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Animals must quickly adapt food-seeking strategies to locate nutrient sources in dy-namically changing environments. Learned associations between food and environ-mental cues that predict its availability promote food-seeking behaviors. However,when such cues cease to predict food availability, animals undergo “extinction” learn-ing, resulting in the inhibition of food-seeking responses. Repeatedly activated setsof neurons, or “neuronal ensembles,” in the dorsal medial prefrontal cortex (dmPFC)are recruited following appetitive conditioning and undergo physiological adapta-tions thought to encode cue-reward associations. However, little is known about howthe recruitment and intrinsic excitability of such dmPFC ensembles are modulated byextinction learning. Here, we used in vivo 2-Photon imaging in male Fos-GFP micethat express green fluorescent protein (GFP) in recently behaviorally activated neu-rons to determine the recruitment of activated pyramidal and GABAergic interneu-ron dmPFC ensembles during extinction. During extinction, we revealed a persistentactivation of a subset of interneurons which emerged from a wider population of in-terneurons activated during the initial extinction session. This activation pattern wasnot observed in pyramidal cells, and extinction learning did not modulate the excit-ability properties of activated pyramidal cells. Moreover, extinction learning reducedthe likelihood of reactivation of pyramidal cells activated during the initial extinction ession. Our findings illuminate novel neuronal activation patterns in the dmPFCunderlying extinction of food-seeking, and in particular, highlight an important rolefor interneuron ensembles in this inhibitory form of learning.

Background In the recent case of R v. Taj, the Court of Appeal of England and Wales upheld the conviction of a defendant who, in a psychotic delusional state, mistook his non-threatening victim to be a terrorist, violently attacking him. The law typically allows honest mistakes (even if unreasonable) as a basis for self-defence (in this case the defence of others). However, because Taj's delusions were found by the court to have been caused by voluntary alcohol consumption, special legal (prior-fault) intoxication rules were applied to block his defence; Taj was convicted and sentenced to 19 years imprisonment for attempted murder. Argument We focus here on the simple question-what does it mean to be intoxicated? On the facts, Taj did not have drugs active in his system at the time of the attack, but the court nonetheless insisted that Taj's delusional mistake was 'attributable to intoxication'; namely, to drink- and drug-taking in the previous days and weeks. This extended conception of intoxication was questionably distinguished from psychosis induced by withdrawal. Furthermore, the court was unreceptive to evidence of a long-standing, underlying mental health disorder. We argue that the court's expanded view of intoxication is problematic, in that intoxication-induced psychosis cannot be sharply distinguished from other causes such as mental disorders; and even if it could be distinguished, it should not give rise to blame and punishment in the same way as does conduct induced by chemically active intoxicants ('drug-on-board'). Conclusion The courts' expansion of the definition of intoxication is both legally and forensically problematic, introducing legal vagaries where the clinical science is already vague; and with intoxication frequently interlocking with historic intoxication and secondary or comorbid mental health conditions, the decision risks inappropriately and/or over-criminalizing defendants.

The article critiques the ‘loss of self-control’ requirement within Loss of Control partial defence, investigating its meaning (legally and scientifically), as well as its theoretical purpose. We contend that the partial defence currently performs a curious and problematic role, promoting questions of self-control, that are most effectively dealt with at a post-conviction stage (ie, at sentencing), into questions for the liability stage. This could be (perhaps best) resolved through the abolition of the mandatory life sentence for murder, and subsequent abolition of the partial defences, but it is accepted that the current political reality weighs heavily against this option. Looking for viable alternatives, we highlight the advantages of an approach that maximises discretion based on a full appraisal of potentially extenuating circumstances; before discussing how the current partial defence, including the requirement for a loss of self-control, should be interpreted to move the current law closer to this goal.

Changes in microglial morphology are powerful indicators of the inflammatory state of the brain. Here, we provide an open-source microglia morphology analysis pipeline that first cleans and registers images of microglia, before extracting 62 parameters describing microglial morphology. It then compares control and ‘inflammation’ training data and uses dimensionality reduction to generate a single metric of morphological change (an ‘inflammation index’). This index can then be calculated for test data to assess inflammation, as we demonstrate by investigating the effect of short-term high-fat diet consumption in heterozygous Cx3CR1-GFP mice, finding no significant effects of diet. Our pipeline represents the first open-source microglia morphology pipeline combining semi-automated image processing and dimensionality reduction. It uses free software (ImageJ and R) and can be applied to a wide variety of experimental paradigms. We anticipate it will enable others to more easily take advantage of the powerful insights microglial morphology analysis provides.

