This work was funded in its entirety by a UK Medical Research Council grant (grant reference: MR/M007006/1) awarded to Sunjeev Kamboj and Ravi Das. The refinement of the memory retrieval procedure and sourcing of cue images was performed by Grace Gale. We would like to thank Katie Walsh and Josie Hanniford for contributions towards piloting the procedure.

All authors have no intellectual, financial, or biomedical conflicts of interest to disclose.

The protocol in this paper describes an alcohol MRM retrieval procedure that explicitly generates prediction error in alcohol delivery (Retrieval + PE) to maximize the probability that naturalistic alcohol MRMs will destabilize. This retrieval procedure takes account of recent experimental work demonstrating the necessity of prediction error for memory destabilization and engaging the reconsolidation process60. It has been shown to produce the most profound changes in indices of alcohol MRMs from subsequent behavioral interventions (counterconditioning and cognitive reappraisal) in two papers28,46 and is currently being further validated in drug models. Since this effect appears to be independent of learning history, the procedure represents a significant improvement over other memory reactivation techniques, which do not incorporate a PE, and should be used as a platform for the assessment of post-destabilization interventions for reducing maladaptive drinking behaviour. Such refinement of techniques is particularly important in light of null findings in the reconsolidation field (see below). Moreover, the RET+PE method has considerable applied significance and may be applicable in combination with a variety of post-retrieval relearning methods. For example, we have demonstrated effects on semantic memory and craving using RET+PE followed by reappraisal, a prototypical adaptive emotion regulation technique commonly used in cognitive behavioural therapy46.
The procedure is easily modified to suit the characterisitics of the participant group. For example, the stimuli used as CS+s during counterconditioning can be changed to wine pictures, if the participants are primarily wine preferring. Alternatively, smoking-related stimuli can be used if the procedure is employed in tobacco smokers.
Effective counterconditioning relies on potent disgust cues. To ensure sufficient relearning during reconsolidation, select visual UCSs that are likely to elicit powerful disgust responses. Although these are visual cues, they should have relevance to gustatory disgust (e.g., contamination-related images; images of food spoilage). If an alternative gustatory UCS (other than denatonium benzoate at the concentrations used here) is used, we recommend a compound that strongly activates bitterness responses.
Although we use an expectancy violation, it is theoretically possible to generate PE using other methods, although their efficacy in destabilizing robustly encoded reward memories has yet to be demonstrated. We have trialed an alternative procedure involving unexpected devaluation of alcohol (using unexpectedly bitter tasting alcohol), although we did not demonstrate convincing memory modifications using this procedure46.
Randomization of participants to groups should prevent baseline differences in trait questionnaire measures. If this is the case, however, these differences should be modelled as random effects in mixed-effects analyses of outcomes. A supplementary approach, which the authors recommend, is to include an extra baseline day prior to the retrieval and counterconditioning day. This allows better assessment of pre-existing group differences, the reliability of these differences, and gives researchers the option of stratified randomization to groups to prevent such differences.
In order to generate an effective PE, it is essential that participants experience a relevant surprising occurrence during retrieval. If an expectancy violation is used, as described here, ensure that participants are observed during this step so that they do not inadvertently consume the alcohol at a point they are instructed not to. Related to this, it is essential that participants are not aware of the experimenter&#39;s intention to withhold alcohol after generating the expectation of alcohol reward. As such, the protocol involves a necessary degree of deception. Upon debriefing, ensure participants are aware that they should not discuss the protocol with others.
While there is evidence that the procedure robustly destabilizes memories and has been designed to maximize the probability of doing so, there is currently no independent means of assessing whether or not destabilization has occurred. This is a limitation of all memory destabilization procedures described to date and as such, null effects of post-destabilization interventions at test are difficult to interpret, as they may be due to low efficacy of the intervention or a failure to sufficiently destabilize MRMs in the first place.
The remarkable generalization of Ret+PE dependent counterconditioning effects to non-trained stimuli makes this approach highly promising for clinical implementation. However, our demonstration of efficacy is limited to an experimental (non-clinical) setting with problem drinkers, who do not have an alcohol use disorder.
Relatedly, although the procedure is easily adapted, it is unknown whether the RET+PE procedure is similarly efficacious (in a modified form) in destabilizing MRMs in different (illicit) substance using populations. We believe it would be at least as effective, as alcohol MRMs are likely to be more overlearned, robust, and cross-contextual than MRMs for most illicit drugs. This remains to be verified experimentally however, and it is unknown how sensitive the efficacy of the procedure is to variations in number of cues presented or their exact nature. We therefore encourage experimentation with the procedure in different drug-using populations and with different retrieval cues, as well as tailoring post-destabilization interventions to population-specific reward maladaptations.
To address the measurement issues outlined above - namely the reliance on efficacy of the post-destabilization intervention to infer effective memory destabilization - independent measures of destabilization are required. We are currently developing electroencephalographic and psychophysiological techniques for resolving this issue and will make these available upon request when completed.
Implementation across the range of severities of problem drinking is required to establish the clinical utility of this technique. Moreover, clinically-oriented studies are essential to determine whether this procedure will be acceptable to treatment seekers.

