Prefrontal Regulation of Punished Ethanol Self-administration

Journal Pre-proof Prefrontal regulation of punished ethanol self-administration Lindsay R. Halladay, Adrina Kocharian, Patrick T. Piantadosi, Michael E. Authement, Abby G. Lieberman, Nathen A. Spitz, Kendall Coden, Lucas R. Glover, Vincent D. Costa, Veronica A. Alvarez, Andrew Holmes PII:

S0006-3223(19)31854-2

DOI:

https://doi.org/10.1016/j.biopsych.2019.10.030

Reference:

BPS 14048

To appear in:

Biological Psychiatry

Received Date: 10 April 2019 Revised Date:

8 October 2019

Accepted Date: 25 October 2019

Please cite this article as: Halladay L.R., Kocharian A., Piantadosi P.T., Authement M.E., Lieberman A.G., Spitz N.A., Coden K., Glover L.R., Costa V.D., Alvarez V.A. & Holmes A., Prefrontal regulation of punished ethanol self-administration, Biological Psychiatry (2019), doi: https://doi.org/10.1016/ j.biopsych.2019.10.030. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

Biological Psychiatry Archival Report

Prefrontal regulation of punished ethanol self-administration

Lindsay R. Halladay 1,2*, Adrina Kocharian 1, Patrick T. Piantadosi 1,5, Michael E. Authement 3,5, Abby G. Lieberman 1, Nathen A. Spitz 1, Kendall Coden 1, Lucas R. Glover 1, Vincent D. Costa 4, Veronica A. Alvarez 3,5, and Andrew Holmes 1

1

Laboratory of Behavioral and Genomic Neuroscience, National Institute on Alcohol Abuse and Alcoholism, NIH, Bethesda, MD, USA 2

3

Department of Psychology, Santa Clara University, Santa Clara, CA, USA

Laboratory on Neurobiology of Compulsive Behaviors, National Institute on Alcohol Abuse and Alcoholism, NIH, Bethesda, MD, USA 4

Department of Behavioral Neuroscience, Oregon Health Sciences University, Portland, OR, USA 5

Center on Compulsive Behaviors, Intramural Research Program, NIH, Bethesda, USA

Running title: Punishment and the prefrontal cortex Keywords: alcohol, compulsive, nucleus accumbens, amygdala, mouse, corticostriatal; infralimbic

*Correspondence to: Lindsay R. Halladay PhD, Alumni Science Hall, Rm 204, Santa Clara University, 500 El Camino Real, Santa Clara, CA 95053, USA, telephone +14085513399, email [email protected]

Abstract Background: A clinical hallmark of alcohol use disorder (AUD) is persistent drinking despite potential adverse consequences. The ventral (vmPFC) and dorsal (dmPFC) medial prefrontal cortex are positioned to exert top-down control over subcortical regions, such as the nucleus accumbens shell (NAcS) and basolateral amygdala (BLA), which encode positive and negative valence of EtOH-related stimuli. Prior rodent studies have implicated these regions in regulation of punished EtOH self-administration (EtOH-SA). Methods: We conducted in vivo electrophysiological recordings in mouse vmPFC and dmPFC to obtain neuronal correlates of footshock-punished EtOH-SA. Ex vivo recordings were performed in NAcS D1-positive MSNs receiving vmPFC input to examine punishment-related plasticity in this pathway. Optogenetic photosilencing was employed to assess the functional contribution of the vmPFC and dmPFC, vmPFC projections to NAcS or BLA, to punished EtOH-SA. Results: Punishment reduced EtOH-lever pressing and elicited aborted presses (lever approach followed by rapid retraction). Neurons in vmPFC and dmPFC exhibited phasic firing to EtOHlever presses and aborts, but only in vmPFC was there a population-level shift in coding from lever presses to aborts with punishment. Closed-loop vmPFC, not dmPFC, photosilencing on a post-punishment probe test negated the reduction in EtOH-lever presses but not aborts. Punishment was associated with altered plasticity at vmPFC inputs to D1-MSNs in NAcS. Photosilencing vmPFC projections to NAcS, not BLA, partially reversed suppression of EtOHlever presses on probe testing. Conclusions: These findings demonstrate a key role for vmPFC in regulating EtOH-SA after punishment, with implications for understanding the neural basis of compulsive drinking in AUDs.

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Introduction Excessive alcohol drinking is the third leading cause of preventable death in the United States (1). By recent estimates, over fifteen million adults are afflicted by alcohol use disorders (AUD), while a quarter of the adult population reports recently engaging in either heavy alcohol use or binge drinking (1). A hallmark of AUDs is that drinking persists despite an awareness of the potential adverse consequences (2). Despite a large body of work describing brain mechanisms underlying alcohol-seeking, the neural circuits that exert control over the drive to use alcohol and other drugs in the face of potential negative outcomes remain poorly understood (3-10). There is growing evidence from human and rodent studies that the medial prefrontal cortex (mPFC) modulates ethanol self-administration (EtOH-SA), and has been shown to either reduce or enable various measures of EtOH-seeking and drinking (4, 5). For instance, cue-induced reinstatement of EtOH consumption is abolished by ablating neuronal ensembles in the ventromedial (vmPFC), but not dorsomedial (dmPFC) PFC (11, 12). The mPFC is also wellpositioned, anatomically, to arbitrate between EtOH-seeking and avoidance via its connections with amygdaloid and striatal regions (13-17). Prior studies have found that the development of resistance to EtOH-SA is associated with plasticity in mPFC projections to the NAc core (NAcC) (18), and that disconnecting dmPFC from NAcC biases rats towards making disadvantageous decisions (19, 20). Moreover, recent data has shown that the shell region of NAc (NAcS) and basolateral amygdala (BLA), as well as mPFC connections with these regions, play a key role in regulating responding to non-drug rewards in the presence of reward-associated cues and potentially aversive outcomes (21-27). These circuits are also affected by EtOH exposure. For example, EtOH dependence was accompanied by insertion of GluA2-lacking AMPA-receptors at glutamatergic synapses in NAc MSNs (28) and increased excitability and amplitude of NMDA receptor-mediated synaptic responses in D1expressing NAc MSNs (29). Furthermore, NMDA receptors in the NAc and medial prefrontal 3

cortex (mPFC) are implicated in punishment-resistant EtOH-drinking in rats (18, 30), and a recent elegant study found activity of NAc-projecting vmPFC neurons predicted lesser cocaineseeking (27). Taken together, these prior studies suggest that vmPFC, BLA, and NAc are part of an integrated circuit regulating the seeking, consumption and, in the face of negative outcomes, avoidance of EtOH and other drugs of abuse (31-36). Nonetheless, understanding of the contributions of the vmPFC and its downstream connections to BLA and NAcS to the regulation of punished EtOH-SA remains incomplete. The aim of the current study was to clarify this role using a combination of in vivo and ex vivo neuronal recordings and optogenetic manipulations in a mouse assay for punished EtOH-SA.

Methods and Materials Subjects Subjects were male (7-8 weeks old) C57BL/6J and B6.Cg-Tg(Drd1a-tdTomato)6Calak/J mice obtained from The Jackson Laboratory, gradually reduced to 85% of their free-feeding body weight before testing.

Ethical considerations All experimental procedures were approved by the NIAAA Animal Care and Use Committee and followed the NIH guidelines outlined in Using Animals in Intramural Research.

Behavioral testing Mice were trained in a Med Associates operant chamber to respond on the left of 2 levers on a continuous schedule of reinforcement to earn sucrose pellets during 40-min sessions (right, ‘inactive-lever’ had no programed consequences) until criterion was met (≥35 rewards) as 4

previously described (37-39). The pellet was then substituted with a progression of 10µL liquidrewards (“sucrose fading”; 10% sucrose, 10% sucrose+10% EtOH, 5% sucrose+10% EtOH, 10% EtOH). Training at each EtOH-concentration stage continued until performance was stable (<20% coefficient between reward-lever responses over 3 sessions). There was then a single punishment session consisting of a baseline-period (10 rewarded/unpunished EtOH-lever presses) immediately followed (after the 10th press) by a 40min punishment period (EtOH-lever presses alternatingly produced 10% EtOH or noEtOH+footshock). A probe test (identical to rewarded training sessions) was conducted 24hr later. For in vivo recordings, a second probe was conducted 24-hr following the first, with data collapsed across probes for analyses. EtOH-lever presses, inactive-presses, and head-entries into the reward receptacle were recorded by Med-PC software. Approaches to the EtOH-lever with an elongated body posture, followed by rapid body-retraction without execution of press were scored manually (Supplemental Video 1) and referred to as ‘aborts,’ keeping with the definition of a similar behavior in rats (40).

In vivo neuronal recordings Following training, fixed microelectrode arrays, were surgically implanted in bilateral vmPFC and dmPFC of the same mouse (41, 42). Recordings were conducted during punishment and probe testing using OmniPlex Neural Data Acquisition System combined with simultaneous CinePlex Behavioral Research Systems (41). Mean firing rate of recorded cells was 6.33±0.90 for vmPFC and 4.12±0.46 for dmPFC. Spike timestamp information was integrated and analyzed using NeuroExplorer. For each unit, spikes were time-locked to the onset of behavioral events. Spiking activity 1s prior to event onset was used as baseline. Using a sliding window (300ms) stepped in 10ms bins from -2.5s to +2.5s around each event, we computed t-tests to determine when the activity surrounding each 5

event deviated from baseline; units with significant deviations were considered responsive to an event for that time bin.

Fluorescence in situ hybridization (FISH) Immediately after probe testing, brains were collected to perform FISH using the RNAscope Multiplex Fluorescent Assay Kit (42) to label vmPFC fos (c-fos), Slc17a7 (vGluT1), Gad1 (Gad67), Drd1a (DR1A), and Drd2 (DR2). We used ImageJ to calculate percentage of DAPIlabeled neurons positive for fos, and of those, the percentage that were Slc17a7-, Gad1-, Drd1a-, and Drd2-positive.