Animals selectively respond to environmental cues associated with food reward to optimize nutrient intake. Such appetitive conditioned stimulus–unconditioned stimulus (CS-US) associations are thought to be encoded in select, stable neuronal populations or neuronal ensembles, which undergo physiological modifications during appetitive conditioning. These ensembles in the medial prefrontal cortex (mPFC) control well-established, cue-evoked food seeking, but the mechanisms involved in the genesis of these ensembles are unclear. Here, we used maleFos-GFPmice that express green fluorescent protein (GFP) in recently behaviorally activated neurons, to reveal how dorsal mPFC neurons are recruited and modified to encode CS-US memory representations using an appetitive conditioning task. In the initial conditioning session, animals did not exhibit discriminated, cue-selective food seeking, but did so in later sessions indicating that a CS-US association was established. Using microprism-basedin vivo2-Photon imaging, we revealed that only a minority of neurons activated during the initial session was consistently activated throughout subsequent conditioning sessions and during cue-evoked memory recall. Notably, usingex vivoelectrophysiology, we found that neurons activated following the initial session exhibited transient hyperexcitability. Chemogenetically enhancing the excitability of these neurons throughout subsequent conditioning sessions interfered with the development of reliable cue-selective food seeking, indicated by persistent, nondiscriminated performance. We demonstrate how appetitive learning consistently activates a subset of neurons to form a stable neuronal ensemble during the formation of a CS-US association. This ensemble may arise from a pool of hyperexcitable neurons activated during the initial conditioning session.SIGNIFICANCE STATEMENTAppetitive conditioning endows cues associated with food with the ability to guide food-seeking, through the formation of a food-cue association. Neuronal ensembles in the mPFC control established cue-evoked food-seeking. However, how neurons undergo physiological modifications and become part of an ensemble during conditioning remain unclear. We found that only a minority of dorsal mPFC neurons activated on the initial conditioning session became consistently activated during conditioning and memory recall. These initially activated neurons were also transiently hyperexcitable. We demonstrate the following: (1) how stable neuronal ensemble formation in the dorsal mPFC underlies appetitive conditioning; and (2) how this ensemble may arise from hyperexcitable neurons activated before the establishment of cue-evoked food seeking.

The hippocampus is essential for spatial and episodic memory but is damaged early in Alzheimer's disease and is very sensitive to hypoxia. Understanding how it regulates its oxygen supply is therefore key for designing interventions to preserve its function. However, studies of neurovascular function in the hippocampus in vivo have been limited by its relative inaccessibility. Here we compared hippocampal and visual cortical neurovascular function in awake mice, using two photon imaging of individual neurons and vessels and measures of regional blood flow and haemoglobin oxygenation. We show that blood flow, blood oxygenation and neurovascular coupling were decreased in the hippocampus compared to neocortex, because of differences in both the vascular network and pericyte and endothelial cell function. Modelling oxygen diffusion indicates that these features of the hippocampal vasculature may restrict oxygen availability and could explain its sensitivity to damage during neurological conditions, including Alzheimer's disease, where the brain's energy supply is decreased.

The hippocampus is essential for spatial and episodic memory but is damaged early in Alzheimer’s disease and is very sensitive to hypoxia. Understanding how it regulates its oxygen supply is therefore key for designing interventions to preserve its function. However, studies of neurovascular function in the hippocampus in vivo have been limited by its relative inaccessibility. Here we compared hippocampal and visual cortical neurovascular function in awake mice, using two photon imaging of individual neurons and vessels and measures of regional blood flow and haemoglobin oxygenation. We show that blood flow, blood oxygenation and neurovascular coupling were decreased in the hippocampus compared to neocortex, because of differences in both the vascular network and pericyte and endothelial cell function. Modelling oxygen diffusion indicates that these features of the hippocampal vasculature may restrict oxygen availability and could explain its sensitivity to damage during neurological conditions, including Alzheimer’s disease, where the brain’s energy supply is decreased., The hippocampus is particularly sensitive to hypoxia but it has been difficult to study blood flow in this region. Here the authors compare the neurovascular function of the hippocampus and cortex and in awake mice, and find differences associated with microvascular structure.

Funder: The University of Sussex Strategic Development Funds, Funder: Sussex Neuroscience 4‐year PhD programme, Exposure to environmental enrichment can modify the impact of motivationally relevant stimuli. For instance, previous studies in rats have found that even a brief, acute (~1 day), but not chronic, exposure to environmentally enriched (EE) housing attenuates instrumental lever pressing for sucrose-associated cues in a conditioned reinforcement setup. Moreover, acute EE reduces corticoaccumbens activity, as measured by decreases in expression of the neuronal activity marker "Fos." Currently, it is not known whether acute EE also reduces sucrose seeking and corticoaccumbens activity elicited by non-contingent or "forced" exposure to sucrose cues, which more closely resembles cue exposure encountered in daily life. We therefore measured the effects of acute/intermittent (1 day or 6 day of EE prior to test day) versus chronic (EE throughout conditioning lasting until test day) EE on the ability of a Pavlovian sucrose cue to elicit sucrose seeking (conditioned approach) and Fos expression in the medial prefrontal cortex (mPFC), orbitofrontal cortex (OFC), and nucleus accumbens (NAc) in mice. One day, but not 6 day or chronic EE , reduced sucrose seeking and Fos in the deep layers of the dorsal mPFC. By contrast, 1 day, 6 day, and chronic EE all reduced Fos in the shallow layers of the OFC. None of the EE manipulations modulated NAc Fos expression. We reveal how EE reduces behavioral reactivity to sucrose cues by reducing activity in select prefrontal cortical brain areas. Our work further demonstrates the robustness of EE in its ability to modulate various forms of reward-seeking across species.

