC) of restraint-inhibited NAc 265

neurons during familiar environment exploration and restraint stress, with line of equality 266

for reference. Positive and negative differences are respectively shaded light red and light 267

orange for clarity. (Right) NAc neurons whose firing rates decreased during restraint 268

showed increased phase-locking to the 2-7 Hz filtered NAc LFP during restraint 269

(***P<0.001, Wilcoxon signed-rank test W=2267, n=76 neurons, below left). Data are 270


d. Same as c, but with NAc single units significantly excited by restraint. NAc neurons 272

whose firing rates increased during restraint did not increase phase locking. (P>0.05, 273

Wilcoxon signed-rank test W=26, n=20 neurons). Positive and negative differences are 274


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respectively shaded light red and light orange for clarity. Bar graph is plotted as mean ± 275

s.e.m. 276

e. (Left) Representative traces of the NAc and VTA LFPs of a mouse freely exploring a 277

familiar environment (grey) or during restraint (red). Black traces are the 2-7 Hz filtered 278

signal. (Centre) Coherence of VTA and NAc LFPs during familiar environment exploration 279

and restraint stress. (Right) Average VTA-NAc coherence in the 2-7 Hz range increases 280

during restraint stress (***P<0.001 paired t-test t(12)= -6.84, n=13 mice). Line and bar 281

graphs are plotted as mean ± s.e.m. 282

f. (Left) Lag analysis of VTA multi-unit activity during familiar environment exploration and 283

restraint stress. Restraint increased synchrony of NAc 2-7 Hz phase with past VTA MUA 284

activity (***P<0.001 Wilcoxon rank-sum test, nFamiliar=138 multi-units, nRestraint=139 multi-285

units). (Centre) Relationship between lag of max PPC between VTA MUA activity and the 286

2-7 Hz filtered NAc LFP during familiar environment exploration and restraint, with line of 287

equality for reference. Positive and negative differences are respectively shaded brown 288

and turquoise for clarity. (Right) During restraint VTA MUA activity reorganized from 289

predominantly lagging NAc phase activity to predominantly leading NAc phase activity 290

(***P<0.001 Wilcoxon signed-rank test, W=-3309, n=139 multi-units). Line and bar graphs 291

are plotted as mean ± s.e.m. 292

Figure 3. VTA GABA activity contributes to the restraint-induced NAc oscillation and 293

stress-induced reward seeking deficits. 294

a. (Left) Example NAc LFP power spectra following infusion of saline (red) or muscimol 295

(violet) into the VTA. (Right) Muscimol infusion reduced the maximum NAc LFP power in 296


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the 2-7 Hz range during restraint (**P<0.01 Wilcoxon signed-rank test W=-45, n=9 mice). 297

Bar graph is plotted as mean ± s.e.m. 298

b. (Top left) Experimental design. (Bottom left) Example raster plot of opto-tagged GABA 299

neuron. (Right) Example immunofluorescent image of Arch expression (green) in the VTA 300

of a Vgat-ires-Cre mouse. Tyrosine hydroxylase counterstain (red) shows expression in 301

non-dopamine neurons. Scale bar is 30 µm. 302

c. (Left) Relationship between lag of max PPC between tagged VTA GABA neurons and 303

the 2-7 Hz filtered NAc LFP during familiar environment exploration and restraint, with 304

line of equality for reference. Positive and negative differences are respectively shaded 305

brown and turquoise for clarity. (Right) During restraint the 2-7 Hz filtered NAc LFP 306

became significantly phase-locked with past VTA GABA neuron activity (*P<0.05 307

Wilcoxon signed-rank test W=-48, n=10 neurons). Data are plotted as mean ± s.e.m. 308

d. The same as c, but for tagged VTA dopamine neurons. There was no change in the 309

direction of phase-locking between the 2-7 Hz filtered NAc LFP and tagged VTA 310

dopamine neurons (P>0.05, paired t-test t(11)=0.754, n=12 neurons). Positive and 311

negative differences were respectively shaded brown and turquoise for clarity. Bar graph 312

is plotted as mean ± s.e.m. 313

e. (Left) Experimental design. (Centre) Representative spectrum of NAc LFP during 314

restraint in light off (black) and light on (green) sessions. (Right) Relationship between 2-315

7 Hz NAc peak power during light off and light on sessions, with line of equality for 316

reference. Positive and negative differences are respectively shaded light green and light 317

blue for clarity. (Far right) Prolonged eNpHR inhibition of VTA GABA neurons reduced 318


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the power of the NAc oscillation (*P<0.05 paired t-test t(6)=3.44, n=7 mice). Data are 319


f. (Left) Experimental design. (Right) Prolonged eNpHR inhibition of VTA GABA neurons 321

during restraint improved latency to retrieve rewards during the reward availability period 322