Building on seminal work of Lewis and colleagues1 over the last twenty years has highlighted an unprecedented potential for plasticity in established (consolidated) memories via the process of memory reconsolidation2,3. Reconsolidation occurs under certain circumstances when memories are retrieved4 and consists of two temporally5 and molecularly6 dissociable stages: initial destabilization7&#160;and subsequent restabilization8. Following retrieval-induced memory destabilization, memories enter an &#39;active&#39;, malleable state, in which they are susceptible to modification through incorporation of novel information9,10,11,12, or weakening through pharmacological interference with the molecular pathways required for restabilization13,14,15,16. This &#39;reconsolidation window,&#39; the interval between destabilization and restabilization, lasts 1 to 6 h and represents a unique opportunity to directly interfere with the maladaptive reward memories (MRMs) that are believed to play a key role in the aetiology17, progression18, and maintenance19,20 of substance use disorders (SUDs) like alcohol use disorder.
MRMS are associations formed through Pavlovian mechanisms that link environmental stimuli or &#39;drug cues&#39; to the availability, and intoxicating and rewarding effects of drugs21. MRMs imbue drug cues with enhanced value, salience22, and motivational properties such that these cues grab attention23, trigger craving and motivate drug seeking-and-using behaviour in people with SUDs. These MRMs are therefore thought to be responsible for triggering relapse when drug cues are encountered24. The primary challenge in the long-term clinical management of SUDs is therefore to reduce the ability of MRMs to promote drug-taking, which, by extension, should reduce the incidence of relapse21. The reconsolidation window offers an exciting opportunity to achieve this aim by updating MRMs while they are unstable to a form that is less likely to contribute to relapse.
Recent research has shown that this can be achieved using purely behavioral interventions10,25. These can produce profound reductions in craving and drug-related reward metrics in heroin addicts26, smokers27, and hazardous drinkers28. By giving a brief reminder of MRMs (typically brief exposure to drug cues) to destabilize them, and subsequently re-associating drug cues with lack of drug (extinction), or aversive outcomes (counterconditioning)29 while MRMs are unstable, the associations between drug cues and outcomes restabilize in an updated, more adaptive form. These rewritten MRMs no longer support relapse. The only difference between these and standard extinction or counterconditioning interventions is the retrieval and destabilization of MRMs - produced by brief exposure to drug cues - prior to the learning intervention. They are therefore typically referred to as &#39;retrieval-extinction&#39; or &#39;retrieval-counterconditioning&#39; interventions.
However, there is now a significant body of research showing inconsistent results with these interventions30,31,32,33. This may be partially explained by individual differences in the expression of plasticity-promoting proteins34. However, since retrieval is necessary but not sufficient4,35,36 for memory destabilization, there are also certain boundary conditions that determine whether a memory destabilizes or not. The extent to which these boundary conditions were operative likely also varied from study-to-study. Chief among these conditions are memory age (chronology)37 and strength (reinforcement history)38,39, duration of the reminder trial5, and the occurrence of mismatch35,40 between predicted and actual outcome during retrieval, known as prediction error (PE)41,42,43. These factors are inter-related, as PE is the central reward &#39;learning signal&#39;44,45 and diminishes over the course of learning. PE encodes the difference between expected outcome encoded in memory and actual outcome, reflecting the inaccuracy of learned predictions. It is by minimizing the occurrence of PE that learning accrues. Old, strongly reinforced memories like MRMs - which have formed over many years - have extremely good predictive power for outcomes across different situations. As such, little PE will be generated when MRMs are retrieved under normal conditions, conferring these traces with destabilization-resistance.
Leveraging this knowledge, we developed a retrieval and PE-generation (Retrieval + PE) procedure for alcohol MRMs that aims to maximize the likelihood of these memories destabilizing by maximizing PE at retrieval. We have shown that subsequent counterconditioning causes comprehensive updating of alcohol MRMs in hazardous drinkers28,46. This manuscript describes the implementation of the Retrieval + PE procedure for destabilizing alcohol MRMs, along with a multisensory counterconditioning technique that rewrites these memories.
The Retrieval + PE procedure involves the use of verbal instructions to maximize participants&#39; expectancy of alcohol reward, then preventing alcohol consumption at the last moment. Without knowledge of the exact learning history, this PE maximization putatively maximizes the likelihood of MRMs destabilization and provides a platform for testing the efficacy of post-destabilization behavioral interventions in improving indices of maladaptive reward memory. Here we describe how to implement this procedure with subsequent disgust-based counterconditioning, which we28 and others29 have shown to be an effective learning modality for updating MRMs. However, the Retrieval + PE procedure could also be used in combination with alternative post-retrieval drug interventions and many other forms of learning-based interventions25.