Changes in microglial morphology are powerful indicators of the inflammatory state of the brain. Here we provide an open-source microglia morphology analysis pipeline that first cleans and registers images of microglia, before extracting 62 parameters describing microglial morphology. It then compares control and “inflammation” training data and uses dimensionality reduction to generate a single metric of morphological change (an ‘inflammation index’). This index can then be calculated for test data to assess inflammation, as we demonstrate by investigating the effect of short-term high fat diet consumption in heterozygous Cx3CR1-GFP mice, finding no significant effects of diet. Our pipeline represents the first open-source microglia morphology pipeline combining semi-automated image processing and dimensionality reduction. It uses free software (ImageJ and R) and can be applied to a wide variety of experimental paradigms. We anticipate it will enable others to more easily take advantage of the powerful insights microglial morphology analysis provides.

The lack of scientific or clinical clarity faced by courts dealing with cases involving intoxicated criminal defendants is unavoidable, but introducing further legal ambiguity to meet policy goals introduces ever greater risk of problematic (including unjust) legal outcomes.

Instrumental conditioning is a crucial substrate of adaptive behaviour, allowing individuals to selectively interact with the stimuli in their environment to maximise benefit and minimise harm. The extent to which complex forms of learning, such as instrumental conditioning, are possible without conscious awareness is a topic of considerable importance and ongoing debate. In light of recent theoretical and empirical contributions casting doubt on the early demonstrations of unconscious instrumental conditioning, we revisit the question of its feasibility in two modes of conditioning. In Experiment 1, we used trace conditioning, following a prominent paradigm (Pessiglione et al., 2008) and enhancing its sensitivity. Success in this task requires participants to learn to approach reward-predictive stimuli and avoid punishment-predictive stimuli through monetary reinforcement. All stimuli were rendered unconscious using forward-backward masking. In Experiment 2, we used delay conditioning to shorten the stimulus-outcome delay, retaining the structure of the original task but presenting the stimuli under continuous flash suppression to allow for an overlap of the stimulus, action, and outcome, as well as replacing monetary reinforcement with primary appetitive reinforcement. In both experiments, we found evidence for absence of unconscious instrumental conditioning, showing that participants were unable to learn to adjust their behaviour to approach positive stimuli and avoid negative ones. This result is consistent with evidence that unconscious stimuli fail to bring about long-term behavioural adaptations, and provides empirical evidence to support theoretical proposals that consciousness might be necessary for adaptive behaviour, where selective action is required.

Criminal responsibility is founded on the volitional control of action, yet many offences are committed whilst intoxicated. Alcohol is a widely-used intoxicant known to impair behavioural control and memory, opening the door to claims of partial or absent responsibility. Criminal law rules have developed to resist such claims. In particular, findings of criminal responsibility in intoxicated offenders frequently call on ‘prior fault’ logic: the intoxicated offender may claim to have been irrational, but culpability can be imported from their earlier, rational choice to consume intoxicants (Robinson, 1985). That drunkenness does not excuse derives from an unforced choice to become drunk. Yet this raises questions over the extent to which an alcoholic’s choice to drink is unforced. Current neurobiological models of addiction stress compulsion as a primary component, bringing into question the volitional nature of continued consumption (Volkow & Fowler, 2000; Dalley, Everitt, & Robbins, 2011). At the same time, some legal scholars have noted that the potentially fatal nature of alcoholic withdrawal could likewise undermine suggestion that consumption is voluntary, drawing analogy to a defence of duress where actions have been forced under the threat of death (Husak, 1999; Yaffe, 2013). We asked 290 UK Magistrates to consider a criminal sentencing scenario in which evidence of a defendant’s state of intoxication at the time of the offence was presented in tandem with information that they were either teetotal, a casual drinker or an alcoholic. We found that intoxication reduced blameworthiness for criminal acts if the offender had no previous experience with alcohol whilst, in direct contrast, intoxication served to aggravate offending if the defendant was an alcoholic. The likelihood of our defendant receiving a reduced sentence was over ten times greater in the event that, though intoxicated, they were not also an alcoholic. Leniency was blocked by alcoholism despite Magistrates’ qualitative responses suggesting its understanding as a generally mitigating factor, revealing a disconnect between expressed opinion and sentencing behaviour in practice. Our results indicate that, far from excusing drunkenness, the state of being an alcoholic is more frequently deemed to aggravate offending, being associated with harsher sentencing even where the offence in question was committed whilst sober.

Most studies that examine responses to food cues use images of actual foods as stimuli. Since foods are rewarding in multiple ways, it then becomes difficult to try and partial out the role of the importance of different aspects of food reward. Here we aimed to evaluate the impact of novel visual cues specifically associated with the immediate sensory reward from a liked sweet taste. In the training phase, one visual cue (CSsweet) was associated with the experience of sweet taste (10%sucrose) and a second, control cue (CSneutral) with a neutral taste (artificial saliva) using a disguised training procedure. In Experiment 1, participants (n = 45) were given an ad libitum snack intake test 30 min post-training, either labelled with CSsweet or CSneutral. Total caloric consumption was significantly higher in the CSsweet (650 ± 47 kcal) than CSneutral (477 ± 45 kcal) condition, but ratings of liking for the snacks did not differ significantly between conditions. In Experiment 2, participants (n = 80) exhibited an overall attentional bias (22.1 ± 9.9 ms) for the CSsweet relative to CSneutral cue (assessed using a dot-probe task), however rated liking for the CSsweet did not change significantly after cue-sweet training. Likewise, measures of expected satiety for drinks labelled with CSsweet did not differ significantly from CSneutral. Overall these two experiments provide evidence that associations between neutral visual cues and the experience of a liked sweet taste leads to cue-potentiated eating in the presence of the CSsweet cue. With no evidence that cue-sweet training altered rated liking for the visual cues, and in keeping with extant literature on the dissociation of hedonic and rewarding properties of food rewards, we propose this potentiation effect to reflect increased incentive salience.