(*P<0.05 Wilcoxon signed-rank test W=-26, n=7 mice), as well as anticipatory lick rate 323

between CS+ onset and reward availability (*P<0.05 Wilcoxon signed-rank test W=28, 324

n=7 mice). Data are plotted as mean ± s.e.m. 325

Figure 4. Activation of NAc-projecting VTA GABA neurons produces a low-frequency 326

oscillation and impairs reward-seeking. 327

a. (Far left) Experimental design. (Centre left) Event-related potential of the NAc LFP, 328

centered on laser onset (average of n=6 mice, shaded area is s.e.m.). Bars indicate onset 329

and duration of laser pulse. (Centre) Representative NAc spectrum of familiar 330

environment exploration with no stimulation (black, Light off) and with rhythmic low-331

frequency stimulation (blue, ChR2). (Centre right) Relationship between max 2-7 Hz NAc 332

power during light off and light stimulation, with line of equality for reference. Positive and 333

negative differences are respectively shaded light green and light blue for clarity. (Far 334

right) ChR2 stimulation increased 2-7 Hz NAc peak power (*P<0.05 Wilcoxon signed-335

rank test W=21, n=6 mice). Data are plotted as mean ± s.e.m. 336

b. (Left) Experimental design. (Right) Rhythmic ChR2 stimulation before the cued-reward 337

task impaired latency to reward retrieval (*P<0.05 Wilcoxon signed-rank test W=21, n=6 338

mice), as well as anticipatory lick rate between CS+ onset and reward availability 339

(*P<0.05 Wilcoxon signed-rank test W=21, n=6 mice). Data are plotted as mean ± s.e.m. 340


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c. (Far left) Experimental design. (Centre left) Example immunofluorescent image of 341

retrograde ChR2 expression (green) in the VTA of a Vgat-ires-Cre mouse. Tyrosine 342

hydroxylase counterstain (red) shows expression in non-dopamine neurons. Scale bar is 343

30 µm. (Centre) Representative NAc spectrum of familiar environment exploration with 344

no stimulation (black, Light off) and with rhythmic low-frequency stimulation (blue, rAAV2). 345

(Centre right) Relationship between max 2-7 Hz NAc power during light off and light 346

stimulation, with line of equality for reference. Positive and negative differences are 347

respectively shaded light green and light blue for clarity. (Far right) Stimulation of NAc-348

projecting VTA GABA cell bodies increased 2-7 Hz NAc peak power (*P<0.05 paired t-349

test t(4)=-3.00, n=5 mice). Data are plotted as mean ± s.e.m. 350

d. (Left) Experimental design. (Right) Rhythmic VTA illumination before the cued-reward 351

task impaired latency to reward retrieval (**P<0.01 paired t-test test t(4)=-4.73, n=5 mice), 352

as well as anticipatory lick rate between CS+ onset and reward availability (**P<0.01 353

paired t-test t(4)=5.40, n=5 mice). Data are plotted as mean ± s.e.m. 354

e. (Top) Representative average CS+ evoked firing rates of putative dopamine and GABA 355

neurons in non-stressed (black) and stressed (red) conditions. (Bottom) Distribution of 356

average CS+ evoked firing rates for putative dopamine and putative GABA neurons 357

recorded during familiar environment and restraint stress conditions (total neurons 358

nFamiliar=155, nRestraint=153). Restraint did not affect the CS+ evoked firing rates of putative 359

dopamine neurons (P>0.05, Wilcoxon rank-sum test W=3144, nFamiliar=82 neurons, 360

nRestraint=81 neurons), but enhanced CS+ evoked firing of putative GABA neurons 361

(*P<0.05, Wilcoxon rank-sum test W=203, nFamiliar=22 neurons, nRestraint=29 neurons). 362


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f. Distribution of putative dopamine and putative GABA neurons recorded during familiar 363

environment and restraint stress conditions (total neurons nFamiliar=155, nRestraint=153). 364

Supplemental Figure 1. Characterization of restraint effects on brain-wide LFP 365

organization and reward behavior. 366

a. (Far left) Example NAc spectra from C57BL/6 strain mice. (Centre left) Distribution of 367

frequencies of peak power of restraint-induced oscillation for C57BL/6 mice (n=22 mice). 368

(Centre right) Example NAc spectra from 129/svev strain mice. (Far right) Distribution of 369

frequencies of peak power of restraint-induced oscillation for 129/svev mice (n=19 mice). 370

b. Box-and-whisker plot summarizing the percent change from familiar to restraint in peak 371

2-7 Hz power of LFP oscillations in the ventral hippocampus (vHPC), basolateral 372

amygdala (BLA), nucleus accumbens (NAc), prefrontal cortex (PFC), ventral tegmental 373

area (VTA) and dorsal hippocampus (dHPC). The percent change of the NAc oscillation 374

was larger than those in the vHPC, BLA, VTA, and dHPC (Kruskal-Wallis Χ2=29.1 375