<table><tbody><tr><td>Multipipette M4 x 1</td><td>Eppendorf</td><td>4982 000.012</td><td>High-accuracy pipette for dosing Bitrex</td></tr><tr><td>Combitips advanced 0.2ml x 1000</td><td>Eppendorf</td><td>0030 089.413</td><td>Syringe tip for use with Micropipette for dosing Bitrex</td></tr><tr><td>Simax 100ml measuring cylinder</td><td>Phillip Harris</td><td>B8A73325</td><td>Any suitable measuring cylinder may be used</td></tr><tr><td>Bitrex (2.5% w/v aqueaous Denatonium Benzoate solution)</td><td>Mcfarlan Smith</td><td>N/A</td><td>Bitter solution for counterconditioning drink UCSs</td></tr><tr><td>International Affective Picture System images</td><td>International Affective Picture System (Lang et al, 2008)</td><td>(http://csea.phhp.ufl.edu/media/iapsmessage.html)</td><td>Upon request from Margaret Bradley&amp;nbsp;</td></tr><tr><td>Stimulus presentation monitor with 1024 x768 colour display minimum</td><td>Dell&amp;nbsp;</td><td>N/A</td><td></td></tr><tr><td>Eyelink 1000 Core System</td><td>SR Research</td><td>Desktop-type mount</td><td>Other eye-tracking systems can be used</td></tr><tr><td>Experiment Builder</td><td>SR Resesarch</td><td>N/A</td><td>Other systems (Presentation/MATLAB/ePrime/Psychopy etc) can be used</td></tr><tr><td>DataViewer</td><td>SR Resesarch</td><td>N/A</td><td>Use software as appropriate to eye tracker setup</td></tr><tr><td>SPSS 21.0 for Windows</td><td>IBM</td><td>Base Package</td><td>Use any appopriate statistical software</td></tr><tr><td>Lion Alcometer 500</td><td>Lion Laboratories</td><td>N/A</td><td>Any accurate alcohol breath testing device may be used</td></tr><tr><td>Terumo 5ml syringe</td><td>MediSupplies</td><td>PMCO595</td><td>Any 5ml syringe may be used</td></tr><tr><td>Mini Table Top Scale</td><td>On Balance</td><td>MTT-200</td><td>Any high-accuracy scales may be used</td></tr><tr><td>Non-alcoholic beer</td><td>Bavaria</td><td>N/A</td><td>Any brand non-acloholic beer may be used</td></tr></tbody></table>

a prediction error-driven retrieval procedure for destabilizing and rewriting maladaptive reward memories in hazardous drinkers

Maladaptive reward memories (MRMs) can become unstable following retrieval under certain conditions, allowing their modification by subsequent new learning. However, robust (well-rehearsed) and chronologically old MRMs, such as those underlying substance use disorders, do not destabilize easily when retrieved. A key determinate of memory destabilization during retrieval is prediction error (PE). We describe a retrieval procedure for alcohol MRMs in hazardous drinkers that specifically aims to maximize the generation of PE and therefore the likelihood of MRM destabilization. The procedure requires explicitly generating the expectancy of alcohol consumption and then violating this expectancy (withholding alcohol) following the presentation of a brief set of prototypical alcohol cue images (retrieval + PE). Control procedures involve presenting the same cue images, but allow alcohol to be consumed, generating minimal PE (retrieval-no PE) or generate PE without retrieval of alcohol MRMs, by presenting orange juice cues (no retrieval + PE). Subsequently, we describe a multisensory disgust-based counterconditioning procedure to probe MRM destabilization by re-writing alcohol cue-reward associations prior to reconsolidation. This procedure pairs alcohol cues with images invoking pathogen disgust and an extremely bitter-tasting solution (denatonium benzoate), generating gustatory disgust. Following retrieval + PE, but not no retrieval + PE or retrieval-no PE, counterconditioning produces evidence of MRM rewriting as indexed by lasting reductions in alcohol cue valuation, attentional capture, and alcohol craving.