Ex vivo whole-cell recordings AAV5/CaMKIIα-hChR2(H134R)-eYFP was bilaterally injected into the vmPFC of D1tdTomato mice. Mice underwent behavioral testing and were scarified on probe day. Cells in NAcS brain sections were voltage-clamped at +40mV (in gabazine). tdTomato-expressing D1MSNs and non-fluorescent putative D2-MSNs were identified using blue light-pulses to optically evoke EPSCs. AMPA-mediated EPSCs were pharmacologically isolated using (R)-CPP, and subtracted from combined EPSCs to obtain the NMDA-mediated EPSC component (28). AMPA:NMDA ratios were calculated by dividing peak receptor-mediated EPSC by peak NMDA-mediated EPSC. For rectification index, AMPA-mediated EPSCs were measured at both negative and positive (-55/+40 mV) holding potentials and spermine was included in the internal solution. Rectification index was calculated by dividing peak current at -55mV by peak current at +40 mV.

In vivo photosilencing

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rAAV8/CAG-ArchT-GFP or rAAV8/CAG-GFP (local PFC-silencing) or AAV5/CaMKIIαeArchT3.0-eYFP or AAV5/CaMKII-eYFP (vmPFC projection-silencing) was bilaterally injected into vmPFC or dmPFC, and optic fiber-containing ferrules were implanted either locally or in terminal regions (NAcS/BLA) (43, 44). One month later, mice underwent reminder-training sessions, then punishment and probe testing. During the probe test, green light was shone when the mouse was in a zone ( 2.75x4 inches) around the EtOH-lever, determined in real-time by CinePlex software.

Retrograde neuronal tracing Retrograde tracers Cholera Toxin B (CTb555/488) were injected into NAcS and BLA of the same mouse. Seven days later, brain mPFC sections were inspected for cell bodies labeled with either or both fluorophore (45).

Statistical analysis Data were analyzed using Student’s t-tests, ANOVA followed by Newman-Keuls and Šidák corrected post hoc tests, and bivariate correlation.

Results Punishment suppresses EtOH-SA We first examined the behavioral profile of mice undergoing in vivo neuronal recordings. Replicating previous observations (37-39), the rate of EtOH-lever pressing was significantly suppressed during punishment and probe testing, compared to the unpunished baseline period of the punishment session (Figure 1B-C). The number of shocks received inversely predicted suppression on probe (i.e., positive correlation between EtOH-SA during punishment and probe; r=.61, P=.003), showing that the degree of suppression during probe testing was not due to the 7

number of shocks received. Head-entries were also significantly reduced following punishment (Figure S2), whereas inactive-lever presses increased slightly during punishment, but returned to low levels on probe. Another important observation was the emergence of EtOH-lever press aborts during punishment and maintained on the probe tests (Figure 1B-C). This behavior reflects the development of a conflict between EtOH-seeking and shock-avoidance, and has been described in rat punishment paradigms (40).

Neuronal correlates of punished EtOH-SA We next tested for in vivo neuronal correlates of behavior by recording the activity of 191 vmPFC and 195 dmPFC neurons in 20 mice (Figure 2A). In both brain regions, we detected examples of individual neurons exhibiting increases or decreases in firing around the time mice made either an EtOH-lever press or an abort (Figure 2B-C). We did not classify these behaviorrelated cells as putative glutamatergic projection neurons verses interneurons based on spiking characteristics. However, FISH-labeling of fos and mRNA markers for glutamatergic and GABAergic phenotypes (Figure 2D-E), showed that of those cells by probe testing, the vast majority were glutamatergic (Figure 2F). This echoes data from other EtOH-SA tasks (e.g., reinstatement) showing that most activated cells are glutamatergic (11).

vmPFC-coding punished of EtOH-SA We next examined population-level mPFC-coding (46) by aligning neuronal firing in a 5-sec window around behavioral events during the pre-punishment baseline, punishment, and probe tests. During baseline, we found clear population-level representation of EtOH-lever presses in vmPFC ( 30% of units, n=65); neuronal engagement increased over the 1.5sec prior to execution of a lever-press and peaked slightly after a press was made (Figure 2G). During the punished phase, press-related activity of vmPFC neurons was minimal (Figure 2H) but there was strong 8

coding of aborts (∼38% of vmPFC units) beginning around 0.5sec prior to the mouse retracting away from the EtOH-lever (Figure 2I). Notably, this vmPFC neuronal coding of aborts was maintained during probe testing, while coding of EtOH-lever presses returned to levels evident during punishment, despite the fact that the number of presses was still much lower (Figure 2JK). To determine whether EtOH-lever and abort events were encoded by distinct neural populations in vmPFC, we conducted a correlational analysis on units that were responsive to either event. Interestingly, only 2 of all units recorded exhibited firing rates during the two events that correlated significantly, suggesting that these behaviors are represented by separate ensembles in vmPFC. These in vivo neuronal data demonstrate that vmPFC neurons exhibit representations of the execution of a response for EtOH and avoidance of that same response in a manner that shifts depending on the prevailing task demands: pre-punishment=press, ongoing punishment=abort. This agrees with previous studies showing the vmPFC can contribute to the promotion or inhibition of reward-seeking across different experimental situations (31, 34, 47-53). The current data also show that during probe testing, when there is prior knowledge of both the reward and punishment outcomes for responding, the vmPFC strongly codes for both lever-pressing and aborts, consistent with a role in arbitrating between opposing actions under conflict, but not a simple response-suppression function.

Silencing vmPFC abolishes punished-suppression of EtOH-SA Our recording data demonstrate vmPFC neuronal correlates of punished-suppression of EtOH-SA, but do not address causal contribution of vmPFC to performance. Therefore, we devised a ‘closed-loop’ optogenetic approach to test the consequences of photosilencing ArchTtransfected vmPFC neurons when mice were near the EtOH-lever during the probe test (Figure 3A-C). 9

We found that silencing vmPFC neurons in this manner abolished punishment-induced suppression of EtOH-SA. Specifically, GFP-expressing control mice exhibited a significant decrease in EtOH-lever pressing during probe testing, relative to the last training day prior to punishment, whereas there was no significant change in ArchT animals such that their probe test lever-pressing was significantly higher than GFP mice (Figure 3D). Notably, despite this silencing-induced normalization of lever-pressing, aborts exhibited by the groups did not differ (Figure 3E), nor did the cumulative or average time spent in the light-on zone proximal to the EtOH-lever (Figure 3F), or inactive-lever presses (Figure 3G). There was a small but significant increase in head-entries in ArchT mice but not GFP controls (Figure 3H), which likely reflects more frequent reward-collection/checking in the higher-pressing ArchT group, rather than greater motivation for the EtOH-reward following vmPFC-silencing. Indeed prior studies in rats report negative (54, 55) or demotivating (56-59) effects on reward-seeking after vmPFC lesioning and pharmacological inactivation. In a control experiment, mice underwent the same behavioral testing and surgical procedures, then on reaching training criterion, were given a pseudo-probe test (i.e., without punishment) during which the vmPFC was silenced (exactly as above). All behavioral parameters were similar between ArchT and GFP groups (Figure S13), demonstrating that vmPFC-silencing was insufficient to alter behavior in the absence of punishment, excluding the possibility that increased EtOH-SA in punished mice following vmPFC-silencing was due to a general increase in motivation for EtOH-SA.

dmPFC-coding of punished EtOH-SA We next determined whether the contribution of the vmPFC to punished EtOH-SA was shared by neighboring dmPFC. Recorded units in the dmPFC did not show the same shifts in task-phase related population-coding seen in vmPFC, though there was moderate dmPFC activity 10

associated with EtOH-lever and abort events during probe testing (Figure 4A-E). Prior work has suggested that dmPFC promotes reward-SA (31), a notion supported by recent reports of in vivo calcium transients evident during sucrose-cue presentation in dmPFC and dmPFC→NAcC cells (60). Indeed, data here indicate an absence of clear neuronal correlates in dmPFC during punishment, but it remains possible that representations are extant but restricted to discrete dmPFC ensembles that are difficult to resolve with neuronal recordings. Related, a recent report found a small subset of shock-activated NAcS-projecting dmPFC cells exhibited calcium correlates during avoidance of punished-reward (22).

dmPFC-photosilencing does not affect punished-suppression of EtOH-SA We next assessed the consequences of photosilencing the dmPFC, employing the same closed-loop design described above (Figure 4F), anticipating that the lack of EtOH-SA correlates in dmPFC would correspond with minimal functional relevance. In contrast to vmPFC effects, following dmPFC-silencing, EtOH-lever press rates in ArchT and GFP groups were similarly decreased following punishment (Figure 4G). Likewise, aborts, time spent in the light-on EtOHlever zone, inactive-lever presses, and head-entries were all unaffected by dmPFC-silencing (Figure 4H-K). These optogenetic data show that, in contrast to the vmPFC, dmPFC is not necessary for the expression of punished-suppression.

Punished-suppression of EtOH-SA produces vmPFC→NAc plasticity Our next step was to identify vmPFC outputs that modulate punished-suppression, focusing on the NAcS. We tested for plastic changes to synaptic inputs selectively from vmPFC to NAcS associated with punished EtOH-SA, focusing on D1-expressing MSNs given the cell-specificity of EtOH-induced changes described in the introduction and the report that pharmacologically 11

blocking D1-receptors in NAcS promotes EtOH-(and sucrose) SA elicited by conditioned cues/contexts (25, 62-66). Indeed, FISH-labeling of fos and mRNA markers for D1- and D2expressing neuronal phenotypes, showed that the large majority of probe testing-activated (fospositive) NAcS MSNs were D1-expressing (Figure S5). After probe testing, glutamatergic synaptic responses (EPSCs) were evoked in D1-expressing MSNs (red cells, D1-tdTomato reporter) and for comparison, putative D2-expressing MSNs (nonred cells) in NAcS by ChR2-mediated light-stimulation of vmPFC inputs (Figure 5A, S7). These recordings revealed a significant decrease in AMPA:NMDA ratio at glutamatergic inputs to D1expressing MSNs in the punished group, compared to unpunished controls (Figure 5B, S7), but no significant difference in AMPA:NMDA ratio at glutamatergic inputs to putative D2expressing MSNs (Figure S7). This suggests preferential synaptic plasticity at vmPFC inputs to NAcS D1-MSNs following punishment and probe testing. Of further note, punishment did not change the rectification index, (i.e., contribution of GluA2 subunit to the AMPA response), in D1R or D2R-expressing MSNs (Figure 5C-D, S7), indicating that AMPA:NMDA ratio reductions were not associated with any obvious change in AMPA receptor subunit composition. These ex vivo recording data show evidence of plasticity at vmPFC→D1 MSNs inputs in the NAcS as a consequence of punishment.