P<0.0001, df=5; post-hoc Tukey-Kramer comparisons *P<0.05, **P<0.01, ***P<0.001, 376

nvHPC=19 mice, nBLA=12 mice, nNAc=10 mice, nPFC=15 mice, nVTA=14 mice, ndHPC=9 mice). 377

Data are plotted with box-and-whisker plots, which give the median, upper and lower 378

quartiles, and 1.5x interquartile range. 379

c. Simultaneous recordings of the restraint-induced oscillations in the PFC and the NAc 380

revealed that 2-7 Hz peak power was larger in the NAc (*P<0.05, Wilcoxon signed-rank 381

test W=71, n=13 mice). Data are plotted as mean ± s.e.m. 382


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d. 2-7 Hz power of the restraint oscillation did not differ between LFP wires targeted to 383

the core and shell of the NAc (P>0.05 Wilcoxon rank-sum test W=58, ncore=13 mice, 384

nshell=10 mice). Data are plotted as mean ± s.e.m. 385

e. Averaged LFP spectra comparing the restraint-induced NAc oscillation to theta 386

oscillations in the dHPC. The restraint-induced NAc oscillation occurs at a lower, distinct 387

frequency band from dHPC theta oscillations (nNAc=10 mice, ndHPC=9 mice). Data are 388


f. (Left) Averaged spectra during periods of immobility during familiar environment 390

exploration (black) and during restraint stress (red). (Right) 2-7 Hz peak NAc power is 391

larger during restraint stress than during immobile periods of familiar environment 392

exploration (***P<0.001 paired t-test t(21)=10.0, n=22 mice). Data are plotted as mean ± 393

s.e.m. 394

g. Averaged spectrogram of NAc LFP during a pre-restraint baseline, 30 minutes of 395

restraint stress, and immediately after release from restraint (n=11 mice). 396

h. (Left) Representative NAc spectrum of a post-social defeat stress-susceptible mouse 397

during exposure to an empty wire cup (black, Cup) or to a wire cup enclosing a CD1 398

mouse (red, CD1). (Right) Exposure to a CD1 increased 2-7 Hz NAc peak power in stress-399

susceptible mice (**P<0.01 Wilcoxon signed-rank test W=45, n=9 mice). Bar graph is 400


i. (Left) Latency to reward retrieval for mice exploring a familiar environment on Day 1 402

(grey) or Day 2 (white) of experiment. Restraint did not have lasting effects on latency to 403

reward retrieval (P>0.05 two-sample t-test; nDay1=8 mice, nDay2=5 mice). (Right) Average 404


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anticipatory lick rate for mice exploring a familiar environment on Day 1 (grey) or Day 2 405

(white) of experiment. Restraint did not have lasting effects on anticipatory lick rate 406

(P>0.05 two-sample t-test; nDay1=8 mice, nDay2=5 mice). Data are plotted as mean ± s.e.m. 407

j. CS+-evoked speed in the cued-reward task. Restraint did not impair mobility (P>0.05 408

paired t-test t(6)=1.53, n=8 mice). Data are plotted as mean ± s.e.m. 409

k. (Left) Average latency to lick during ‘reward’ phase of CS- trials. Restraint stress did 410

not impair lick latency on CS- trials (P>0.05 paired t-test t(12)=0.813, n=13 mice). (Right) 411

Average anticipatory lick rate during CS- trials. Stress did not affect anticipatory lick rate 412

on CS- trials (P>0.05 Wilcoxon signed-rank test W=-48, n=13 mice). Data are plotted as 413

mean ± s.e.m. 414

415

Supplemental Figure 2. Identification of the VTA as possible source of the NAc restraint 416

oscillation. 417

a. An example of an NAc neuron whose activity was uniformly distributed across phase 418

angles in the 2-7 Hz filtered NAc LFP during familiar environment exploration (left), but 419

phase-locked during restraint (right). Positive and negative differences are respectively 420

shaded light red and light orange for clarity. 421

b. (Left) Relationship between the PPC of all recorded NAc single units during familiar 422

environment exploration and restraint stress, with line of equality for reference. Positive 423

and negative differences are respectively shaded light red and light orange for clarity. 424

(Right) Overall there was an increase in phase-locking between NAc single units and the 425

2-7 Hz filtered NAc LFP during restraint (***P<0.001, Wilcoxon signed-rank test W=5017, 426


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n=126 neurons). Data are plotted with box-and-whisker plots, which give the median, 427

upper and lower quartiles, and 1.5x interquartile range. 428

c. (Left) Relationship between the PPC of PFC single units with the 2-7 Hz filtered NAc 429