Compare group means at baseline for subjective measures. Groups should be equivalent on baseline intake of alcohol, disgust sensitivity/propensity, SOCRATES and NAEQ scores, and momentary craving (ACQ-NOW). If there are differences in these measures at baseline, analysis should continue using mixed-effects models and these measures should be included as random-effects59. Performance of these analyses are not described here. Assess frequency of positive alcohol breathalyzer readings in each group and report this as an outcome.
Pleasantness ratings of CS+s and CS-s did not differ at baseline (before counterconditioning), but diverged over trials of counterconditioning, with pleasantness ratings for beer CS+s reducing significantly through repeated pairing with the pictorial and bitter drink UCSs. If CS+s and CS-s differ at baseline, use a mixed-effects model with random intercepts and slopes to analyze these data. The CS-s are never paired with UCSs, so do not show a decline in pleasantness. This is represented by a CS Type (CS+ vs. CS-) x Trial (Baseline, Trial 1, 2, 3 &#38; 4) interaction if counterconditioning has been successful, as indicated in Figure 2 (representative data from Das et al.28)
Evidence of rewriting of MRM networks is provided by a generalized reduction in pleasantness ratings to alcohol stimuli, but not neutral stimuli at follow up (Day 8). In the RET+PE group, reduced pleasantness ratings were evident in response to CS+s used in the retrieval/counterconditioning task (Beer CS+s), as well as to novel beer and wine images rated for the first time at follow up. Statistically this was evident in a CS Type (Beer CS+, Neutral CS-, Novel Beer, Novel Wine) x Group ( No RET+PE, RET+PE, RET no PE) interaction. Example results showing this pattern taken from Das et al.28 are shown in Figure 3.
Examine this pattern of results in the optional attentional bias task. Attentional bias is calculated as dwell time on a target alcohol image - dwell time on its composition-matched non-target pair image. A score of 0 indicates no bias, positive score attentional bias towards alcohol stimuli, and a negative score an oculomotor aversion to alcohol pictures. Representative results taken from Das et al.28 are shown in Figure 4. It can be seen that an oculomotor aversion to alcohol images at follow up (Day 8) was produced in the RET+PE group only, indicated by a significant Group x CS Type interaction.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56097/56097fig1.jpg" /> 
Figure 1: A Schematic of the three retrieval groups and prediction error procedures. 
Key to abbreviations: RET + PE = Retrieval + Prediction Error; the group who retrieve alcohol memories and have expected alcohol withheld. RET + No PE = Retrieval + No Prediction Error; the group who retrieve alcohol memories and consume alcohol as expected. No RET + PE = No retrieval + Prediction Error; this group does not retrieve alcohol memories and has expected orange juice withheld. In vivo drink: A 150 mL glass of beer in RET + PE and RET + No PE or a 150 mL glass of orange juice in the no RET group. 1) In vivo drink: The drink relevant to the particpants&#39; group is placed in front of participants and they are told that they will consume this drink according to prompts they will see on screen; after rating a series of pictures for pleasantness. 2) Cue image rating: Participants in RET + PE and RET + No PE rate four images of beer and two images of soft drinks (coffee and cola) for pleasantness. Brief in vivo exposure to beer and rating of beer images triggers retrieval of drinking memories in these groups. The No RET group rates four images of orange juice and the soft drink images for pleasantness and thus do not retrieve alcohol memories. 3) Prediction error: As all participants are told that they will drink the in vivo drink at the start of the procedure, PE is engendered by preventing participants in RET + PE and No RET + PE from consuming the drink in front of them. In RET + No PE, the drink is consumed as expected, engendering no PE. 4) Distractors: High working-memory tasks are performed (prose recall, digit span, and category and verbal fluency) to distract participants and disengage them from the retrieval task. 5) Intervention: a counterconditioning task where beer images are paired with disgust-inducing images and very bitter drinks. This procedure aims to create an association between beer images and the experience of disgust that overwrites existing associations in the RET + PE group due to destabilization of alcohol memories. <a href="https://cloudflare.jove.com/files/ftp_upload/56097/56097fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56097/56097fig2.jpg" /> 
Figure 2: Mean pleasantness ratings (&#177; SEM) of neutral and beer cues during counterconditioning. Representative changes in pleasantness ratings of CS+s (beer images paired with disgusting outcomes) and CS-s (neutral images paired with neutral outcomes) over the course of counterconditioning, taken from Das et al.28 As counterconditioning aims to invoke conditioned disgust in response to beer images, perceived pleasantness of these images should reduce during counterconditioning. Ratings do not differ at baseline, but reduce to CS+s over the course of counterconditioning. ** = CS+ &#62; CS- at p&#60; 0.001. <a href="https://cloudflare.jove.com/files/ftp_upload/56097/56097fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56097/56097fig3.jpg" /> 