Silencing vmPFC→NAcS, not vmPFC→BLA, attenuates punished-suppression of EtOH-SA We next asked whether the punishment-related plastic changes in the vmPFC→NAcS reflect a causal contribution of this pathway. Employing the same closed-loop photosilencing procedure used for local PFC-silencing, we shone green light into the NAcS to inhibit ArchT-transfected fibers originating from vmPFC during probe testing (Figure 5E). YFP controls showed a significant decrease in EtOH-lever pressing, but the corresponding decrease in ArchT animals was comparably smaller and non-significant, such that YFP and ArchT groups differed 12

significantly (Figure 5F). The rate of aborts, inactive-lever presses, and time spent in the EtOHlever zone did not differ between groups, but head-entry rate was slightly higher in the ArchT group, in line with their slightly higher rate of EtOH-lever pressing (Figure 5G-H, S9). We compared the behavioral effects of vmPFC→NAcS-silencing with those of a parallel pathway, vmPFC→BLA (Figure 5I). Silencing vmPFC→BLA projections did not affect any measure for YFP or ArchT groups (Figure 5J-L, S11). These data extend our recording and optogenetic data implicating the vmPFC in punishedsuppression of EtOH-SA by showing that vmPFC interacts with NAcS to some extent to modulate this behavior.

Minimal collateralization of vmPFC neurons to NAcS and BA While optogenetics experiments were designed to selectively target vmPFC→NAcS/BLA, it is possible that non-selective effects occurred through inhibition of vmPFC neurons sending collaterals to both regions. To address this, Alexa-conjugated CTb (555/488) were injected into NAcS and BLA to retrogradely-label neurons in mPFC. Consistent with recent, methodologically similar work quantifying vmPFC collateralization to NAcS and BLA (45), we saw very few double-labeled cells in vmPFC or dmPFC (Figure S12), suggesting the attenuated punishedsuppression effect of vmPFC→NAcS-silencing is unlikely to stem from effects on vmPFC→NAcS/BLA collaterals, though we cannot exclude the contribution of vmPFC collaterals to other brain regions.

Discussion Collectively, these data provide correlative and causal evidence supporting an important contribution of the vmPFC in regulating EtOH-SA in the face of potential punishment.

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Two key findings in the current study were that 1) following punishment, vmPFC neurons coded EtOH-lever pressing and were behaviorally required for the suppression of lever-pressing, and 2) while vmPFC neurons also strongly coded for aborting EtOH-lever pressing after punishment, vmPFC was not necessary for expression of this avoidance beahvior. These data fit with the long-standing view of the importance of this region for inhibitory control over various processes at play in the current task, most pertinently reward-seeking and threat-avoidance (61, 62). As noted, a striking observation was that despite clear evidence for neural representation of abort-behavior within vmPFC, optogenetically silencing vmPFC did not reduce the number of aborts made during probe testing. This dichotomy suggests that despite the disinhibited EtOHseeking induced by vmPFC-silencing, the association between the EtOH-lever response and the punished outcome was retained and expressed. A related conclusion is that brain regions other than vmPFC (e.g., periaqueductal gray or certain amygdaloid nuclei) either normally mediate abort expression, or can maintain abort-behavior when the vmPFC is non-functional. These data support a model in which rather than being a simple substrate for inhibiting EtOH-SA, the role of vmPFC is more complex in signaling changes in response-outcome relationships after punishment, which, in turn, enables appropriate modification of some (lever-pressing) but not all (aborts) behaviors. It is becoming increasingly clear that the vmPFC and dmPFC can contribute to reward-related behaviors in complementary or contrasting ways, depending on factors not limited to the testing context and history with or type of drug/reward (53). Further speaking to this complexity, we found that, in contrast to vmPFC-silencing, silencing the dmPFC using the same experimental design was without effect. On the one hand, these negative findings concur with recent work showing that pharmacologically inactivating rat dmPFC did not affect punished EtOH-lever pressing (55) or conditioned avoidance-behavior (63). On the other hand, a lack of dmPFC14

silencing effects is perhaps surprising in view of the neural correlates of probe performance we detected in dmPFC, as well as earlier data from rats showing that dmPFC-inactivation or photosilencing increased punished licking for water (64) and promoted punished cocaine-seeking (65, 66). There may be technical reasons for these discrepancies, such as species idiosyncrasies or differences in drug or behavioral assays used. A more interesting possibility, however, is that dmPFC does indeed contribute to punished EtOH-SA, but this effect is difficult to uncover due to functional heterogeneity across dmPFC neurons: our neuronal recordings indicated the presence of both inhibitory and excitatory coding of presses and aborts in dmPFC during probe testing. In this regard, a recent study found that photoexcitation of dmPFC→NAcS only reduced punished responding for a milkshake reward (albeit partially) when shock-activated dmPFC neurons were selectively manipulated (22). Thus, similarly targeted manipulations of task-related neuronal ensembles may be required to unmask a role for the dmPFC in punished EtOH-SA. Providing initial insight into downstream targets of the vmPFC that are recruited to regulate punished EtOH-SA, we found evidence of plasticity of vmPFC inputs to NAcS D1-MSNs suggestive of a punishment-induced change in synaptic strength of these inputs. This plasticity appeared restricted to input onto D1-MSNs, but not putative D2-MSNs. Incubation of cocaineseeking has been associated with strengthening of vmPFC inputs onto NAcS D1-MSNs, as evidenced by the formation of silent synapses, which when optogenetically reversed, produces either increased or decreased cocaine-responding (67, 68). If the vmPFC reduced EtOH-lever pressing after punishment via downstream connections to NAcS, the most parsimonious prediction would be behavioral suppression associated with strengthening of vmPFC inputs to NAcS D1-MSNs. Our finding of reduced D1-MSN AMPA:NMDA ratios, however, appears more in-line with a depotentiation in this pathway. One possible explanation is that this change is indicative of a postsynaptic homeostatic change in 15

NAcS MSNs that could relate to the recruitment of the vmPFC and increased drive onto these cells with punishment. Equally plausible is the interpretation that the changes result from altered inputs from other regions, or in microcircuits within the NAcS itself. Further work will be needed to parse these possibilities. Prior work has shown that ablating mPFC→NAcS neurons reduces EtOH-reinstatement (17), while activating vmPFC→NAc reduces cocaine-seeking (27), and disconnecting the vmPFC→NAcS pathway produces heroin-relapse (69). We found that silencing the vmPFC→NAcS pathway attenuated a punishment-induced decrease in EtOH-lever pressing. Of note, this effect was characterized by a partial reversal of suppression, rather the full reversal seen with local vmPFC-silencing. This difference could be explained in various ways. One reason could be the use of a CaMKII-promoter to express ArchT in vmPFC. This promoter prefers, but is not exclusive to, glutamatergic neurons, leaving open the possible contribution of silencing longrange GABAergic interneurons, which could have different or opposite effects to those produced by glutamatergic projection-silencing (70). Another explanation is that vmPFC→NAcS silencing does not fully recapitulate the effects of local vmPFC-silencing because of the contribution of other efferent projections of vmPFC. Although the identity of these is unclear, our negative optogenetic data indicate that vmPFC→BLA does not critically mediate punished-suppression in the current task, despite evidence supporting a role for the BLA in other assays of punished reward-seeking (23, 71). A third, particularly intriguing possibility again raises the issue of functional heterogeneity at the level of the NAcS itself. If distinct subpopulations of NAcS cells promote EtOH-SA, while others mediate suppression, it would not be surprising that the net effect of non-discriminately silencing vmPFC inputs to NAcS would be mixed. In this context, vmPFC-inactivation has been found to disinhibit a subpopulation of NAcS neurons encoding reward-seeking suppression, while simultaneously inhibit another population of reward-encoding NAcS cells (72). Recent work also 16

shows that with VTA-DA inputs to NAcS, effects on reward-seeking and aversion can be opposite, depending on the sub-compartment and cell-type targeted (26, 73). Finally, in terms of their outputs, NAcS cells targeting the VTA versus lateral hypothalamus have opposite effects on context-induced EtOH-relapse (74). Thus, the vmPFC→NAcS circuit is positioned to exert inhibitory and excitatory influences on consumption and responding for rewards including EtOH (75-78). An important future question will be defining the contribution of inputs from vmPFC,(as well as BLA) (17, 79, 80) to specific functional classes of NAcS neurons involved in punishedsuppression of EtOH-SA. Functional perturbation of the PFC has been consistently reported in AUDs and in animal models of EtOH-exposure, which can lead to a shift towards less-flexible behaviors mediated by subcortical structures (81-83). These shifts are thought to contribute to the progression from moderate drinking to uncontrolled, compulsive alcohol abuse. Consistent with this, the current study shows that the vmPFC codes for behaviors associated with the avoidance of a punished response for EtOH and is required for the suppression of EtOH-SA after punishment. Further, the current results show that vmPFC interacts with the NAcS to modulate punished EtOH-SA behavior. Collectively, these findings advance our understanding of the neural circuits that may underlie compulsive drinking.