LFP during familiar environment exploration and restraint stress, with line of equality for 430

reference. Positive and negative differences are respectively shaded light red and light 431

orange for clarity. (Right) Restraint had no effect on phase-locking strength between PFC 432

single units and the 2-7 Hz NAc LFP (P>0.05 Wilcoxon signed-rank test W=92, n=47 433

neurons). 434

d. Change in the average 2-7 Hz coherence of the NAc LFP with LFPs located in the 435

vHPC, BLA, PFC, VTA, and dHPC from familiar environment exploration to restraint. The 436

increase in VTA-NAc coherence was larger than the coherence change of all other 437

recorded brain regions (Kruskal-Wallis Χ2=32.0 P<0.0001, df=4; post-hoc Tukey-Kramer 438

comparisons ***P<0.001, *P<0.05; nVHPc=17 mice, nBLA=17 mice, nPFC=13 mice, nVTA=13 439

mice, ndHPC=7 mice). Data are plotted as mean ± s.e.m. 440

441

Supplemental Figure 3. eNpHR is capable of prolonged inhibition of restraint oscillation. 442

a. (Top left) Experimental design. (Top tight) Example immunofluorescent image of Arch 443

expression (green) in the VTA of a Dat-ires-Cre mouse. Tyrosine hydroxylase 444

counterstain (red) shows expression in dopamine neurons. (Bottom) Example raster plot 445

of opto-tagged dopamine neuron. 446

b. (Left) Relationship between the firing rate of opto-tagged VTA DA single units during 447

familiar environment exploration and restraint, with line of equality for reference. Positive 448


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and negative differences are respectively shaded light green and light purple for clarity. 449

(Right) Restraint inhibited VTA DA firing rate (***P<0.001 Wilcoxon sign-rank test W=-78, 450


c. (Left) Relationship between the firing rate of opto-tagged VTA GABA single units during 452

familiar environment exploration and restraint, with line of equality for reference. Positive 453

and negative differences are respectively shaded light green and light purple for clarity. 454

(Right) Restraint inhibited VTA GABA firing rate (*P<0.05 Wilcoxon sign-rank test W=-43, 455


d. (Far Left) Experimental design. (Centre left) Representative spectrum of NAc LFP 457

during restraint in light off (black) and light on (green) epochs. (Centre right) Relationship 458

between 2-7 Hz NAc peak power during light off and light on epochs, with line of equality 459

for reference. Positive and negative differences are respectively shaded light green and 460

light blue for clarity. (Far right) Arch inhibition of VTA dopamine neurons had no effect on 461

the maximum power of the NAc LFP (P>0.05, Wilcoxon signed-rank test W=-7, n=6 mice). 462

Data are plotted as mean ± s.e.m. 463

e. Same as d., but for VTA GABA inhibition. Arch inhibition of VTA GABA neurons 464

reduced the power of the NAc oscillation (*P<0.05 Wilcoxon signed-rank test W=-21, n=6 465

mice). Positive and negative differences were respectively shaded light green and light 466

blue for clarity. Bar graph is plotted as mean ± s.e.m. 467

f. (Far left) Experimental design. (Centre left) Representative spectrum of NAc LFP during 468

restraint in light off (black) and light on (green) epochs. (Centre right) Relationship 469

between 2-7 Hz NAc peak power during light off and light on epochs, with line of equality 470


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light blue for clarity. (Far right) Illumination of the VTA had no effect on the maximum 472

power of the NAc LFP (P>0.05 paired t-test t(3)=-2.57, n=4 mice). Bar graph is plotted as 473

mean ± s.e.m. 474

g. (Left) Example traces of efficacy of Arch and eNpHR inhibition of VTA GABA neurons 475

in decreasing the 2-7 Hz NAc oscillation during restraint. (Right) Efficacy of Arch and 476

eNpHR inhibition of VTA GABA neurons in decreasing the 2-7 Hz NAc oscillation during 477

different times in the light on period. Arch inhibition of VTA GABA neurons decreases the 478

2-7 Hz oscillation during the first 10 s of illumination (*Bonferroni adjusted P<0.05 one-479

sample t-test t(5)=5.21, n=6 mice) but not during the last 10 s of illumination (Bonferroni 480

adjusted P>0.05 one-sample t-test t(5)=3.06). By contrast, eNpHR inhibition of VTA 481

GABA neurons successfully decreases the 2-7 Hz restraint oscillation during the first 10 482

s of illumination (***Bonferroni adjusted P<0.001 one-sample t-test t(6)=8.72, n=7 mice), 483

50-60s after illumination onset (*Bonferroni adjusted P<0.05 one-sample t-test t(6)=4.18), 484

and 1740-1800 s after illumination onset (**Bonferroni adjusted P<0.01 one-sample t-test 485

t(6)=5.87). Bar graphs are plotted as mean ± s.e.m. 486

h. (Left) Example traces of the efficacy of Arch and eNpHR inhibition of VTA GABA 487

neurons in decreasing the firing rate of opto-tagged neurons during restraint. (Right) 488

Efficacy of Arch and eNpHR inhibition of VTA GABA neurons in decreasing opto-tagged 489

GABA neuron firing rates during different times in the light on period. Arch significantly 490

reduced the firing rates of tagged GABA neurons 50-60 s after illumination onset 491