Figure 3: Mean pleasantness ratings (&#177; SEM) of counterconditioned, novel alcohol cues and neutral cues at test. Representative generalized reductions in pleasantness ratings (liking) of alcohol images seven days after counterconditioning is performed following retrieval (RET) with prediction error (PE) (RET +PE) from Das et al.28 This pattern is consistent with rewriting of reward associations relating to alcohol in RET + PE. Key: Neutral CS- = coffee and cola images paired with neutral images during counterconditioning. Beer CS+ = beer images paired with disgusting outcomes during counterconditioning. Novel Beer = previously unseen beer images. Wine = previousy unseen wine images. * = significant pairwise comparisons at p&#60;0.05, Bonferroni corrected. <a href="https://cloudflare.jove.com/files/ftp_upload/56097/56097fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56097/56097fig4.jpg" /> 
Figure 4: Mean attentional bias to counterconditioned, novel alcohol cues and neutral cues at test. Representative generalized abolition of attentional bias to all alcohol stimuli in the group that underwent counterconditioning after RET + PE from Das et al28. Bars indicate mean attentional bias scores, calculated as the averaged difference in total time looking at a &#39;target image&#39; containing visible alcohol vs. a composition-matched &#39;non target&#39; imagelacking visible alcohol. Error bars are SEMs. Neutral CS- = coffee and cola images paired with neutral images during counterconditioning. Beer CS+ = beer images paired with disgusting outcomes during counterconditioning. Novel Beer = previously unseen beer images. Wine = previously unseen wine images. * = significant pairwise comparisons at p&#60;0.05, Bonferroni corrected. <a href="https://cloudflare.jove.com/files/ftp_upload/56097/56097fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>TRIAL NUMBER</td>
			<td colspan="2">CS TYPE</td>
			<td colspan="2">UCS TYPE</td>
			<td colspan="2">UCS FILE NAME</td>
		</tr>
		<tr>
			<td>1</td>
			<td colspan="2">Beer CS+1</td>
			<td colspan="2">GUSTATORY</td>
			<td colspan="2">DRINK_SCREEN.PNG</td>
		</tr>
		<tr>
			<td>2</td>
			<td colspan="2">Neutral CS-1</td>
			<td colspan="2">NEUTRAL</td>
			<td colspan="2">7020.jpg</td>
		</tr>
		<tr>
			<td>3</td>
			<td colspan="2">Beer CS+1</td>
			<td colspan="2">GUSTATORY</td>
			<td colspan="2">DRINK_SCREEN.PNG</td>
		</tr>
		<tr>
			<td>4</td>
			<td colspan="2">Neutral CS-2</td>
			<td colspan="2">NEUTRAL</td>
			<td colspan="2">7019.jpg</td>
		</tr>
		<tr>
			<td>5</td>
			<td colspan="2">Beer CS+4</td>
			<td colspan="2">PICTORIAL</td>
			<td colspan="2">rotten_foot.jpg</td>
		</tr>
		<tr>
			<td>6</td>
			<td colspan="2">Beer CS+3</td>
			<td colspan="2">PICTORIAL</td>
			<td colspan="2">9325.jpg</td>
		</tr>
		<tr>
			<td>7</td>
			<td colspan="2">Beer CS+2</td>
			<td colspan="2">GUSTATORY</td>
			<td colspan="2">DRINK_SCREEN.PNG</td>
		</tr>
		<tr>
			<td>8</td>
			<td colspan="2">Beer CS+4</td>
			<td colspan="2">PICTORIAL</td>
			<td colspan="2">9325.jpg</td>
		</tr>
		<tr>
			<td>9</td>
			<td colspan="2">Beer CS+3</td>
			<td colspan="2">PICTORIAL</td>
			<td colspan="2">rotten_foot.jpg</td>
		</tr>
		<tr>
			<td>10</td>
			<td colspan="2">Neutral CS-1</td>
			<td colspan="2">NEUTRAL</td>
			<td colspan="2">7020.jpg</td>
		</tr>
		<tr>
			<td>11</td>
			<td colspan="2">Beer CS+1</td>
			<td colspan="2">GUSTATORY</td>
			<td colspan="2">DRINK_SCREEN.PNG</td>
		</tr>
		<tr>
			<td>12</td>
			<td colspan="2">Neutral CS-2</td>
			<td colspan="2">NEUTRAL</td>
			<td colspan="2">7019.jpg</td>
		</tr>
		<tr>
			<td>13</td>
			<td colspan="2">Neutral CS-1</td>
			<td colspan="2">NEUTRAL</td>
			<td colspan="2">7019.jpg</td>
		</tr>
		<tr>
			<td>14</td>
			<td colspan="2">Beer CS+2</td>
			<td colspan="2">GUSTATORY</td>
			<td colspan="2">DRINK_SCREEN.PNG</td>
		</tr>
		<tr>
			<td>15</td>
			<td colspan="2">Beer CS+4</td>
			<td colspan="2">PICTORIAL</td>
			<td colspan="2">9405.jpg</td>
		</tr>
		<tr>
			<td>16</td>
			<td colspan="2">Beer CS+1</td>
			<td colspan="2">GUSTATORY</td>
			<td colspan="2">DRINK_SCREEN.PNG</td>
		</tr>
		<tr>
			<td>17</td>
			<td colspan="2">Beer CS+4</td>
			<td colspan="2">PICTORIAL</td>
			<td colspan="2">9301.jpg</td>
		</tr>
		<tr>
			<td>18</td>
			<td colspan="2">Neutral CS-2</td>
			<td colspan="2">NEUTRAL</td>
			<td colspan="2">7019.jpg</td>
		</tr>
		<tr>
			<td>19</td>
			<td colspan="2">Beer CS+3</td>
			<td colspan="2">PICTORIAL</td>
			<td colspan="2">9405.jpg</td>
		</tr>
		<tr>
			<td>20</td>
			<td colspan="2">Beer CS+2</td>
			<td colspan="2">GUSTATORY</td>
			<td colspan="2">DRINK_SCREEN.PNG</td>
		</tr>
		<tr>
			<td>21</td>
			<td colspan="2">Beer CS+3</td>
			<td colspan="2">PICTORIAL</td>
			<td colspan="2">9301.jpg</td>
		</tr>
		<tr>
			<td>22</td>
			<td colspan="2">Neutral CS-2</td>
			<td colspan="2">NEUTRAL</td>
			<td colspan="2">7019.jpg</td>
		</tr>
		<tr>
			<td>23</td>
			<td colspan="2">Beer CS+2</td>
			<td colspan="2">GUSTATORY</td>
			<td colspan="2">DRINK_SCREEN.PNG</td>
		</tr>
		<tr>
			<td>24</td>
			<td colspan="2">Neutral CS-1</td>
			<td colspan="2">NEUTRAL</td>
			<td colspan="2">7020.jpg</td>
		</tr>
	</tbody>
</table>
Table 1: Pseudorandomized trial order for counterconditioning task used in Das et al.28 
CS Type = Conditioned stimulus type. There are four beer images used in the counterconditioning. These are arbitrarily numbered Beer CS+ 1 to 4. Two neutral images of coffee and cola are also used and these are arbitrarily numbered Neutral CS-1 and Neutral CS-2. UCS Type = Unconditioned Stimulus type. Gustatory unconditioned stimuli are the words &#34;DRINK NOW&#34; appearing on-screen, and participants consuming 15 mL 0.067% Denatonium Benzoate solution. Pictorial UCSs are unpleasant/disgusting images sourced from the International Affective Picture System (IAPS) database and internet. Neutral UCSs are two images sourced from the IAPS database that are rated neutrally. UCS file name: this column gives the specific images to be displayed as outcomes on each trial. Numeric file names refer to IAPS database numbers. These images are available upon request to the IAPS database. Other images are available from the authors on request.