Acknowledgements Work supported by the NIAAA Intramural Research Program (ZIA- AA000401) to AH and the Intramural Programs of NIAAA and NINDS (ZIA- AA000421) to VAA, Postdoctoral Research Associate Training fellowship from the Center on Compulsive Behaviors to PTP and MEA, and NIH Director’s Challenge Award and DDIR Innovation Award to VAA. Financial Disclosures The authors report no biomedical financial interests or potential conflicts of interest. 17

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Figure legends Figure 1: Punished-suppression of EtOH self-administration. (A) Mice were first trained to reliably press 1 of 2 levers for EtOH reward. Following training, there was a punishment session in which, after the first 10 rewarded/unpunished presses (=pre-punishment baseline), presses alternatingly produced either EtOH-reward and no shock, or EtOH-reward and footshock. The day after punishment, there was a probe test in which all presses were rewarded, without shock (i.e., same procedure as during training). (B) Example behavioral raster plots showing patterns of EtOH-lever presses, inactive-lever presses and aborts during the last session of training, punishment session (including the pre-shock baseline period) and probe test. (C) The average rate of EtOH-lever presses was significantly lower on the punishment session and probe tests, relative to the pre-punish baseline of the punishment session (repeated measures ANOVA: F(2,40)=29.00, P<0.001, followed by post hoc tests). The rate of inactive-lever presses was slightly but significantly elevated on the punishment session, but not probe tests, relative to prepunishment baseline (repeated measures ANOVA: F(2,38)=3.31, P=0.047, followed by post hoc tests). Aborts were evident on the punishment session and were maintained, at a significantly lower rate, during the probe tests (paired t-test: t(19)=4.83, P<0.001). For corresponding headentry rates, see Figure S1. Data in panel C are means ± SEM from n=19-20 mice. *P<.05 versus pre-punishment baseline or, in the case of aborts, versus punishment.

Figure 2: vmPFC encoding of punished-suppression. (A) Location of multi-channel electrode arrays implanted in the vmPFC (open circles) and dmPFC (open circles, for corresponding data, see Figure 4) for in vivo neuronal recordings. Example raster plots and peri-event histograms of PFC neurons exhibiting increased or decreased firing as mice made (B) EtOH-lever presses and (C) aborts. (D-E) Following probe testing, vmPFC neurons were labeled for fos (c-fos), Gad1 (Gad67) and Slc17a7 (vGluT1) mRNA via fluorescence in situ hybridization (RNAscope). (F) 28

Around 6% of DAPI-labeled neurons in the vmPFC were fos-positive after punishment. Of the fos-positive cells, the vast majority (91%) were positive for the glutamatergic marker, Slc17a7, rather than the GABAergic maker, Gad1 (7%). Around 2% of fos-positive cells were not clearly labeled (n/a) for either marker. (G) vmPFC neurons displayed population-level coding for EtOHlever presses during the pre-punishment baseline period. (H) During the punishment period, there was minimal coding of presses, as a significantly smaller proportion of neurons encoded EtOHlever pressing during punishment compared to the baseline period (Z=3.36, P<.001). (J) vmPFC neurons preferentially encoded aborts during the punishment period; a significantly greater proportion of vmPFC neurons encoded abort versus EtOH-lever pressing (Z=4.22, P<.0001). There was vmPFC representation of EtOH-lever pressing (I) and aborts (K) during the probe tests. Data are means ± SEM from a total of n=129 (punished session) and 124 (probe tests) neurons in n=20 mice.

Figure 3: Closed-loop vmPFC-silencing reverses punished-suppression. (A) Optical fibers were bilaterally directed at vmPFC neurons transfected with rAAV8/CAG-ArchT-GFP (ArchT). (B) In vivo recordings showing vmPFC neuronal inhibition in response to green light. (C) Green light was shone to silence vmPFC neurons when mice approached the EtOH-lever during probe testing. (D) Difference in EtOH-SA between ArchT and GFP groups (two-way ANOVA, group main effect: F(1,12)=5.91, P=.03). Significant suppression of EtOH-lever pressing during the probe test in GFP controls (mean=22.6 train, 9.3 punishment, 6.6 probe) (paired t-test: t(6)=4.09, P<0.01), but not vmPFC-silenced ArchT group (mean=29.5 train, 7.4 punishment, 30.6 probe) (paired t-test: t(6)=0.08, P=0.93), such that the rate on the probe test was significantly higher in the ArchT group than the controls (unpaired t-test: t(12)=2.30, P=0.04). GFP and ArchT groups did not differ in (E) aborts (unpaired t-test: t(11)=0.52, P=0.61) or (F) cumulative duration in the light-on zone (unpaired t-test: t(12)=0.88, P=0.40). (G) The rate of inactive-lever pressing was 29

unaffected by punishment (two-way ANOVA, session main effect: F(1,12)=.001, P=.98), but the ArchT group pressed the inactive-lever more than GFP (group main effect: F(1,12)=7.93, P=.02). (H) The rate of head-entries into the reward-receptacle was unaffected by punishment (two-way ANOVA, session main effect: F(1,12)=2.75, P=.12) or group (group main effect: F(1,12)=2.75, P=.12). Data are means ± SEM from n=7 mice/group. *P<.05 versus train in the ArchT group; #P<.05 versus probe in GFP controls.

Figure 4. dmPFC encoding of events related to punished-suppression. dmPFC neurons did not display clear representations of EtOH-lever presses (A) during the baseline period before punishment, nor coding of presses (B) or aborts (C) in the punishment period itself, though there was evidence of some level of press-related coding during the probe trial (D-E). (F) Optical fibers were bilaterally directed at dmPFC neurons transfected with rAAV8/CAG-ArchT-GFP (ArchT). Green light was shone to silence vmPFC neurons when mice approached the EtOH-lever during probe testing. (G) Significant suppression of EtOH-lever pressing during the probe test (two-way ANOVA, session main effect: F(1,12)=25.50, P=.0003) that did not differ between GFP and ArchT groups (GFP mean= 33.7 train, 8.2 punishment, 9.4 probe; ArchT mean=33.3 train, 6.6 punishment, 6.7 probe) (group main effect: F(1,12)=.053, P=.82). (H) GFP and ArchT groups did not differ in the rate of aborts (unpaired t-test: t(12)=0.43, P=0.67) or (I) time in the light-on zone (unpaired t-test: t(12)=1.09, P=0.30). (J) The rate of inactive-lever presses was higher in GFP versus ArchT groups (two-way ANOVA, group main effect: F(1,12)=14.37, P=.003) and decreased in all animals following punishment (session main effect: F(1,12)=5.10, P=.04) (K) Rate of head-entries decreased for both groups following punishment (two-way ANOVA, session main effect: F(1,12)=14.21, P=.049), but did not differ between groups (group main effect: F(1,12)=.78, P=.39). Data are means ± SEM from a total of n=129 (punished session) and 124

30

(probe tests) neurons in n=20 mice for A-E, and n=7 mice/group for F-K. *P<.05 versus train in the ArchT group; #P<.05 versus probe in GFP controls.

Figure 5: Punishment-related plastic changes in the vmPFC

NAcS pathway. (A) Following probe testing, ex vivo recordings measured responses of tdTomato-labeled NAcS D1-positive medium spiny neurons to blue light-evoked, ChR2-mediated stimulation of vmPFC inputs. (B) There was a significant decrease in AMPA:NMDA ratio in the punished group, as compared to unpunished controls (unpaired t-test: t(17)=2.32, P=0.03). (C) The rectification index of the AMPA receptor mediated response was not different between groups (unpaired t-test: t(13)=1.09, P=0.29). (D) Representative traces of AMPA and NMDA synaptic currents evoked by ChR2 stimulation. (E) Optical fibers were bilaterally directed at the NAcS to silence fibers originating from vmPFC neurons transfected with AAV5/CamKII-eArchT3.0-eYFP (ArchT) when mice approached the EtOH-lever during probe testing. (F) Punishment reduced EtOH-lever pressing (two-way ANOVA, session main effect: F(1,12)=8.38, P=.01). YFP-expressing controls showed a significant decrease in EtOH-lever pressing rate on the probe test, relative to training (mean=36.4 train, 5.5 punishment, 8.3 probe) (paired t-test: t(5)=5.41, P<0.01), whereas the decrease in the ArchT group was modest and non-significant (mean=32.46 train, 8.4 punishment, 21.3 probe) (paired t-test: t(7)=1.02, P=0.34). Further, probe test pressing rates differed significantly between YFP-expressing and ArchT mice (unpaired t-test(12)=1.69, P=.034). Neither the rate of (G) aborts (unpaired t-test: t(11)=1.07, P=0.31) nor the (H) time spent in the EtOH-lever zone (unpaired t-test: t(11)=0.54, P=0.60) was different between groups. (I) Optical fibers were bilaterally directed at the BLA to silence fibers originating from vmPFC neurons transfected with AAV5/CamKII-eArchT3.0-eYFP (ArchT) when mice approached the EtOHlever during probe testing. (J) The rate of EtOH-lever pressing was significantly lower, as compared to training (two-way ANOVA, session main effect: F(1,10)=17.14, P=.002) in both 31

YFP-expressing controls (mean=35.8 train, 12.5 punishment, 7.6 probe) (paired t-test: t(3)=3.31, P<0.05) and the ArchT-expressing group (mean=27.0, 7.6 punishment, 14.7 probe) (paired t-test: t(7)=2.32, P=0.05). There were no group differences in the rate of (K) aborts (unpaired t-test: t(10)=0.12, P=0.91) or (L) the time spent in the EtOH-lever zone (unpaired t-test: t(10)=1.33, P=0.21). Data are means ± SEM from n=7-12 cells in n=4-9 mice/group in A-D, n=6-8 mice/group for E-H and n=4-8 mice/group in I-L.