(***Bonferroni adjusted P<0.001 one-sample t-test t(9)=10.3, n=10 neurons), but not 0-492

10 s after illumination onset (Bonferroni adjusted P>0.05 one-sample t-test t(9)=2.35, 493


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n=10 neurons). eNpHR successfully inhibited tagged VTA GABA neurons 0-10 s, 50-60 494

s, and 1740-1800 s after illumination onset (all ***Bonferroni adjusted P<0.001 one-495

sample t-test; respectively t(7)=15.7, t(7)=13.4, and t(7)=22.0; n=8 neurons). Bar graphs 496

are plotted as mean ± s.e.m. 497

i. (Left) Experimental design. (Centre) Example trace of effect of eNpHR inhibition on 2-498

7 Hz NAc peak power during familiar environment exploration. (Right) VTA GABA 499

inhibition during familiar environment exploration has no effect on peak 2-7 Hz NAc power 500

(P>0.05 paired t-test t(3)=-0.090, n=4 mice). Bar graphs are plotted as mean ± s.e.m. 501

Extended Figure 4. eYFP control for prolonged VTA illumination. 502

a. (Far left) Experimental design. (Centre left) Representative spectrum of NAc LFP 503

during restraint in light off (black) and light on (green) sessions. (Centre right) Relationship 504

between 2-7 Hz NAc peak power during light off and light on sessions, with line of equality 505


light blue for clarity. (Far right) Prolonged VTA illumination during restraint had no effect 507

on peak 2-7 Hz NAc power (P>0.05 paired t-test t(3)=1.62, n=4 mice). Bar graphs are 508


b. (Left) Experimental design. (Right) Prolonged VTA illumination during restraint had no 510

effect on latency to retrieve rewards during the reward availability period (P>0.05 paired 511

t-test t(3)=1.95, n=4 mice), or anticipatory lick rate between CS+ onset and reward 512

availability (P>0.05 paired t-test t(3)=-2.02, n=4 mice). Bar graphs are plotted as mean ± 513

s.e.m. 514


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Extended Figure 5. Rhythmic VTA illumination does not have non-specific effects on NAc 515

physiology or reward-seeking behavior. 516

a. (Far left) Experimental design. (Centre left) Representative NAc spectrum of familiar 517

environment exploration with no stimulation (black, Light off) and with rhythmic low-518

frequency illumination (blue, eYFP). (Centre right) Relationship between max 2-7 Hz NAc 519

power during light off and rhythmic light illumination, with line of equality for reference. 520

Positive and negative differences are respectively shaded light green and light blue for 521

clarity. (Far right) Rhythmic light illumination had no effect on peak 2-7 Hz NAc LFP power 522

(P>0.05 paired t-test t(3)= -0.633, n=4 mice). Bar graphs are plotted as mean ± s.e.m. 523

b. (Left) Experimental design. (Right) Rhythmic VTA illumination before the cued-reward 524

task had no effect on latency to retrieve rewards during the reward availability period 525

(P>0.05 paired t-test t(3)=0.518, n=4 mice), or anticipatory lick rate between CS+ onset 526

and reward availability (P>0.05 paired t-test t(3)=-0.982, n=4 mice). Data are plotted as 527

mean ± s.e.m. 528

c. (Far left) Experimental design. (Centre left) Representative NAc spectrum of familiar 529

environment exploration with no stimulation (black, Light off) and with rhythmic high-530

frequency stimulation (blue, 20 Hz). (Centre right) Relationship between max 2-7 Hz NAc 531

power during light off and rhythmic light illumination, with line of equality for reference. 532

Positive and negative differences are respectively shaded light green and light blue for 533

clarity. (Far right) 20 Hz stimulation did not affect 2-7 Hz NAc peak power (P>0.05 paired 534

t-test t(3)= -0.830, n=4 mice). Bar graphs are plotted as mean ± s.e.m. 535


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d. (Left) Experimental design. (Right) Rhythmic high-frequency VTA illumination before 536

the cued-reward task had no effect on latency to retrieve rewards during the reward 537

availability period (P>0.05 paired t-test t(3)=-2.56, n=4 mice), or anticipatory lick rate 538

between CS+ onset and reward availability (P>0.05 paired t-test t(3)=1.51, n=4 mice). 539

Data are plotted as mean ± s.e.m. 540

METHODS 541

Subjects. 4-6-month-old male and female Vgat-ires-Cre homozygous mice, Dat-ires-Cre 542

heterozygous mice, C57BL/6J mice, and 129/svev mice (The Jackson Laboratory, stock 543

numbers 028862, 006660, 0006664, and 002448 respectively) were used as 544

experimental subjects. Mice were housed with a standard 12 hour light-dark cycle and 545

were given ad libitum access to food and water (when not being water restricted for the 546

cued-reward task). 547

Surgical Procedures. Mice were anesthetized with 1%–3% vaporized isoflurane in 548

oxygen (1 L/min) and placed in a stereotaxic apparatus. Cre mice were injected with Cre-549

inducible Archaerhodopsin (AAV5-EF1a-DIO-eArch3.0-EYFP, UNC vector core), Cre-550

inducible Halorhodopsin (AAV5-Ef1a-DIO eNpHR 3.0-EYFP, Addgene), Cre-inducible 551