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A Prediction Error-driven Retrieval Procedure for Destabilizing and Rewriting Maladaptive Reward Memories in Hazardous Drinkers

This manuscript describes a memory retrieval procedure for destabilizing robust reward memories and rewriting them with counterconditioning prior to their reconsolidation.

Maladaptive reward memories (MRMs) can become unstable following retrieval under certain conditions, allowing their modification by subsequent new ...

a-prediction-error-driven-retrieval-procedure-for-destabilizing

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9.7K Views.  University College London. This manuscript describes a memory retrieval procedure for destabilizing robust reward memories and rewriting them with counterconditioning prior to their reconsolidation.

Video: A Prediction Error-driven Retrieval Procedure for Destabilizing and Rewriting Maladaptive Reward Memories in Hazardous Drinkers

A Procedure to Observe Context-induced Renewal of Pavlovian-conditioned Alcohol-seeking Behavior in Rats

Environmental contexts in which drugs of abuse are consumed can trigger craving, a subjective Pavlovian-conditioned response that can facilitate drug-seeking behavior and prompt relapse in abstinent drug users. We have developed a procedure to study the behavioral and neural processes that mediate the impact of context on alcohol-seeking behavior in rats. Following acclimation to the taste and pharmacological effects of 15% ethanol in the home cage, male Long-Evans rats receive Pavlovian discrimination training (PDT) in conditioning chambers. In each daily (Mon-Fri) PDT session, 16 trials each of two different 10 sec auditory conditioned stimuli occur. During one stimulus, the CS+, 0.2 ml of 15% ethanol is delivered into a fluid port for oral consumption. The second stimulus, the CS-, is not paired with ethanol. Across sessions, entries into the fluid port during the CS+ increase, whereas entries during the CS- stabilize at a lower level, indicating that a predictive association between the CS+ and ethanol is acquired. During PDT each chamber is equipped with a specific configuration of visual, olfactory and tactile contextual stimuli. Following PDT, extinction training is conducted in the same chamber that is now equipped with a different configuration of contextual stimuli. The CS+ and CS- are presented as before, but ethanol is withheld, which causes a gradual decline in port entries during the CS+. At test, rats are placed back into the PDT context and presented with the CS+ and CS- as before, but without ethanol. This manipulation triggers a robust and selective increase in the number of port entries made during the alcohol predictive CS+, with no change in responding during the CS-. This effect, referred to as context-induced renewal, illustrates the powerful capacity of contexts associated with alcohol consumption to stimulate alcohol-seeking behavior in response to Pavlovian alcohol cues.

A procedure to study the capacity of an alcohol associated environmental context to trigger the renewal of alcohol-seeking behavior in rats is described.

Environmental contexts in which drugs of abuse are consumed can trigger craving, a subjective Pavlovian-conditioned response that can facilitate ...

Behavior

A Method for Evaluating the Reinforcing Properties of Ethanol in Rats without Water Deprivation, Saccharin Fading or Extended Access Training

Operant oral self-administration methods are commonly used to study the reinforcing properties of ethanol in animals. However, the standard methods require saccharin/sucrose fading, water deprivation and/or extended training to initiate operant responding in rats. This paper describes a novel and efficient method to quickly initiate operant responding for ethanol that is convenient for experimenters and does not require water deprivation or saccharin/sucrose fading, thus eliminating the potential confound of using sweeteners in ethanol operant self-administration studies. With this method, Wistar rats typically acquire and maintain self-administration of a 20% ethanol solution in less than two weeks of training. Furthermore, blood ethanol concentrations and rewards are positively correlated for a 30 min self-administration session. Moreover, naltrexone, an FDA-approved medication for alcohol dependence that has been shown to suppress ethanol self-administration in rodents, dose-dependently decreases alcohol intake and motivation to consume alcohol for rats self-administering 20% ethanol, thus validating the use of this new method to study the reinforcing properties of alcohol in rats.

This protocol describes a novel and efficient method to quickly initiate operant responding for ethanol in rats that, contrary to standard methods, does not require water deprivation or saccharin/sucrose fading to initiate responding.

Operant oral self-administration methods are commonly used to study the reinforcing properties of ethanol in animals. However, the standard methods ...

A Conflict Model of Reward-seeking Behavior in Male Rats

The present protocol describes a novel conflict task as a model of inhibitory control in rats. In this model, a natural rewarding stimulus (sexual stimulus) that represents a high-value reward, and the aversive stimuli (pins), are concurrently presented. The male rats have to climb or jump over the obstacle full of pins to approach and investigate the sexual partner. If the animal persists in their approaching behavior regardless of the aversive stimuli, it is considered as a maladaptive or risky reward-seeking behavior. The conflict task permits the evaluation of deficit in inhibitory control resulting from exposure to abused drug, such as morphine, or a stressful event. 
The main advantage of this model is that it provides a simple and quick way to discover the deficit in inhibitory control after exposure to opiate drugs or other stressful events. In addition to opiates, this behavioral model would also be useful for quickly discovering the inhibitory control deficits induced by other addictive drugs. However, the limitation is that the male rats' performance may be subject to exercising effects with repeated testing under this conflict task. In the future, one can hope that the individuals with the compulsive phenotype of reward-seeking behavior after exposure to opiates will be identified based on modifying this conflict model.

This conflict model is used to measure the impairment of inhibitory control after exposure to addictive drugs, or other factors that may influence inhibitory control. A sexual stimulus and an aversive obstacle are concurrently presented, thus male rats have to conquer the obstacle to approach the sexual reward.

The present protocol describes a novel conflict task as a model of inhibitory control in rats. In this model, a natural rewarding stimulus (sexual ...