32

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Deposited Data; Public Database Genetic Reagent

Advanced probes: cfos, vGluT1, gad67, Drd1a, Drd2, Dapb, Polar2a, Cell Ppib,Diagnostics Ubc

c-fos; gene ID 14281, cat#316921, vGluT1; gene ID 72961, cat#416631, Gad67); gene ID 14415, cat#400951, Polr2a/Ppib/Ubc; 3-plex Positive Contr

Organism/Strain

Jackson Laboratory male C57BL/6J; B6.Cg-Tg(Drd1a-tdTomato)6Calak/J mice

Peptide, Recombinant Protein

retrograde tracers Cholera Toxin B-555 and 488

RRID:IMSR_JAX:000664; RRID:IMSR_JAX:016204 cat# C34776, 0.5% dilution

Thermo Fisher Scientific

Recombinant DNA Sequence-Based Reagent Software; Algorithm

CinePlex Behavioral Research Systems; Med-PC softwarePlexon Inc; Med Associates

Transfected Construct

AAV5/CaMKIIα-hChR2(H134R)-eYFP; rAAV8/CAG-AAddgene; UNC Vector Core (Chapel Hill, NC, USA). RRID:Addgene_26969

Other

ID 14415, cat#400951, Polr2a/Ppib/Ubc; 3-plex Positive Control Probe, cat#320881; Drd1a: gene ID 13488, cat# 406491, and Drd2: gene ID 13489, cat# 406501

Key Resource Table The journals of the Society of Biological Psychiatry support efforts in the biomedical research community to improve transparency and reproducibility in published research. Thus, Biological Psychiatry and Biological Psychiatry: Cognitive Neuroscience and Neuroimaging are pleased to participate in the initiative to include a Key Resources Table in published articles. Authors are asked to submit this table at first revision, which may be uploaded using the "Key Resources Table" item type. This table will then be published as supplemental information. The Key Resources Table is designed to promote reproducibility and thus, should include the resources and relevant details necessary to reproduce the study's results. It does not need to be exhaustive. Extensive lists (e.g., oligonucleotides, etc.) may be supplied in a supplementary table and the table referenced here. We strongly encourage the use of RRID identifiers that provide persistent, unique identifiers to key study resources. Search for RRIDs at https://scicrunch.org/resources. Resource categories Note: For all categories, indicate sex and species when applicable ●

Antibody - include host organism common name and clonality (e.g., “mouse monoclonal”)

●

Biological sample - any other biological entity, ranging from isolated tissue to defined population

●

Cell line - if a primary cell line, describe in Additional Information

●

Chemical compound, drug - commercially available reagents

●

Commercial assay/kit - detection assays; labeling and sample preparation kits

●

Deposited data or public database - include both raw data from this paper deposited into a repository and public repository databases (postmortem tissue; genetic consortia data; etc.)

●

Genetic reagent - applies to mutations and variants in whole organism, including transgenically introduced constructs

●

Peptide, recombinant protein - commercially available reagents

●

Recombinant DNA reagent - traditional cultured clones, plasmids, cDNAs, etc., including recombinant DNA libraries

●

Sequence-based reagent - oligonucleotides, primers, etc.; indicate sequence

●

Software, algorithm - include version number and URL for download

●

Organism/Strain - applies to whole organism

●

Transfected construct - in cell line; indicate species of cell line or construct component

●

Other - miscellaneous other categories, including histological stains

EXAMPLE KEY RESOURCES TABLE Resource Type

Specific Reagent or Resource

Add additional rows as needed for each Include species and sex when applicable. resource type

Source or Reference

Identifiers

Additional Information

Include name of manufacturer, company, repository, individual, or research lab. Include PMID or DOI for references; use “this paper” if new.

Include catalog numbers, stock numbers, database IDs or accession numbers, and/or Include any additional information or RRIDs. RRIDs are highly encouraged; search notes if necessary. for RRIDs at https://scicrunch.org/resources.

Antibody

rabbit anti-E2F2

Abcam

Abcam Cat# ab50917, RRID:AB_869541

Antibody

total actin

MP Biomedicals

Cat#8691002, RRID:AB_2335304

Antibody

E2F3

Bacterial or Viral Strain

C-18, Cat#SC-878, RRID:AB_2096807 N/A

Bacterial or Viral Strain

AAV-hSyn-DIO-hM3D(Gq)-mCherry HSV-wtSmurf1

Santa Cruz University of North Carolina Vector Core PMID: 10458166

Biological Sample

postmortem brain tissue

Addgene plasmid # 11752 RRID:SCR_003316

Cell Line

control 03231 iPSC line

Harvard Brain Tissue Resource Center National Institute of Neurological Disorders and Stroke repository

Chemical Compound, Drug

Terazosin

Sigma-Aldrich

N/A

Commercial Assay Or Kit

Bio-Rad DC Protein Assay

Bio-Rad Laboratories, Inc.

# 5000111

Commercial Assay Or Kit

Illumina, Inc.

Deposited Data; Public Database

TruSeq Stranded mRNA GSE35978, Sample Prep Kit v2 GSE17806, GSE53987, GSE13564, GSE80655, and GSE25219

NCBI GEO DataSets

Cat. No. RS-122-2101 RRID:SCR_005012; https://www.ncbi.nlm.nih.gov/gds

Organism/Strain

Mouse: C57BL/6J, male

The Jackson Laboratory

RRID:IMSR_JAX:000664

Sequence-Based Reagent

Primers for RT-qPCR, see Table S1

This paper

N/A

Software; Algorithm

HTSeq Python package

https://doi.org/10.1093/bioinformatics/btp120; https://doi.org/10.1093/bioinformatics/btu638 RRID:SCR_005514

Software; Algorithm

MATLAB v9.1

Mathworks

NINDS # ND03231; RRID:SCR_004520

RRID:SCR_001622

B

A

C

Light (λ=532 nm)

vmPFC silencing

Training to criterion

Punishment EtOH Inactive -lever EtOH -lever

EtOH Inactive -lever EtOH -lever

rAAV8/CAGArchT-GFP

EtOH -lever

EtOH -lever

EtOH

1s

8 0

40

F 1000

32 24 16 8

0

GFP ArchT

800 600 400

200 0

Probe

Probe

G Number of inactivelever presses

GFP ArchT

Time in EtOH-lever zone (s)

16

*

Number of aborts

Number of EtOH-lever presses

24

48

#

40

32

E

GFP ArchT

1 day

48 32 16 0

EtOH

EtOH -lever

Shock/no EtOH

GFP ArchT

H 320

Number of head entries

Firing (Hz)

optical fiber

48

EtOH Inactive -lever EtOH -lever Light ON

1 day

D

Probe + silencing

240 160 80 0

EtOH

GFP ArchT

C

B

2.0

AMPA:NMDA ratio

Post-probe test vmPFCNAcS ex vivo recordings AAV5-CamKIIahChR2(H134R)-eYFP D1+ MSN

Blue light (473 nm)

1.0

0.5

Control 50 pA

2.0

50 msec

1.5 1.0 Punished

0.5

50 pA 50 msec

0.0

0.0

recording electrode

Optical fiber

*

40

G 48

32 24 16

8 0

YFP ArchT

40

H 1000

Time in EtOH-lever zone (s)

AAV5/CamKIIaeArchT3.0-eYFP

48

YFP ArchT

Number of aborts

vmPFCNAcS silencing

F Number of EtOH-lever presses

D1-tdTomato

E

1.5

D 2.5

* Rectification index

A

32 24 16 8

0

800 600 400

200 0

Probe

Optical fiber

48 40 32

24 16 8 0

*

YFP ArchT

*

K

48

YFP ArchT

40

Probe

L

1000

Time in EtOH-lever zone (s)

AAV5/CamKIIaeArchT3.0-eYFP

J

Number of aborts

vmPFCBA silencing

Number of EtOH-lever presses

I

YFP ArchT

32 24 16 8

0

YFP ArchT

800 600 400

200 0

Probe

Probe

A

Training

Punishment

Probe

EtOH Inactive -lever EtOH -lever

EtOH Inactive -lever EtOH -lever

1-2 days

1 day

EtOH -lever

EtOH -lever

EtOH

pre-shock baseline

B

EtOH Inactive -lever EtOH -lever

EtOH

EtOH -lever

Shock/no EtOH

EtOH

shock

EtOH-lever presses Inactive-lever presses

End

Start

Start

Start

End

Aborts

16 24 32 40 48

0

C

*

*

EtOH-lever presses

*

Inactive-lever presses

*

Aborts

0

8

16 24 32 40 48 0

8

Number per session

8

16 24 32 40 48

B

A

D Train

lateral 0.24 mm

5

Firing (Hz)

Firing (Hz)

EtOH-lever press

0

-2 -1 0 +1 +2 Time from EtOH-lever (s)

15 min

Punish

FISH (RNAScope)

E

8

0

1 day

-2 -1 0 +1 +2 Time from EtOH-lever (s)

C fos lateral 0.36 mm

7

Firing (Hz)

0

lateral 0.48 mm

15

-1 0 +1 +2 Time from abort (s)

H

-1 0 +1 +2 Time from abort (s)

DAPI

I

Punish

vmPFC population-coding (% phasic neurons)

vmPFC population-coding (% phasic neurons)

Pre-punish baseline

-2

-1.0

0

+1.0

-2.0

+2.0

Time from EtOH-lever press (s)

Probe

-1.0

0

+1.0

+2.0

-2.0

Time from EtOH-lever press (s)

-1.0

0

+1.0

+2.0

Time from EtOH-lever press (s)

K

Punish

vmPFC population-coding (% phasic neurons)

J

n/a

Slc17a7

Probe

vmPFC population-coding (% phasic neurons)

-2.0

Gad1

fos

vmPFC

0

vmPFC population-coding (% phasic neurons)

Firing (Hz)

F

-2

G

Slc17a7

Gad1

Abort

-2.0

-1.0

0

+1.0

Time from abort (s)

+2.0

-2.0

-1.0

0

+1.0

Time from abort (s)

+2.0

C

B Pre-punish baseline

Probe

-2.0

-1.0

0

+1.0

dmPFC population-coding (% phasic neurons)

dmPFC population-coding (% phasic neurons)

dmPFC population-coding (% phasic neurons)

Punish

+2.0

-2.0

Time from EtOH-lever press (s)

-1.0

0

+1.0

-2.0

+2.0

Time from EtOH-lever press (s)

D

-1.0

0

+1.0

-2.0

+2.0

40 32 24 16

*

*

1250

48

40

1000

32 24 16

8

8

0

0

GFP ArchT

I

750 500 250 0

Probe

-1.0

0

+1.0

+2.0

Time from abort (s)

J Number of inactivelever presses

optical fiber

Number of EtOH-lever presses

rAAV8/CAGArchT-GFP

48

Number of aborts

dmPFC silencing

GFP ArchT

H

Time in EtOH-lever zone (s)

GFP ArchT

+2.0

Probe

Time from abort (s)

G

+1.0

dmPFC population-coding (% phasic neurons)

dmPFC population-coding (% phasic neurons)

-2.0

F

0

Time from EtOH-lever press (s)

E

Punish

-1.0

Probe

48 32 16 0

GFP ArchT

K 320

Number of head entries

A

240 160 80 0

GFP ArchT

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Prefrontal Regulation of Punished Ethanol Self-administration

Supplemental Information

Figure S1: Behavioral apparatus for testing effects of footshock punishment on EtOH-SA. A Med Associates mouse operant chamber was equipped with metal-rod flooring for delivering footshock. Rewards were delivered into a receptacle located between two ultra-sensitive response levers; a response on the left, ‘EtOH lever,’ resulted in an EtOH reward accompanied by a 2-second tone cue (except during punishment - here when left-lever responding resulted in alternating footshock or EtOH+tone cue). Responding on the right, ‘inactive lever,’ did not have any programmed consequences. Note, training, punishment, and probe sessions took place in the same chamber to minimize the influence of changes in context on performance.