Channelrhodopsin-2 (AAV5-Ef1a-DIO hChR2(E123T/T159C)-EYFP, Addgene), or 552

control EYFP virus (AAV5-EF1a-DIO-EYFP, UNC vector core) at two locations within the 553

VTA bilaterally (+/-0.5 ML, -3.4 AP, 4.5 depth below brain surface; 0.5 µL virus per 554

injection. For projection-specific experiments, mice were injected with retrogradely 555

transported Cre-inducible Channelrhodopsin-2 (rAAV2-EF1a-DIO-hChR2(H134R)-556

EYFP-WPRE-HGHpA, Addgene) at eight locations within the NAc bilaterally (±1.8 mm 557

ML, 0.98 mm AP, -4.12 mm DV; ±0.9 mm ML, 1.5 mm AP, -4.0 mm DV; ±0.6 mm ML, 1.5 558


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mm AP, -3.4 mm DV; ±1.0 mm ML, 1.0 mm AP, -3.4 mm DV; 0.4 µL virus per injection). 559

For microdrive implantation experiments, animals underwent a second surgery to implant 560

the microdrive about 4 weeks after initial viral injection. Animals were again anesthetized 561

and placed in a stereotaxic apparatus. Craniotomies were made to allow for implantation 562

of a bundle of 15 stereotrodes (13 micron tungsten wire, California Fine Wire) and a 200 563

micron optical fiber in the left VTA (-0.5 ML, -3.4 AP, 4.45 below brain surface); a 200 564

micron optical fiber in the right VTA; a local field potential (LFP) wire (76 micron tungsten 565

wire, California Fine Wire) in the NAc (1.25 ML, 1.25 AP, 4.0 below brain surface); a 566

ground screw over the cerebellum; and a reference screw over the orbitofrontal cortex. 567

Electrodes were connected to a 32 channel electrode interface board (EIB-36 Narrow, 568

Neuralynx), and the board and wires were fixed to an advanceable custom microdrive. 569

Electrode placements were confirmed by passing current through an electrode at each 570

site (5 mA, 10 s) to generate an electrothermolytic lesion. Mice were anesthetized with 571

ketamine/xylazine prior to generating the lesions and were then perfused. The brains 572

were extracted, cryoprotected, sectioned, mounted, stained, and examined under a 573

microscope to determine lesion placements and characterize eYFP, Arch, eNpHR, and 574

ChR2 expression in VTA. 575

Optogenetics. 561 nm wavelength light (Opto Engine, LLC), 532 nm wavelength light 576

(OEM laser) or 473 nm wavelength (LaserGlow Technologies) light was delivered at 10 577

mW via 200 mm diameter, 0.22 NA optical fibers. For Arch experiments, 532 nm light was 578

delivered in 60 second ON/60 second OFF epochs as mice explored a familiar 579

environment and while mice were restrained. For eNpHR experiments, 532 nm or 561 nm 580

light was delivered constantly for 30 minutes while mice were restrained. In addition, 532 581


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nm or 561 nm light was delivered 1 second ON/10 second OFF as mice explored a 582

familiar environment for optogenetic tagging. For ChR2 experiments, mice either received 583

no illumination or 4 Hz stimulation (10 ms pulse width) while exploring a familiar 584

environment. For both stimulation and non-stimulation days, mice received 50 intermittent 585

light pulses at the end of the recording for optogenetic tagging. 586

Immunohistochemistry. At the conclusion of experiments (6-8 weeks after viral 587

injection), mice were first anesthetized with a ketamine (100 mg/kg) and xylazine (7 588

mg/kg) mixture then perfused with 4% paraformaldehyde (Thermo Fisher Scientific), and 589

the brains were extracted and cryoprotected in 30% sucrose in 1X PBS until they sank. 590

40 µm sections were taken in a cryostat and stored in 1X PBS at 4°C until they were used 591

for immunohistochemistry experiments. The following antibodies and dilutions were used: 592

chicken polyclonal anti-GFP (1:500, Abcam, ab13970), Cy3 donkey anti-sheep (1:200, 593

JacksonImmunoResearch, 713-165-147), sheep polyclonal anti-TH (1:1000, Abcam, 594

ab113), Cy2 donkey anti-chicken (1:200, Jackson ImmunoResearch 703-225-155). 595

Cued-reward task. After 5–7 days of post-surgical recovery, the mice were water 596

restricted to 90% of baseline body weight. Prior to experimental recordings, the mice 597

underwent 3 days of habituation to the recording setup: an opto/electrical tether was 598

connected to the head stage preamplifier while the mice explored a small, rectangular, 599

wooden box (24.5 × 34 cm) in 200 lux lighting conditions in 15-20 min daily sessions. 600