The Motivation for Alcohol Reward: Predictors of Progressive-Ratio Intravenous Alcohol Self-Administration in Humans

The Progressive Ratio (PR) self-administration paradigm is a common pre-clinical method used to examine the motivation for a drug attributed to a craving, reward, or the relief of negative affect. The Computer-assisted Alcohol Infusion System (CAIS) enables intravenous alcohol self-administration behavior in humans. This system provides the investigator with control over the trajectory of each incremental breath alcohol concentration (BrAC) reward and the maximum BrAC allowed in a session. This paradigm allows participants to earn these alcohol rewards using a sequence of button presses specified by the investigator. The system employs a physiologically-based pharmacokinetic model-based algorithm to achieve the same incremental BrAC exposure in every participant. Participants (n = 11) took part in two identical sessions to examine test-retest reliability, and an additional group (n = 73) completed a single session. Sessions began with a 25 min priming phase: participants were instructed to press a button an increasing number of times per reward, accumulating four standardized incremental BrAC trajectories. The second phase comprised an ad-lib, PR paradigm lasting 125 min. Each reward required an increasing number of button presses. Measures of self-administration included: average and peak BrAC, total rewards earned, total grams of ethanol consumed per unit of total body water, the total number of button presses, and the average rate of button pressing. Self-administration measures were highly correlated both between and within sessions, demonstrating test-retest reliability and internal consistency. Recent drinking history was strongly associated with self-administration measures; heavier drinkers chose greater alcohol self-administration. These results indicate the reliability and sensitivity of this progressive-ratio intravenous alcohol self-administration method for assessing the motivational properties of alcohol, with the potential for improved testing of the efficacy of new medications thought to reduce consumption of alcohol. This method can be used to understand the genetic and environmental determinants of alcohol self-administration in humans.

This study aims to show that the Progressive-Ratio Computer-assisted Alcohol-Infusion System (CAIS) paradigm is a reliable and sensitive method that can be used to examine the motivating properties associated with alcohol self-administration in humans.

The Progressive Ratio (PR) self-administration paradigm is a common pre-clinical method used to examine the motivation for a drug attributed to a craving, ...

A Procedure to Study Stress-Induced Relapse of Heroin Seeking after Punishment-Imposed Abstinence

The punishment-imposed abstinence procedure models the self-imposed abstinence that humans initiate due to the adverse consequences associated with drug-taking. This model has been implemented in experiments using different types of substances of abuse such as methamphetamine, cocaine, and alcohol. However, punishment-induced abstinence in heroin-trained animals has not been demonstrated. Furthermore, acute stress is a key trigger for relapse in humans and animal models. It was previously demonstrated that acute food deprivation robustly induced reinstatement of extinguished cocaine and heroin seeking. The procedure described here can be used to assess the effects of acute stress exposure on heroin seeking after punishment-imposed abstinence. A total of 8 rats were implanted with chronic intravenous (i.v.) catheters and trained to self-administer heroin (0.1 mg/kg/infusion) for 18 days under a seek-take chained schedule. Completing the seek link gave access to the take lever, which was paired with a heroin infusion. The seek lever was programmed with a variable interval 60 schedule of reinforcement (VI60), and the take lever was programmed with a fixed-ratio 1 reinforcement schedule (FR1). Following self-administration training, a mild foot shock was delivered on 30% of the completed seek links instead of the extension of the take lever. Footshock intensity was increased by 0.1 mA per daily session from 0.2 mA to 1.0 mA. Heroin-seeking tests were performed after 24 h of food deprivation (FD) or sated conditions. Rats under acute food deprivation condition robustly increased heroin seeking after punishment-imposed abstinence.

A procedure that demonstrates a robust acute food deprivation-induced relapse to heroin seeking after punishment-imposed abstinence is described. A punishment-imposed abstinence model was successfully implemented using the seek and take chain schedule for heroin self-administration. Heroin-seeking tests are then performed following 24 h of food-deprivation stress.

The punishment-imposed abstinence procedure models the self-imposed abstinence that humans initiate due to the adverse consequences associated with ...

The Use of Trace Eyeblink Classical Conditioning to Assess Hippocampal Dysfunction in a Rat Model of Fetal Alcohol Spectrum Disorders

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain undergoes rapid organizational change and is similar to accelerated brain changes that occur during the third trimester in humans. This model of fetal alcohol spectrum disorders (FASDs) produces severe brain damage, mimicking the amount and pattern of binge-drinking that occurs in some pregnant alcoholic mothers. We describe the use of trace eyeblink classical conditioning (ECC), a higher-order variant of associative learning, to assess long-term hippocampal dysfunction that is typically seen in alcohol-exposed adult offspring. At 90 days of age, rodents were surgically prepared with recording and stimulating electrodes, which measured electromyographic (EMG) blink activity from the left eyelid muscle and delivered mild shock posterior to the left eye, respectively. After a 5 day recovery period, they underwent 6 sessions of trace ECC to determine associative learning differences between alcohol-exposed and control rats. Trace ECC is one of many possible ECC procedures that can be easily modified using the same equipment and software, so that different neural systems can be assessed. ECC procedures in general, can be used as diagnostic tools for detecting neural pathology in different brain systems and different conditions that insult the brain.

Trace eyeblink classical conditioning (ECC) was used to assess hippocampal-dependent associative learning in adult rats that were administered a high concentration (11.9% v/v) of alcohol during early neonatal brain development. In general, ECC procedures are sound diagnostic tools for detecting brain dysfunction across many psychological and biomedical settings.

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain ...