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Figure S2: Punishment reduced rates of head-entries into the reward-receptacle. Relative to the last day of training, the rate of head-entries was significantly lower on the punishment period of the punishment session and the probe tests (repeated measures ANOVA: F(2,38)=21.03, P<0.001, followed by post hoc tests). Data are means±SEM.

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Figure S3: Optic fiber placements and virus localization for vmPFC photosilencing. (A) Estimated location of bilateral fiber placements in vmPFC (GFP, gray circles, n=7; ArchT, green circles, n=7). (B) Representative example from the left hemisphere, with corresponding atlas for orientation, of bilaterallyexpressed virus.

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Figure S4: Optic fiber placements and virus localization for dmPFC photosilencing. (A) Estimated location of bilateral fiber placements in dmPFC (GFP, gray circles, n=7; ArchT, green circles, n=7). (B) Representative example from the left hemisphere, with corresponding atlas for orientation, of bilaterallyexpressed virus.

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Figure S5: NAcS cells activated by punishment are primarily D1-expressing MSNs. (A, B) Following probe testing, NAcS neurons were labeled for fos (c-fos), Drd1a (dopamine receptor D1A) and Drd2 (dopamine receptor 2) mRNA via fluorescence in situ hybridization (RNAscope). (C) Around 7% of DAPIlabeled neurons in the NAcS were fos-positive after punishment. Of the fos-positive cells, the majority (75%) were positive for the Drd1a, rather than Drd2 (25%).

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Figure S6: Behavioral data from the ex vivo vmPFCNAcS neuronal recording experiment. (A). Compared to the final training session, EtOH-seeking during the probe test was unchanged in control mice (P=0.1964), but was diminished in punished mice (P<0.0001; corrected post hoc Sidak’s tests following significant repeated measures ANOVA interaction, F(1,28)=8.20,P=0.0079). (B) During the probe session, aborts did not change in the control group (P=0.9933), but were dramatically increased in punished mice, as compared to the final training session (P=0.0001; corrected post hoc Sidak’s tests following significant repeated measures ANOVA interaction, F(1,28)=20.62,P=0.0001). (C) Inactive lever-presses did not change as a function of punishment history or task phase (repeated measures ANOVA, all F-values < 3, all P-values > 0.1). (D) The number of head entries did not change as a function of punishment, although they did tend to be decreased during the probe session, as compared to the final training session (repeated measures ANOVA, main effect of task phase, F(1,28)=5.55,P=0.0257). *P<.05 versus train. White bars: Control (n=14), Black bars: Punished (n=16). Data are means±SEM.

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Figure S7: No change in synaptic responses from vmPFCD2 MSNs NAcS neurons following punishment. (A) Following probe testing, ex vivo recordings measured responses of non-fluorescent putative D2 medium spiny neurons in NAcS evoked by blue light- ChR2-mediated stimulation of vmPFC inputs. (B) No significant change in AMPA:NMDA ratio of vmPFC inputs to D2-MSNs in the punished group, as compared to unpunished controls (unpaired t-test: t(21)=1.848, P=0.07). (C) The rectification index of the AMPA receptor mediated response was not different between groups (unpaired t-test: t(21)=0.868, P=0.39). (D) Representative traces of AMPA and NMDA currents.

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Figure S8: Optic fiber placements and virus localization for vmPFCNAcS photosilencing. (A) Estimated location of bilateral fiber placements in the NAcS (GFP, gray circles, n=6; ArchT, green circles, n=8). (B) Representative example from the left hemisphere of bilaterally-expressed virus.

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Figure S9: vmPFCNAcS silencing did not affect head-entry or inactive-lever press rates. (A) Relative to the last day of training, the rate of head-entries was not significantly different on the probe test (two-way ANOVA, session main effect: F(1,12)=.09, P=.77) in either the YFP (paired t-test: t(5)=2.39, P=0.06) or ArchT (paired t-test: t(7)=1.35, P=0.22) group. (B) Relative to the last day of training, the rate of inactive-lever pressing was not significantly different on the probe test (two-way ANOVA, session main effect: F(1,12)=<.001, P=.99) in the YFP (paired t-test: t(5)=0.22, P=0.83) or ArchT (paired t-test: t(7)=0.42, P=0.97) group. Data are means±SEM.

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Figure S10: Optic fiber placements and virus localization for vmPFCBLA photosilencing. (A) Estimated location of bilateral fiber placements in the BLA (GFP, gray circles, n=4; ArchT, green circles, n=8). (B) Representative example from the right hemisphere of bilaterally-expressed virus.

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Figure S11: vmPFCBLA silencing did not affect head-entry or inactive-lever press rates. (A) Relative to the last day of training, the rate of head-entries was not significantly different on the probe test (two-way ANOVA, session main effect: F(1,13)=4.37, P=.06) in either the YFP (paired t-test: t(3)=2.67, P=0.08) or ArchT (paired t-test: t(7)=1.21, P=0.27) group. (B) Relative to the last day of training, the rate of inactive-lever pressing was not significantly different on the probe test (two-way ANOVA, session main effect: F(1,13)=.43, P=.52) in the YFP (paired t-test: t(3)=0.19, P=0.86) or ArchT (paired t-test: t(7)=0.41, P=0.70) group. Data are means±SEM.

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Figure S12: Minimal collateralization of mPFC neurons to NAcS and BLA. (A) Fluorescentlyconjugated CTb488 and CTb555 was unilaterally injected into the BLA and NAcS, respectively, of the same mouse to label projection neurons in the dmPFC and vmPFC. Images depicting injection site of CTb488 in the (B) BLA and CTb555 in the (C) NAcS. (D) There were very few examples (indicated by yellow-orange arrow heads) of putative double-labeled cells in dmPFC and vmPFC. Scale bar=200 µm.

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Figure S13: vmPFC silencing does not affect EtOH-seeking in the absence of punishment. (A) Optic fibers were bilaterally directed at vmPFC neurons transfected with rAAV8/CAG-ArchT-GFP (ArchT) (top). Representative viral expression and fiber placement in vmPFC. Scale bar=200 µm (bottom). (B) Cartoon schematic of closed-loop silencing design during EtOH-seeking (no punishment). (C) Mean number of EtOH lever-presses (unpaired t-test: t(10)=0.31, P=0.76), aborts (unpaired t-test: t(10)=0.11, P=0.91), inactive presses (unpaired t-test: t(10)=1.16, P=0.27), and head entries (unpaired t-test: t(10)=0.03, P=0.98) made by control (n=7) and ArchT-infused (n=5) mice. Data are means±SEM.

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Supplemental Methods and Materials Subjects Subjects were, with one exception, male C57BL/6J mice obtained from The Jackson Laboratory (Bar Harbor, ME, USA) at 7-8 weeks of age. For the ex vivo physiology experiment, mice were male, C57BL/6Jbackground, ‘D1-tdTomato’ (B6.Cg-Tg(Drd1a-tdTomato)6Calak/J, JAX stock #016204) mice, expressing the DsRed fluorescent protein, tdTomato, under the control of the Drd1a gene. All mice were housed (in pairs prior to surgery, singly post-surgery) in a temperature (72 ± 5 °F) and humidity (45 ± 15%) controlled vivarium, under a 12-h light/dark cycle (lights on at 0630 h). Approximately 1 week prior to training, mice were gradually reduced to 85% of their free-feeding body weight, and maintained at this level through behavioral testing.

Ethical considerations All experimental procedures carried out were approved by the NIAAA Animal Care and Use Committee and followed the NIH guidelines outlined in Using Animals in Intramural Research.

Behavioral testing Apparatus Testing took place in 21.6 × 17.8 × 12.7 cm operant chambers housed within sound and lightattenuating enclosures, equipped with metal-rod flooring for shock delivery (Med Associates, St. Albans, VT, USA). Reinforcements were delivered into a receptacle located between 2 ultra-sensitive response levers. Med Associates Med-PC software-controlled tone generation and reward delivery. Training, punishment and probe testing were conducted in the same context in view of the critical importance of context to punished suppression of EtOH-seeking and other forms of EtOH-seeking (1-4). For a photograph of the apparatus, see Figure S1.

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Training Mice were first trained to respond for EtOH using a sucrose-fading procedure, based on previously described methods (2, 5-6). To establish operant responding, mice were initially trained to respond on the left of 2 levers on a fixed-ratio 1 [FR1] continuous schedule of reinforcement to earn 14-mg sucrose pellets (BioServ, Frenchtown, NJ USA) during 40-minute daily sessions (presses on the right, ‘inactive,’ lever had no programed consequences). To reinforce the association between the reward-lever and reward delivery, presses on the reward-lever also produced a 3-kHz pure tone (throughout training and testing). This continued until a minimum of 35 rewards were earned during a single session. During subsequent sessions, the pellet was then replaced with 10 µL 10% sucrose solution (w/v in tap water, infused over 0.3 seconds into a food cup within the reward-receptacle) until responding was stable (<20% coefficient between the number of reward-lever responses over 3 consecutive daily sessions). This procedure was repeated with 10% EtOH introduced into the solution (10% sucrose + 10% EtOH, v/v), then with the sucrose content reduced (5% sucrose + 10% EtOH, v/v), and finally with the sucrose removed entirely (10% EtOH, v/v).