Upon reaching their target weight, the mice were trained on a cued-reward task using a 601

custom-built, Arduino-driven rectangular, wooden box in which tones (1 kHz or 5-10 kHz 602

white noise; 1 second duration) produced by a piezo buzzer (Digi-Key) predicted water 603

rewards (15 µL) delivered with a solenoid valve (The Lee Company). The mice were 604


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presented with 150 trials of pseudo-randomly interleaved CS+ (predicting reward) or CS- 605

(non-reward predicting). CS+/CS- identity was counterbalanced across mice. After the 606

CS+ ended there was 0.5 second delay, followed by a 5 second period during which 607

reward would be delivered upon detection of a lick at the reward port by an infrared 608

photointerruptor (SparkFun). This reward retrieval period was followed by a 10 second 609

intertrial interval (ITI). Mice were trained until they retrieved rewards on at least 70% of 610

CS+ trials for two consecutive days. Lick detection at the reward port was used to 611

measure the first lick during the reward availability period and the rate at which mice 612

would lick during reward anticipation and reward delivery. 613

Restraint. Mice were placed in tapered plastic film restraint bags (Decapicone) punctured 614

with breathing holes before entering 50 mL plastic conical tubes, modified with a slit to 615

allow the headstage and recording tether to pass. The mice used to characterize restraint-616

induced physiology in Figure 1 and Extended Figure 1 had previously undergone a 5-617

minute social interaction test not analyzed in this study. 618

Social Defeat. CD1 retired breeder males (Charles River laboratories) were used as 619

aggressors. We prescreened CD1 males to ensure that they attacked male mice in under 620

1 min. For 10 days, we placed the experimental mouse in a different aggressive mouse’s 621

homecage for 10 minutes. The mice then spent the remaining 24 hours across a 622

perforated, transparent divider from the aggressor. 623

Social Interaction. Mice explored a chamber (40 cm x 40.5 cm) containing an empty 624

enclosure (10 cm diameter wire cup) for 2.5 min, then explored the same chamber for an 625

additional 2.5 min after we introduced a novel mouse (CD1 male) into the enclosure. We 626

defined the interaction zone using a 7 cm radius around the enclosure. Susceptible mice 627


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were defined as those that spent less time interacting with the CD1 mouse than they did 628

with the empty enclosure. 629

Pharmacological inactivation. Guide cannulas (26 gauge; Plastics One) were bilaterally 630

implanted into the VTA as described above. An LFP wire was glued to the side of the 631

cannula so that it extended 1.5 mm past the cannula tip. LFP wires were also implanted 632

in the NAc. After recovery from surgery, the mice were habituated to the familiar 633

environment for 3 days. Saline or muscimol (Tocris Bioscience) dissolved in saline (8.8 634

mM concentration) was microinfused into the VTA while the mice were in the home cage 635

by back loading into a 33-gauge infusion cannula and into polyethylene (PE 20) tubing 636

connected to 1.0 µL Hamilton microsyringe. The infusion cannula protruded 1 mm beyond 637

the guide cannula tip. An infusion volume of 0.13 µL was delivered using a Harvard 11 638

Plus syringe pump (Harvard Apparatus) at a rate of 0.13 μL/min and the infusion cannula 639

was left in place for 5 minutes post-infusion. After a 20–30 min post-infusion interval, 640

neural activity was recorded as the mice were placed in the familiar environment and then 641

the restraint tube. 642

Neural Recordings. A Digital Lynx system (Neuralynx, Bozeman, MT) was used to 643

amplify, band-pass filter (1-1000 Hz for LFPs and 600-6000 Hz for spikes), and digitize 644

the electrode recordings at sampling rates of 2 and 32 kHz for LFPs and spikes, 645

respectively. Single units were clustered based on the first two principal components 646

(peak and energy) from each channel using Klustakwik (Ken Harris) and visualized in 647

Spike Sort 3D (Neuralynx). Clusters were then visually inspected and included or 648

eliminated based on waveform appearance, inter-spike interval distribution, isolation 649

distance, and L-ratio. 650


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Power, Coherence, Phase-Locking Analysis, and Directionality Analysis. To 651

calculate power, after normalizing the raw LFP data to the root mean square of the voltage 652

signal while the mice were in the familiar environment, the power spectra and coherence 653

of the LFPs were calculated with MATLAB wavelet functions (cwt and wcoherence). 654