Debates continue over whether the prevailing neuroscientific model of addiction as a brain disease informs questions around moral and criminal responsibility, but little empirical work has been conducted with those tasked to address this question in practical terms on a daily basis. We have explored this point over two studies, respectively sampling 110 and 276 Magistrates active in the UK. In the first study we asked them to consider a criminal sentencing scenario in which evidence of a defendant’s brain damage and impaired impulse control was presented. This neurological damage was attributed to either a (fictional) disease or to addiction. When the same neuropsychiatric profile resulted from disease, rather than heroin use and addiction, custodial sentences were significantly reduced. The pivotal factor denying addiction the mitigating power of disease was perceived choice in the initial acquisition; removing choice from addiction dramatically increased the odds of sentence reduction, while attaching choice to disease tended to aggravate or reverse earlier leniency. The second study presented another criminal sentencing scenario in which the defendant exhibited similar neurological impairment, but additionally included ‘mixed’ aetiologies in which either disease led to addiction or addiction led to disease. Our results confirm the dramatic effect which the aetiology of impairment can have on judgements of criminal responsibility, whilst moreover give suggestion that drug-use tips the balance in favour of the punitive element when weighing criminal sentencing decisions.

The hippocampus is essential for spatial and episodic memory but is damaged early in Alzheimer’s disease and is very sensitive to hypoxia. Understanding how it regulates its oxygen supply is therefore key for designing interventions to preserve its function. However, studies of neurovascular function in the hippocampus in vivo have been limited by its relative inaccessibility. Here we compared hippocampal and visual cortical neurovascular function in awake mice, using two photon imaging of individual neurons and vessels and measures of regional blood flow and haemoglobin oxygenation. We show that blood flow, blood oxygenation and neurovascular coupling were decreased in the hippocampus compared to neocortex, because of differences in both the vascular network and pericyte and endothelial cell function. Modelling oxygen diffusion indicates that these features of the hippocampal vasculature could explain its sensitivity to damage during neurological conditions, including Alzheimer’s disease, where the brain’s energy supply is decreased.

Visual Abstract, Animals must learn relationships between foods and the environmental cues that predict their availability for survival. Such cue–food associations are encoded in sparse sets of neurons or “neuronal ensembles” in the nucleus accumbens (NAc). For these ensemble-encoded, cue-controlled appetitive responses to remain adaptive, they must allow for their dynamic updating depending on acute changes in internal states such as physiological hunger or the perceived desirability of food. However, how these neuronal ensembles are recruited and physiologically modified following the update of such learned associations is unclear. To investigate this, we examined the effects of devaluation on ensemble plasticity at the levels of recruitment, intrinsic excitability, and synaptic physiology in sucrose-conditioned Fos-GFP mice that express green fluorescent protein (GFP) in recently activated neurons. Neuronal ensemble activation patterns and their physiology were examined using immunohistochemistry and slice electrophysiology, respectively. Reward-specific devaluation following 4 d of ad libitum sucrose consumption, but not general caloric devaluation, attenuated cue-evoked sucrose seeking. This suggests that changes in the hedonic and/or incentive value of sucrose, and not caloric need, drove this behavior. Moreover, devaluation attenuated the size of the neuronal ensemble recruited by the cue in the NAc shell. Finally, it eliminated the relative enhanced excitability of ensemble (GFP+) neurons against non-ensemble (GFP−) neurons observed under non-devalued conditions, and did not induce any ensemble-specific changes in excitatory synaptic physiology. Our findings provide new insights into neuronal ensemble mechanisms that underlie the changes in the incentive and/or hedonic impact of cues that support adaptive food seeking.

Cues that predict the availability of food rewards influence motivational states and elicit food-seeking behaviors. If a cue no longer predicts food availability, then animals may adapt accordingly by inhibiting food-seeking responses. Sparsely activated sets of neurons, coined “neuronal ensembles,” have been shown to encode the strength of reward–cue associations. Although alterations in intrinsic excitability have been shown to underlie many learning and memory processes, little is known about these properties specifically on cue-activated neuronal ensembles. We examined the activation patterns of cue-activated orbitofrontal cortex (OFC) and nucleus accumbens (NAc) shell ensembles using wild-type andFos-GFPmice, which express green fluorescent protein (GFP) in activated neurons, after appetitive conditioning with sucrose and extinction learning. We also investigated the neuronal excitability of recently activated, GFP+ neurons in these brain areas using whole-cell electrophysiology in brain slices. Exposure to a sucrose cue elicited activation of neurons in both the NAc shell and OFC. In the NAc shell, but not the OFC, these activated GFP+ neurons were more excitable than surrounding GFP− neurons. After extinction, the number of neurons activated in both areas was reduced and activated ensembles in neither area exhibited altered excitability. These data suggest that learning-induced alterations in the intrinsic excitability of neuronal ensembles is regulated dynamically across different brain areas. Furthermore, we show that changes in associative strength modulate the excitability profile of activated ensembles in the NAc shell.SIGNIFICANCE STATEMENTSparsely distributed sets of neurons called “neuronal ensembles” encode learned associations about food and cues predictive of its availability. Widespread changes in neuronal excitability have been observed in limbic brain areas after associative learning, but little is known about the excitability changes that occur specifically on neuronal ensembles that encode appetitive associations. Here, we reveal that sucrose cue exposure recruited a more excitable ensemble in the nucleus accumbens, but not orbitofrontal cortex, compared with their surrounding neurons. This excitability difference was not observed when the cue's salience was diminished after extinction learning. These novel data provide evidence that the intrinsic excitability of appetitive memory-encoding ensembles is regulated differentially across brain areas and adapts dynamically to changes in associative strength.