Punishment and probe testing After completing training, there was a single punishment session which began with a baseline period identical to the training sessions. Beginning on the 11th press, and continuing throughout the session, EtOHlever presses alternatingly produced either 10% EtOH or no EtOH plus a 0.3 mA, 0.75-second footshock delivered through the grid floor. The session continued in this manner for 40 minutes. One day later, a probe test was conducted, during which all EtOH-lever responses were rewarded with 10% EtOH without concomitant punishment.

Dependent variables EtOH-lever presses, inactive lever presses and head entries into the reward receptacle were recorded by the Med-PC software. Approaches to the EtOH-lever with an elongated body posture, followed by rapid body retraction without a lever press were scored manually from video by an observer blind to experimental

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condition (Supplemental Video 1). This behavior resembles the stretched-attend posture previously observed in mouse anxiety-related assays (7) and in a rat punished operant test (8). We refer to this behavior as an ‘abort’ in keeping with the prior study by Hunt & Brady (8). All behavioral measures were expressed as the rate per minute.

In vivo neuronal recordings Electrode implantation Electrode arrays were implanted for in vivo neuronal recordings after training criterion was attained, prior to punishment, using previously described methods (9, 10). Mice were anaesthetized with 2% isoflurane (Baxter Healthcare, Deerfield, IL, USA) and placed in a stereotaxic apparatus (David Kopf Instruments, Tujunga, CA) to chronically implant fixed microelectrode arrays. Each array (Innovative Neurophysiology, Inc., Durham, NC, USA) contained 2 rows of 8 35-µm tungsten electrodes, with 150-µm electrode spacing and 200-µm row spacing. The array was bilaterally implanted with electrodes in the vmPFC and dmPFC (A/P +1.5 to +2.5 mm, M/L ± 0.5 mm, D/V -2.1 to -2.7 mm relative to bregma, at a +10° angle on the A/P axis). To verify electrode placement at the completion of testing, electrolytic lesions were made by passing 40 µA of current to each electrode for 2 seconds using a Square Pulse Stimulator (Grass Technologies, West Warwick, RI, USA), under isoflurane anesthesia. One day later, mice were terminally overdosed with ketamine/xylazine and transcardially perfused with 4% paraformaldehyde. Sagittal (50 µm-thick) sections were cut on a vibratome (Leica Biosystems, Buffalo Grove, IL, USA) mounted and stained for acetylcholinesterase. Following visual inspection using an Olympus BX41 microscope (Olympus, Center Valley, PA, USA), mice with inaccurate placements were excluded from further data analyses (electrode placement maps are shown in the Supplemental Figures).

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Neuronal recordings Recordings were conducted during punishment and probe testing using the OmniPlex D Neural Data Acquisition System (Plexon Inc., Dallas, TX, USA), with simultaneous behavioral analysis via CinePlex Behavioral Research Systems (Plexon). Extracellular waveforms exceeding a voltage threshold digitized at 40 kHz were manually sorted, with the aid of Plexon Offline Sorter software, using principal component analysis of spike clusters and visual inspection of waveforms and inter-spike intervals. Neuronal activity was timestamped to EtOH-lever presses and aborts. Because footshock interfered with the recording signal, neuronal data for EtOH-lever presses during punishment comprise only the EtOH-rewarded/non-shocked presses. Spike and timestamp information was integrated and analyzed using NeuroExplorer (NEX Technologies, Littleton, MA, USA). Neuronal activity in the ±2.5 seconds around each behavioral event was Z-score normalized to a 1-second pre-event period (i.e., -3.5 to -2.5 seconds pre-event) and presented in 300-millisecond bins stepped in 5-millisecond increments (11). A significant change in the populationcoding for a given event was determined by comparing, via Student’s t-test, the proportion of cells with a Z-score >2.0 during the 5-second peri-event-window to the 1-second baseline. To determine whether EtOH-lever and abort events were encoded by distinct populations of neurons, we conducted a correlational analysis on any unit that was responsive to either lever pressing or aborted presses (defined by having at least 3 baseline-normalized 100 ms bins in a 2 s window around the event that had a Z score of ±2.58). There was only 1 unit each for punishment and probe testing that exhibited firing rates in the 2 s window around EtOH-lever press that significantly correlated (P>.05) with firing rates during the same window around aborted presses.

Fluorescence in situ hybridization (RNAScope) A cohort of mice underwent training, punishment and a probe test as described above. Immediately after the probe test, mice were sacrificed via cervical dislocation and rapid decapitation. Brains were immediately extracted and frozen in 2-methylbutane (Sigma-Aldrich, St. Louis, MO) on dry ice and stored

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at -80°C. The next day, 16-µm coronal sections were cut at -20°C on a cryostat (Microm International GmbH, Waldorf, Germany), mounted, and fixed in 4°C 10% buffered formalin for 20 minutes. The slides were then rinsed in phosphate buffered saline (PBS), dehydrated in a sequential series of EtOH dilutions (50%, 70%, and 100% X 2), and stored overnight in 100% EtOH at -20°C. The next day, samples were processed using RNAscope Multiplex Fluorescent Assay Kit (Advanced Cell Diagnostics, Hayward, CA, USA) according to the manufacturer’s protocol and previous procedures in our laboratory (10). The target probes were Mm-fos (c-fos; gene ID 14281, cat#316921), Mm-Slc17a7 (vesicular glutamate transporter 1 (vGluT1); gene ID 72961, cat#416631), Mm-Gad1 (glutamate decarboxylase 1 (aka Gad67); gene ID 14415, cat#400951), Drd1a (dopamine receptor D1A; gene ID 13488, cat# 406491) and Drd2 (dopamine receptor 2; gene ID 13489, cat# 406501). After sections had been treated with an amplifier and fluorescent probes to separately label each gene, slides were incubated with DAPI for 20 seconds at room temperature and cover-slipped with Vectashield fluorescent mounting medium (Vector Laboratories, Burlingame, CA). For each brain, additional sections were processed as either a) a negative control to confirm the absence of background labeling on each channel using a bacterial mRNA (DapB of Bacillus subtilis strain; 3-plex Negative Control Probe, cat#320871), or b) a positive control to confirm the ability to detect the presence of labeling on each channel, using 3 housekeeping genes (RNA polymerase II subunit A (Polr2a), peptidylprolyl isomerase B (Ppib), ubiquitin C (Ubc); 3-plex Positive Control Probe, cat#320881). Images containing the entire vmPFC were obtained in each hemisphere on each of 3 fluorescent channels (405, 488, and 550 nm) (plus DAPI) with a Zeiss LSM 700 confocal microscope under a Plan-Apochromat 20x/0.8 M27 objective (Carl Zeiss Microscopy, Thornwood, NY, USA) and Zen lite digital imaging software (Zeiss). All DAPI-labeled neurons within a region estimated to comprise the entire vmPFC were marked with a circle drawn around the soma using ImageJ (NIH). For each fluorescent channel, the number of pixels within each neuron’s somatic area was calculated by ImageJ to determine whether a given cell was fos plus

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Slc17a7 or Gad1 positive. Data were expressed as the percentage of DAPI cells that were fos-positive and the number of those fos-positive cells that were also either Slc17a7 or Gad1 positive.

Ex vivo neuronal recordings For virus delivery, D1-tdTomato mice were anesthetized and placed in a stereotaxic apparatus to infuse 0.2 µL per hemisphere of AAV5/CaMKIIα-hChR2(H134R)-eYFP (1.1 x13) - to transfect CaMKIIpositive vmPFC projection neurons with ChR2 (UNC Vector Core). Infusions were bilaterally directed at the vmPFC (coordinates as above). After at least 1-week post-surgery, mice were trained and tested as above. Because we observed that mice without indwelling cranial implants (for in vivo recordings and optogenetics) exhibited less pronounced suppression following the 0.3 mA shock level, the intensity was increased slightly to 0.35 mA to ensure robust suppression in this experiment. Immediately following the probe test, mice were anesthetized with isoflurane and quickly decapitated. Brains were dissected and transferred to ice-cold artificial cerebrospinal fluid (ACSF) containing (in mM): 225 sucrose, 13.9 NaCl, 26.2 NaHCO3, 1 NaH2PO4, 1.25 glucose, 2.5 KCl, 0.1 CaCl2, 4.9 MgCl2, and 3 kynurenic acid saturated with 95% O2-5% CO2. Coronal slices containing the NAcS were cut at 240 μm and incubated in ACSF containing (in mM): 124 NaCl, 1 NaH2PO4, 2.5 KCl, 1.3 MgCl2, 2.5 CaCl2, 20 glucose, 26.2 NaHCO3, and 0.4 ascorbic acid saturated with 95% O2-5% CO2 at 33°C for at least 30 minutes. Slices were shielded from light to prevent photobleaching, at room temperature, before being transferred to the recording chamber. Individual tdTomato-expressing (D1+) and td-Tomato-non-expressing (D1-) cells were identified under an Olympus BX51WI microscope and voltage-clamped at +40 mV in whole-cell configuration in an ACSF perfusate containing 5 μM gabazine to block GABAA receptors. Patch pipettes were filled with an intracellular solution containing (in mM): 120 cesium methanesulfonate, 20 CsCl, 10 Hepes, 0.2 EGTA, 10 sodium phosphocreatine, 4 Na2-ATP, and 0.4 Na-GTP, 100 spermine tetrahydrochloride (pH, 7.25; 290– 310 mOsm). (R)-CPP (5 μM) was added to the perfusion to block NMDA currents. Combined EPSCs were optically evoked with a 470 nm LED (1 pulse every 15 sec, pulse width 0.6-2.0 ms). AMPA-mediated 19

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EPSCs were subtracted from the combined EPSCs to obtain the NMDA-mediated EPSC component. AMPA:NMDA ratios were calculated by dividing the peak receptor-mediated EPSC by the peak NMDAmediated EPSC. AMPA-mediated EPSCs were measured at both positive and negative holding potentials of +40 mV and -55mV, respectively to assess rectification. AMPA rectification was calculated by dividing the peak AMPA EPSC amplitudes recorded at -55 mV by the EPSC amplitudes recorded at +40 mV. Data was acquired with a Multiclamp 700B amplifier and digitized using IGOR Pro 7 (WaveMetrics, Lake Oswego, OR, USA) running the mafPC program (courtesy of M. A. Xu-Friedman).