Power and coherence were calculated on 1-minute segments of data and averaged 655

across the entire experimental session. To remove mechanical artifacts and EMG noise, 656

indices with voltage values 3 standard deviations from the mean were omitted when 657

averaging power. To speed-filter data, indices corresponding to when the animal was 658

moving 0-5 cm were included when averaging power. To capture the animal-specific 659

frequency of the restraint-induced oscillation, we measured the peak restraint-induced 660

signal within a 2-7 Hz range and compared it with the corresponding signal in the familiar 661

environment. 662

For phase locking analysis, we digitally band-pass filtered the raw LFPs using a zero 663

phase delay filter (K. Harris and G. Buzsaki). The phase was calculated using a Hilbert 664

transform, and a corresponding phase was assigned to each spike. We limited our 665

analysis to units that fired at least 100 times over the period analyzed. Functional 666

directionality was calculated based on the pairwise phase consistency (PPC) of VTA 667

multiunit spikes assigned based on the corresponding phases of the 2-7 Hz filtered NAc 668

LFP. We calculated PPC of VTA spikes that were shifted in 2.5 ms steps ± 80 ms to 2-7 669

Hz NAc signals. We compared the lags of the maximum PPC value to determine VTA-to-670

NAc directionality, where negative lags correspond to VTA-to-NAc directionality. 671

Single-unit analyses. We identified significantly light-modulated units by bootstrapping. 672

Laser ON and laser OFF spikes were combined, binned, and randomly shuffled 30,000 673


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times. We considered units light-responsive if the observed laser OFF vs. laser ON firing 674

rate difference was greater than 95% of the firing rate differences from the shuffled data. 675

To determine the effects of restraint effects on firing rate, average firing rate was 676

compared between a 5-minute baseline period during which mice explored a familiar 677

environment and restraint during the same recording session. For task-evoked single unit 678

activity, spikes were binned in 10-ms bins and averaged across trials per neuron. CS+ 679

evoked single unit activity was calculated as the average firing rate per neuron during 680

CS+ presentation. Identification of putative VTA dopamine and VTA GABA neurons was 681

adapted from Cohen et al26. Neurons were first filtered Neurons were first filtered based 682

on whether they were significantly task-modulated by performing a one-way ANOVA of 683

firing during a 1 second baseline period, during the 1 second CS+ presentation, and 684

during the 0.5 second delay period. Significantly task-modulated units were then 685

classified as putative VTA dopamine neurons if they had significantly higher reward-686

evoked firing in the 0-0.15 second period after reward retrieval relative to pre-CS+ 687

baseline firing rate, as well as no difference between pre-CS+ baseline firing rate and 688

delay period firing rate. Significantly task-modulated units were classified as putative VTA 689

GABA neurons if they exhibited a higher firing rate during the delay period than the pre-690

CS+ baseline firing rate. 691

Quantification and statistical analysis. Statistical analysis was performed in Graphpad 692

Prism 7 or MATLAB. Two-tailed tests were used throughout. Data was tested for normality 693

to determine whether to use parametric or non-parametric tests. For behavioral 694

experiments, Wilcoxon signed-rank tests or paired t-test were used to assess the effects 695

of stress and optogenetic manipulations within animals. For between-animal 696


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comparisons, Wilcoxon rank-sum tests or two-sample t-tests were used. For calculation 697

of the efficacy of Arch and eNpHR on oscillation inhibition, Bonferroni-corrected one-698

sample t-tests were used to compare each time period to 100%. For all analyses, the 699

alpha level was 0.05. Error bars and shaded error bands represent the standard error of 700

the mean. 701

702

Funding and Disclosure 703

This work was supported by a grant from the NIMH to A.Z.H (K08 MH109735), by a 704

NARSAD grant from the Brain Behavior Research Foundation (AZH), and by the Hope 705

for Depression Research Foundation (J.A.G., A.Z.H.). A.Z.H. is an advisory board 706

member of Genetika+, incorporated. 707

708

Acknowledgments 709

We would like to thank René Hen, Francis Lee, and Christian Lüscher for their feedback. 710

We would also like to thank Mihir Topiwala, Joseph Stujenske and Ekaterina Likhtik for 711

their early technical and analytic training as well as members of the Gordon and Hen labs 712

for technical assistance and discussions. D.C.L. and A.Z.H. designed and analyzed the 713

experiments. D.C.L., L.A.C, L.K, E.S.H., A.I.A., A.J.P., L.Y., Z.H.B, A.F. and A.Z.H. 714

conducted the experiments and analyzed the data. J.A.G. assisted with the design, 715

implementation, analysis and interpretation of experiments. D.C.L and A.Z.H. interpreted 716

the results and wrote the paper. 717

Data availability 718


https://doi.org/10.1101/2020.05.28.121905


The data that support the findings of this study are available from the corresponding 719

author upon reasonable request. 720

Code availability 721

The custom-written code used to analyze data is available from the corresponding author 722

upon reasonable request. 723

724

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840

841


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https://doi.org/10.1101/2020.05.28.121905


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https://doi.org/10.1101/2020.05.28.121905


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Ventral tegmental area GABA neurons mediate stress-induced … · GABA neurons (Fig. 3b) 39. Restraint stress reduced the firing rates of both VTA DA and . 137. GABA neurons (Extended