Several theoretical frameworks have been developed to understand putative processes and mechanisms involved in addiction. Whilst these ‘theories of addiction’ disagree about importance and/or nature of a number of key psychological processes (e.g. the necessity of craving and/or the involvement of drug-value representations), a number of commonalities exist. For instance, it is widely accepted that Pavlovian associations between cues and environmental contexts and the drug effects acquired over the course of addiction play a critical role, especially in relapse vulnerability in detoxified addicts. Additionally, all theories of addiction (explicitly or implicitly) propose that chronic drug exposure produces persistent neuroplastic changes in neurobiological circuitries underlying critical emotional, cognitive and motivational processes, although disagreement exists as to the precise nature of these neurobiological changes and/or their psychological consequences. The present review, rather than limiting itself to any particular theoretical stance, considers various candidate psychological, neurobiological and/or behavioral processes in addiction and outlines conceptual and procedural approaches for the experimental medicine laboratory. The review discusses (1) extinction, renewal and (re)consolidation of learned associations between cues and drugs, (2) the drug reward value, (3) motivational states contributing to drug seeking and (4) reflective (top-down) and sensory (bottom-up) driven decision-making. In evaluating these psychological and/or behavioral processes and their relationship to addiction we make reference to putative underlying brain structures identified by basic animal studies and/or imaging studies with humans.

An environmental stimulus paired with reward (a conditioned stimulus; CS) can acquire predictive properties that signal reward availability and may also acquire incentive motivational properties that enable the CS to influence appetitive behaviors. The neural mechanisms involved in the acquisition and expression of these CS properties are not fully understood. The metabotropic glutamate receptor, mGluR5, contributes to synaptic plasticity underlying learning and memory processes. We examined the role of mGluR5 in the acquisition and expression of learning that enables a CS to predict reward (goal-tracking) and acquire incentive properties (conditioned reinforcement). Mice were injected with vehicle or the mGluR5 antagonist, MTEP (3 or 10 mg/kg), before each Pavlovian conditioning session in which a stimulus (CS+) was paired with food delivery. Subsequently, in the absence of the primary food reward, we determined whether the CS+ could reinforce a novel instrumental response (conditioned reinforcement) and direct behavior toward the place of reward delivery (goal-tracking). MTEP did not affect performance during the conditioning phase, or the ability of the CS+ to elicit a goal-tracking response. In contrast, 10 mg/kg MTEP given before each conditioning session prevented the subsequent expression of conditioned reinforcement. This dose of MTEP did not affect conditioned reinforcement when administered before the test, in mice that had received vehicle before conditioning sessions. Thus, mGluR5 has a critical role in the acquisition of incentive properties by a CS, but is not required for the expression of incentive learning, or for the CS to acquire predictive properties that signal reward availability.

Cannabinoid CB1 receptor is abundantly expressed throughout the CNS and is implicated in numerous physiological and behavioral functions, including appetite and feeding. In the present study, wild-type and CB1 heterozygous and homozygous knockout mice were tested on an instrumental outcome-selective devaluation task to assess changes in acquired instrumental response levels for a distinct food reward following selective satiation. Deletion of CB1 receptor, as well as reduction in CB1 expression (HET), produced deficits in outcome-selective instrumental devaluation. These results identify a critical role for CB1 receptor in the ability of animals to represent, update, and/or use sensory-specific outcome representations to alter appetitive behaviors.

It is widely thought that regulation of post-synaptic AMPA receptors is a critical component in changes in synaptic efficacy underlying learning and memory. The regulation of AMPA receptors occurs through trafficking of the receptor and/or modulation of the receptor's channel properties and both of these processes depend on phosphorylation of the receptor. Using homologous recombination (knock-in) techniques we targeted two phosphorylation sites on the AMPA-GluR1 receptor: the CaMKII/PKC Ser 831 site and the PKA Ser 845 site. Mice with either or both of these sites mutated were then tested on an incentive learning task that assessed their ability to acquire a simple association between a cue and reward and to then use this cue as a reinforcer to guide their behavior (conditioned reinforcement). We report that, whereas WT mice showed enhanced responding for the reward-associated cue, mice with mutations of both phosphorylation sites or the Ser 831 site alone, failed to show such a conditioned reinforcement effect. By contrast, mice with only the Ser 845 site deficient showed normal CS+ reinforced responding. Thus, action at the Ser 831 phosphorylation site was necessary for normal conditioned reinforcement. Finally, the behavioral deficit was highly specific: performance on a number of other measures of motivated performance, including responding reinforced by the food itself, was unaffected by the mutations. Our findings provide novel evidence for a molecular mechanism in a form of appetitive incentive learning critical in regulating normal motivated behavior, as well as maladaptive forms such as addiction and eating disorders.

Alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor regulation has been shown to be critically involved in synaptic plasticity underlying learning and memory. This regulation occurs through trafficking of the receptor and modulation of the receptor's channel properties, both of which depend on protein phosphorylation. Using homologous recombination (knock-in) techniques we targeted two phosphorylation sites on the AMPA-GluR1 receptor: the Ser831 site, phosphorylated by calcium calmodulin-dependent protein kinase II/protein kinase C, and the Ser845 site, phosphorylated by protein kinase A. Mice with mutations that prevented phosphorylation at one or both of these sites were tested on a single-outcome Pavlovian-instrumental transfer task often used to assess the acquisition of incentive motivation by cues for food reinforcement. Mice were separately trained to associate a Pavlovian cue with food and to perform an instrumental lever-press response to earn that same reward. During a transfer test, the cue was presented while the mice were lever-pressing under extinction conditions. Whereas wild-type control mice showed substantial enhancement of lever-pressing when the cue was presented (i.e. showed Pavlovian-instrumental transfer), mice with mutations at both of these phosphorylation sites showed no evidence of such transfer. By contrast, mice with either serine site mutated alone showed normal transfer. These results suggest critical roles for GluR1 phosphorylation pathways in a form of incentive learning that can play an important part in regulating normal motivated behavior as well as maladaptive behaviors such as addiction and eating disorders.

Repeated cocaine administration to rats outside their home cage induces behavioral sensitization that is strongly modulated by the drug administration environment. We hypothesized that stimuli in the drug administration environment activate specific sets of striatal neurons, called neuronal ensembles, for further cocaine-enhanced activation, and that repeated activation of these neuronal ensembles underlies context-specific sensitization. In the present study, we repeatedly administered cocaine or saline to rats on alternate days in two distinct environments outside the home cage, one paired with cocaine and the other with saline. On test day, cocaine challenge injections in the cocaine-paired environment produced strongly enhanced levels of locomotor activity, while cocaine challenge injections in the saline-paired environment did not. The corresponding record of past neuronal activation in nucleus accumbens and caudate-putamen during repeated drug administration was assessed using FosB immunohistochemistry, while acute neuronal activation on test day was assessed using c-fos in situ hybridization. Although only 2% of striatal neurons were FosB labeled, 87% of these FosB-labeled neurons were co-labeled with c-fos when cocaine was injected in the cocaine-paired environment. The degree of co-labeling was significantly less following cocaine or saline challenge injections in the saline-paired environment. Furthermore, the total number of c-fos-labeled neurons was greater with either cocaine or saline challenge injections in the cocaine-paired environment than in the saline-paired environment. These findings demonstrate that the drug administration environment partly determines which striatal neuronal ensembles are activated, and to what extent, following context-specific sensitization to cocaine.

We examined the reinforcer-specificity of Pavlovian conditioning in the control of appetitive and consummatory behaviors in Pavlovian-to-instrumental transfer, cue-potentiated eating, and devaluation procedures. Rats received pairings of one conditioned stimulus with sucrose and another conditioned stimulus with maltodextrin. In Experiment 1, rats were also trained to earn sucrose for one instrumental response and maltodextrin for another. In a transfer test, the Pavlovian cues enhanced the rate of instrumental responding more when the food reinforcer predicted by the instrumental response and the Pavlovian cue were consistent than when they were inconsistent, but both cues enhanced both responses. In Experiment 2, sated rats' consumption of each food was potentiated in the presence of a cue for that food, but not in the presence of a cue for the other food. In Experiment 3, one food was devalued by pairing it with lithium chloride, prior to testing food consumption and food-cup directed behaviors. The food cues selectively controlled food-cup related behaviors, regardless of the presence of the devalued or nondevalued foods in the food cup. Together, these results are consistent with the view that conditioned cues modulate appetitive and consummatory behaviors with increasing levels of specificity. The closer an action comes to ingestion, the more it is controlled by sensory properties conveyed by learned cues. These data are discussed in the context of allostatic regulation of food foraging and intake.

BackgroundResilience is the capacity of individuals to resist mental disorders despite exposure to stress. Little is known about its neural underpinnings. The putative variation of white-matter microstructure with resilience in adolescence, a critical period for brain maturation and onset of high-prevalence mental disorders, has not been assessed by diffusion tensor imaging (DTI). Lower fractional anisotropy (FA) though, has been reported in the corpus callosum (CC), the brain's largest white-matter structure, in psychiatric and stress-related conditions. We hypothesized that higher FA in the CC would characterize stress-resilient adolescents.MethodThree groups of adolescents recruited from the community were compared: resilient with low risk of mental disorder despite high exposure to lifetime stress (n = 55), at-risk of mental disorder exposed to the same level of stress (n = 68), and controls (n = 123). Personality was assessed by the NEO-Five Factor Inventory (NEO-FFI). Voxelwise statistics of DTI values in CC were obtained using tract-based spatial statistics. Regional projections were identified by probabilistic tractography.ResultsHigher FA values were detected in the anterior CC of resilient compared to both non-resilient and control adolescents. FA values varied according to resilience capacity. Seed regional changes in anterior CC projected onto anterior cingulate and frontal cortex. Neuroticism and three other NEO-FFI factor scores differentiated non-resilient participants from the other two groups.ConclusionHigh FA was detected in resilient adolescents in an anterior CC region projecting to frontal areas subserving cognitive resources. Psychiatric risk was associated with personality characteristics. Resilience in adolescence may be related to white-matter microstructure.