In vivo photosilencing vmPFC and dmPFC neurons Virus infusion and optical fiber implantation. For the punishment experiments, viral vector infusions were done after training criterion was attained, prior to punishment, using previously described methods (12, 13). For the non-punishment control experiments, viral vector infusions were conducted prior to training (see below). For virus delivery, mice were anesthetized and placed in a stereotaxic apparatus to infuse 0.3 µL per hemisphere of either rAAV8/CAG-ArchT-GFP (1 x 10^12 vp/ml) – to transfect cells with the light-responsive inhibitory opsin, archaerhodopsin, ArchT - or a light-unresponsive control (rAAV8/CAG-GFP), obtained from the UNC Vector Core (Chapel Hill, NC, USA). Viral vectors were infused using a 33-gauge Hamilton syringe at a rate of 0.02 µL per minute, which remained in place for 10 minutes after infusion to ensure diffusion. Infusions were bilaterally directed at either the vmPFC (A/P +1.65 mm, M/L ±0.95 mm, D/V -2.9 mm relative to bregma, infused at +20° angle on the M/L axis) or, in a separate experiment, the dmPFC (A/P +1.85 mm, M/L ±0.95 mm, D/V -2.2 mm relative to bregma, infused at +20° angle on the M/L axis). During the same surgery, ferrules (numerical aperture 0.37) containing optic fibers were chronically implanted 0.1 mm above the infusion site.

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Closed-loop photosilencing For the punishment experiments, at least 4 weeks after viral infusion, mice received 1-2 training ‘reminder’ sessions to ensure EtOH-seeking remained robust. Punishment occurred the following day. A probe test was conducted the next day, during which green light (λ = 532 nm, 10 mW) was delivered through 65.5 μm bifurcated patch cable (Fiber Optics For Sale Co, Fremont, CA, USA) coupled to a 200-mW, 532nm, laser system (Opto Engine, Midvale, UT, USA) when the mouse entered a zone (⁓2.75 x 4 inches) around the EtOH-lever, as determined in real-time by the CinePlex software, until exiting. The location and size of this zone was chosen after examination of behavioral patterns of the mice used in recording experiments. Laser power at the tip of the fiber was measured before each test session using a power meter (PM20, Thorlabs, Newton, NJ, USA) and adjusted to achieve 10 mW. The estimated irradiance at 0.5 mm from the fiber tip was 4.87 mW/mm2 based on 10 mW at the fiber tip, N.A. 0.37 and 561 nm wavelength light (http://www.stanford.edu/group/dlab/cgi-bin/graph/chart.php). For the non-punishment control experiments, mice were trained as described above and then underwent a training EtOH-seeking session in the absence of punishment whereby entries into the EtOH-lever zone triggered green light delivery in an identical manner as above.

Neuronal recordings during photosilencing To validate efficacy of the viral construct used for closed-loop photosilencing experiments, mice (n=2) received a unilateral microinfusion of AAV5/CaMKII-eArchT3.0-eYFP (3 x 10^12 vp/ml) directed at the vmPFC (A/P +1.65 mm, M/L ±0.95 mm, D/V -2.9 mm relative to bregma, infused at +20° angle on the M/L axis). During the same surgery, a ferrule (numerical aperture 0.37) containing an optic fiber, which was affixed to an electrode array (Innovative Neurophysiology, Inc., Durham, NC, USA) so that the tip of the optic fiber was sitting adjacent to the center of the electrode array tips, was chronically implanted 0.1 mm above the infusion site. At least 4 weeks after recovery from surgery, mice underwent in vivo recordings in their home cage, during which green light (λ = 532 nm, 10 mW) was delivered through a 65.5 μm patch cable (Fiber Optics For Sale Co, Fremont, CA, USA) coupled to a 200-mW, 532-nm, laser system (Opto 21

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Engine, Midvale, UT, USA) to record single unit responses to light delivery (Figure 3B). Neural spike sorting procedures were carried out as described above.

Photosilencing the vmPFCNAcS and vmPFCBLA pathways Procedures were the same as described above for the local PFC-silencing experiments with the exception that surgery was performed prior to, rather than after, training, in order to avoid a prolonged gap in behavioral testing during the period (at least 5 weeks) necessary to ensure virus expression in projections. For virus delivery, mice were anesthetized and placed in a stereotaxic apparatus to infuse 0.3 µL per hemisphere of either AAV5/CaMKII-eArchT3.0-eYFP (3 x 10^12 vp/ml) - to transfect CaMKII-positive vmPFC projection neurons with ArchT - or a light-unresponsive control (AAV5/CaMKII-eYFP), obtained from the UNC Vector Core. Infusions were bilaterally directed at the vmPFC (coordinates as above) and ferrules implanted bilaterally in either the NAcS (A/P +1.7 mm, M/L ±1.75 mm, D/V -4.75 mm relative to bregma; +10° mediolateral axis) or, in a separate cohort of mice, the BLA (A/P -1.5 mm, M/L ±3.3 mm, D/V -4.55 mm relative to bregma).

Photosilencing the vmPFC during non-punished EtOH-seeking To assess vmPFC contributions to EtOH-seeking in the absence of punishment, a separate cohort of mice was infused with virus (as described above in Photosilencing the vmPFCNAcS and vmPFCBLA pathways) and implanted with fiber optic ferrules into the vmPFC (as described above in In vivo photosilencing vmPFC and dmPFC neurons). Photosilencing of the vmPFC was conducted during an EtOH-seeking session (no punishment).

Virus expression and fiber placement verification. Mice were terminally overdosed to obtain brain sections using the same methods described above. Sections were cover-slipped with Vectashield mounting medium containing 4’,6-diamidino-2-phenylindole

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(DAPI) (Vector Laboratories, Burlingame, CA, USA) and visually inspected with the aid of an Olympus BX41 microscope. Mice found to have poor or inaccurate virus expression or fiber placements were excluded from further data analyses (optrode placement maps are shown in the Supplemental Figures). (Figure 3B).

Retrograde neuronal tracing The retrograde tracer Cholera Toxin B (CTb), conjugated to Alexa Fluor 555 (cat# C34776, 0.5% dilution, Thermo Fisher Scientific, Waltham, MA, USA) or Alexa Fluor 488 (cat# C4775, 0.5% dilution, Thermo Fisher Scientific) was injected into the NAcS and BLA, respectively (coordinates as above), at a volume of 0.2 µl. Seven days later, mice were deeply anaesthetized with pentobarbital and transcardially perfused with PBS followed by 4% PFA. Coronal (50-µm thick) sections were cut on a vibratome (as above) and imaged using a with a Zeiss LSM 700 confocal microscope under a Plan-Apochromat 20x/0.8 NA M27 objective (Carl Zeiss Microscopy, Thornwood, NY, USA).

Statistical analysis Group differences were analyzed using Student’s t-tests or analysis of variance (ANOVA) followed by Newman-Keuls and Šidák corrected post hoc tests using the IGRO Pro 7, Prism and StatView programs. The threshold for statistical significance was set at P<.05.

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Supplemental References 1. Marchant NJ, Campbell EJ, Pelloux Y, Bossert JM, Shaham Y (2018): Context-induced relapse after extinction versus punishment: similarities and differences. Psychopharmacology. 2. Halladay LR, Kocharian A, Holmes A (2017): Mouse strain differences in punished ethanol selfadministration. Alcohol. 58:83-92. 3. Bouton ME, Schepers ST (2015): Renewal after the punishment of free operant behavior. J Exp Psychol Anim Learn Cogn. 41:81-90. 4. Pelloux Y, Hoots JK, Cifani C, Adhikary S, Martin J, Minier-Toribio A, et al. (2018): Contextinduced relapse to cocaine seeking after punishment-imposed abstinence is associated with activation of cortical and subcortical brain regions. Addiction biology. 23:699-712. 5. Radke AK, Jury NJ, Kocharian A, Marcinkiewcz CA, Lowery-Gionta EG, Pleil KE, et al. (2017): Chronic EtOH effects on putative measures of compulsive behavior in mice. Addiction biology. 22:423-434. 6. Jury NJ, Pollack GA, Ward MJ, Bezek JL, Ng AJ, Pinard CR, et al. (2017): Chronic Ethanol During Adolescence Impacts Corticolimbic Dendritic Spines and Behavior. Alcoholism, clinical and experimental research. 41:1298-1308. 7. Holmes A, Rodgers RJ (2003): Prior exposure to the elevated plus-maze sensitizes mice to the acute behavioral effects of fluoxetine and phenelzine. Eur J Pharmacol. 459:221-230. 8. Hunt HF, Brady JV (1955): Some effects of punishment and intercurrent anxiety on a simple operant. J Comp Physiol Psychol. 48:305-310. 9. Gamble-George JC, Baldi R, Halladay L, Kocharian A, Hartley N, Silva CG, et al. (2016): Cyclooxygenase-2 inhibition reduces stress-induced affective pathology. eLife. 5. 10. Gunduz-Cinar O, Brockway E, Lederle L, Wilcox T, Halladay LR, Ding Y, et al. (2018): Identification of a novel gene regulating amygdala-mediated fear extinction. Molecular psychiatry. 11. Seo M, Lee E, Averbeck BB (2012): Action selection and action value in frontal-striatal circuits. Neuron. 74:947-960. 12. Bukalo O, Pinard C, Silverstein S, Brehm C, Hartley N, Whittle N, et al. (2015): Prefrontal inputs to the amygdala instruct fear extinction memory formation. Science Advances. 1:e1500251. 13. Bergstrom HC, Lipkin AM, Lieberman AG, Pinard CR, Gunduz-Cinar O, Brockway ET, et al. (2018): Dorsolateral Striatum Engagement Interferes with Early Discrimination Learning. Cell reports. 23:2264-2272.  

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