A Protocol for Measuring Cue Reactivity in a Rat Model of Cocaine Use Disorder

Cocaine use disorder (CUD) follows a trajectory of repetitive self-administration during which previously neutral stimuli gain incentive value. Cue reactivity, the sensitivity to cues previously linked with the drug-taking experience, plays a prominent role in human craving during abstinence. Cue reactivity can be assessed as the attentional orientation toward drug-associated cues that is measurable as appetitive approach behavior in both preclinical and human studies. Herein describes an assessment of cue reactivity in rats trained to self-administer cocaine. Cocaine self-administration is paired with the presentation of discrete cues that act as conditioned reinforcers (i.e., house light, stimulus light, infusion pump sounds). Following a period of abstinence, lever presses in the cocaine self-administration context accompanied by the discrete cues previously paired with cocaine infusion are measured as cue reactivity. This model is useful to explore neurobiological mechanisms underlying cue reactivity processes as well as to assess pharmacotherapies to suppress cue reactivity and therefore, modify relapse vulnerability. Advantages of the model include its translational relevance, and its face and predictive validities.&#160;The primary limitation of the model is that the cue reactivity task can only be performed infrequently and must only be used in short duration (e.g., 1 hour), otherwise rats will begin to extinguish the pairing of the discrete cues with the cocaine stimulus. The model is extendable to any positively reinforcing stimulus paired with discrete cues; though particularly applicable to drugs of abuse, this model may hold future applications in fields such as obesity, where palatable food rewards can act as positively reinforcing stimuli.

Cue reactivity is conceptualized as sensitivity to cues linked with drug-taking experiences that contribute to craving and relapse in abstinent humans. Cue reactivity is modeled in rats by measuring attentional orientation toward drug-associated cues that results in appetitive approach behavior in a cue reactivity test following self-administration and forced abstinence.

Cocaine use disorder (CUD) follows a trajectory of repetitive self-administration during which previously neutral stimuli gain incentive value. Cue ...

Murine Drinking Models in the Development of Pharmacotherapies for Alcoholism: Drinking in the Dark and Two-bottle Choice

Alcohol Use Disorder (AUD) is a major problem with more than an estimated 76 million people worldwide meeting the diagnostic criteria. Current treatments are limited to three FDA-approved medications that are largely ineffective even when combined with psychosocial intervention, as is evident by the high relapse rate. As such, the search for more novel treatments represents an important public health goal. To this end, the following protocol utilizes two simple rodent drinking models to assess the preclinical efficacy of lead anti-alcohol compounds: two-bottle choice (TBC) and drinking in the dark (DID). The former allows mice to voluntary drink in moderation while the latter induces mice to voluntary consume a large amount of alcohol in a short period that mimics binge drinking. The simple and high throughput nature of both of these paradigms allow for rapid screening of pharmacological agents or for identifying strains of mice that exhibit certain voluntary drinking behavior.

Alcohol Use Disorder (AUD) is a major national health problem and the development of more effective treatments is&#160;required to offset the needs of this patient population. To this end, the following protocol utilizes two simple rodent drinking models to assess the preclinical efficacy of lead anti-alcohol compounds.

Alcohol Use Disorder (AUD) is a major problem with more than an estimated 76 million people worldwide meeting the diagnostic criteria. Current treatments ...

Medicine

Investigating Drivers of Antireward in Addiction Behavior with Anatomically Specific Single-Cell Gene Expression Methods

Increasing rates of addiction behavior have motivated mental health researchers and clinicians alike to understand antireward and recovery. This shift away from reward and commencement necessitates novel perspectives, paradigms, and hypotheses along with an expansion of the methods applied to investigate addiction. Here, we provide an example: A systems biology approach to investigate antireward that combines laser capture microdissection (LCM) and high-throughput microfluidic reverse transcription quantitative polymerase chain reactions (RT-qPCR). Gene expression network dynamics were measured and a key driver of neurovisceral dysregulation in alcohol and opioid withdrawal, neuroinflammation, was identified. This combination of technologies provides anatomic and phenotypic specificity at single-cell resolution with high-throughput sensitivity and specific gene expression measures yielding both hypothesis-generating datasets and mechanistic possibilities that generate opportunities for novel insights and treatments.

The combination of laser capture microdissection and microfluidic RT-qPCR provides anatomic and biotechnical specificity in measuring the transcriptome in single neurons and glia. Applying creative methods with a system's biology approach to psychiatric disease may lead to breakthroughs in understanding and treatment such as the neuroinflammation antireward hypothesis in addiction.

Increasing rates of addiction behavior have motivated mental health researchers and clinicians alike to understand antireward and recovery. This shift ...

Modeling Alcohol Use Disorder Using Chronic Intermittent Ethanol Vapor Exposure

Chronic Intermittent Ethanol Vapor Exposure Paired with Two-Bottle Choice to Model Alcohol Use Disorder

Alcohol use disorder (AUD) is a chronic alcohol-related disorder that typically presents as uncontrolled drinking and preoccupation with alcohol. A key component of AUD research is using translationally relevant preclinical models. Over the past several decades, a variety of animal models have been used to study AUD. One prominent model of AUD is the chronic intermittent ethanol vapor exposure (CIE) model, which is a well-established approach for inducing alcohol dependence in rodents through repeated cycles of ethanol exposure via inhalation. To model AUD in mice, the CIE exposure is paired with a voluntary two-bottle choice (2BC) of alcohol drinking and water to measure the escalation of alcohol drinking. The 2BC/CIE procedure involves alternating weeks of 2BC drinking and CIE, which repeat until the escalation of alcohol drinking is achieved. In the present study, we outline the procedures for performing 2BC/CIE, including the daily use of the CIE vapor chamber, and provide an example of escalated alcohol drinking in C57BL/6J mice using this approach.

Presented here is a protocol for the 2BC/CIE model of alcohol dependence in mice to study alcohol use disorder.

Alcohol use disorder (AUD) is a chronic alcohol-related disorder that typically presents as uncontrolled drinking and preoccupation with alcohol. A key ...