There is a wealth of data showing a large impact of food cues on human ingestion, yet most studies use pictures of food where the precise nature of the associations between the cue and food is unclear. To test whether novel cues which were associated with the opportunity of winning access to food images could also impact ingestion, 63 participants participated in a game in which novel visual cues signalled whether responding on a keyboard would win (a picture of) chocolate, crisps, or nothing. Thirty minutes later, participants were given an ad libitum snack-intake test during which the chocolate-paired cue, the crisp-paired cue, the non-winning cue and no cue were presented as labels on the food containers. The presence of these cues significantly altered overall intake of the snack foods; participants presented with food labelled with the cue that had been associated with winning chocolate ate significantly more than participants who had been given the same products labelled with the cue associated with winning nothing, and in the presence of the cue signalling the absence of food reward participants tended to eat less than all other conditions. Surprisingly, cue-dependent changes in food consumption were unaffected by participants' level of contingency awareness. These results suggest that visual cues that have been pre-associated with winning, but not consuming, a liked food reward modify food intake consistent with current ideas that the abundance of food associated cues may be one factor underlying the 'obesogenic environment'.

Neuroadaptations induced by high-dose cocaine treatment have been hypothesized to persist after the cessation of drug treatment and mediate the expression of sensitization and tolerance to cocaine. We looked for evidence of these neuroadaptations in rats receiving more modest behaviorally effective cocaine treatments. Rats were exposed to either a sensitizing regimen of seven once-daily injections of 15 mg/kg cocaine or a tolerance-producing regimen involving a continuous infusion of the same daily dose. We assessed enzyme activity levels of protein kinase A and adenylate cyclase, and protein levels of tyrosine hydroxylase, cdk5 and neurofilaments in the nucleus accumbens and ventral tegmental area. Only protein kinase A activity levels were altered by cocaine treatment, but this alteration persisted for only 7 days, whereas a sensitized locomotor response was still evident at 21 days. Although behavioral tolerance to cocaine was seen the day after the termination of treatment, none of the molecular measures was altered on this or any other day. Thus, although increased protein kinase A activity can temporarily modulate sensitized responses to cocaine, alterations in total levels of the molecules assessed in our study do not correlate with the expression of sensitized or tolerant locomotor responses to cocaine.

The effects of psychoactive drugs are not just a function of their pharmacological actions, but are due to complex interactions among pharmacological, psychological, and environmental factors. We discuss here how drug-environment interactions determine the likelihood that addictive drugs produce a persistent form of neurobehavioral plasticity (sensitization) thought to be involved in the pathophysiology of addiction and relapse.

Women are at greater risk for cocaine addiction than are men, and female rats similarly show a greater propensity to self-administer cocaine than do male rats. This could be due to the intrinsic sex differences in brain organization, to the activational effects of circulating gonadal hormones, or both. For example, estradiol enhances dopamine release in the striatum and nucleus accumbens, and facilitates the behavioral sensitization induced by repeated cocaine exposure. We report here that independent of circulating gonadal hormones, female rats acquire cocaine self-administration behavior more rapidly, and they self-administer more cocaine at a faster rate than do male rats. This indicates that there is an intrinsic difference between male and female subjects in the neural systems mediating drug-seeking behavior due to sexual differentiation of the brain. Furthermore, we find that the administration of estradiol to gonadectomized female subjects greatly facilitates the acquisition of cocaine self-administration. These data demonstrate that both intrinsic sex differences in brain organization and the actions of circulating estradiol contribute to increased vulnerability for cocaine use in female subjects.

Incentive motivation theory ascribes a critical role to reward-associated stimuli in the generation and maintenance of goal-directed behavior. Repeated psychomotor stimulant treatment, in addition to producing sensitization to the psychomotor-activating effects, can enhance the incentive salience of reward-associated cues and increase their ability to influence behavior. In the present study, we sought to investigate this incentive sensitization effect further by developing a model of conditioned reinforcement (CR) in the mouse and investigating the effects of a sensitizing treatment regimen of amphetamine on CR. Furthermore, we assessed the role of contextual stimuli in amphetamine-induced potentiation of CR. We found that mice responded selectively on a lever resulting in the presentation of a cue previously associated with 30% condensed milk solution, indicating that the cue had attained rewarding properties. Prior treatment with amphetamine (4 x 0.5 mg/kg i.p.) resulted in psychomotor sensitization and enhanced subsequent responding for the CR. Furthermore, this enhancement of responding for the cue occurred independent of the drug-paired context, whereas the sensitized locomotor response was only observed when mice were tested in the same environment as that in which they had received previous amphetamine. These results demonstrate that the CR paradigm previously developed in the rat can be successfully adapted for use in the mouse, and suggest that behavioral sensitization to amphetamine increases the rewarding properties (incentive salience) of reward-paired cues, independent of the drug-paired context.

Behavioral sensitization following repeated intermittent cocaine administrations is thought to involve alterations in cocaine regulation of neural activity within the accumbens and caudate brain regions. Although Fos immunohistochemistry and c-fos in situ hybridization have frequently been used to assess changes in cocaine-induced neural activity following prior cocaine exposure, these techniques have rarely been used to examine neural activity in the accumbens of behaviorally sensitized animals. In the present experiment, we compared the ability of increasing doses of cocaine to induce Fos in the accumbens and caudate of rats following a treatment procedure (7 once daily injections of 15 mg/kg of cocaine or the saline vehicle) shown to produce robust and persistent (1 week) locomotor sensitization. In sensitized animals, there was a leftward shift in the dose-response curve for cocaine induction of Fos in the accumbens, but not in the caudate. These results provide the first parametric evidence for sensitization of cocaine-induced Fos expression in the accumbens.

Treatment of cocaine addiction is hampered by high rates of relapse even after prolonged drug abstinence. This relapse to compulsive cocaine use can be triggered by re-exposure to cocaine, by re-exposure to stimuli previously associated with cocaine or by exposure to stress. In laboratory rats, similar events reinstate cocaine seeking after prolonged withdrawal periods, thus providing a model to study neuronal mechanisms underlying the relapse to cocaine. The endocannabinoid system has been implicated in a number of neuropsychiatric conditions, including drug addiction. The active ingredient of marijuana, Delta9-tetrahydrocannabinol, activates the mesolimbic dopamine (DA) reward system and has rewarding effects in preclinical models of drug abuse. We report here that the synthetic cannabinoid agonist, HU210 (ref. 13), provokes relapse to cocaine seeking after prolonged withdrawal periods. Furthermore, the selective CB1 receptor antagonist, SR141716A (ref. 14), attenuates relapse induced by re-exposure to cocaine-associated cues or cocaine itself, but not relapse induced by exposure to stress. These data reveal an important role of the cannabinoid system in the neuronal processes underlying relapse to cocaine seeking, and provide a rationale for the use of cannabinoid receptor antagonists for the prevention of relapse to cocaine use.

The environmental context in which psychostimulant drugs are administered can have a large effect on both their acute psychomotor activating effects and their ability to induce the psychomotor sensitization associated with repeated drug administration. For example, the acute effects of amphetamine and the development of psychomotor sensitization to amphetamine and cocaine are enhanced when they are administered in a distinct and relatively novel test environment, relative to when they are given in the home cage, in the absence of any environmental stimuli predictive of drug administration. The experiments reported here were designed to further examine this phenomenon and to test the hypothesis that the ability of a distinct context to promote robust psychomotor sensitization is due to its ability to reliably signal (cue) drug administration. Specifically, we compared the ability of contextual cues (a distinct test environment) and discrete cues (light, tone and/or odor), which both reliably predict drug administration, to promote the induction of sensitization. The psychomotor stimulant effects (rotational behavior) of repeated intravenous infusions of 0. 5 mg/kg amphetamine were assessed in rats for whom drug treatments were signaled either: (1) by placement into a distinct test environment; (2) by presentation of discrete cues; or (3) rats for whom drug treatments were given in the home environment in the absence of any environmental cues. Amphetamine produced robust sensitization when given in association with placement into a distinct test environment. The same treatment failed to produce sensitization when the drug was given unsignaled in the animal's home cage. Most importantly, signaling drug administration by presenting discrete cues was not sufficient to promote the robust sensitization seen when treatments were given in a distinct test environment. These results confirm that the induction of psychomotor sensitization can be powerfully modulated by environmental context and further establish that, although contextual stimuli associated with a distinct test environment promote robust sensitization, discrete cues that merely predict drug administration do not have this property. Possible reasons for the difference in the ability of contextual versus discrete environmental cues to promote sensitization are discussed.

We have previously reported that intravenous (i.v.) administrations of 0.5-1.0 mg/kg of amphetamine in the absence of any environmental stimuli predictive of drug administration failed to induce psychomotor sensitization whereas the same drug did produce robust sensitization when given in association with environmental novelty. These results were obtained by studying rotational behavior in animals with a unilateral 6-OHDA lesion of the mesostriatal dopamine system. The purpose of this study was to determine if environmental novelty has a similar effect on sensitization to the locomotor activating effects of amphetamine in neurologically intact rats. Rats were implanted with i.v. catheters and divided in four groups. Two groups were housed in locomotor activity cages and given seven consecutive i.v. infusions of either saline (SAL-HOME group) or 0.375 mg/kg of amphetamine (AMPH-HOME group), using a remotely activated delivery system. Simultaneously, the other two groups were transported to the test cages and given the same treatment (SAL-NOVEL and AMPH-NOVEL groups). After one week withdrawal, all groups were given an amphetamine challenge (0.375 mg/kg, i.v.). Amphetamine sensitization developed when the drug was administered under NOVEL conditions, as indicated by a progressive increase in amphetamine-induced locomotor activity over test sessions and by a greater response to the amphetamine challenge in the AMPH-pretreated versus the SAL-pretreated group. In contrast, no sensitization was observed under HOME conditions. Similar results were obtained with the analysis of vertical activity.

This paper presents data concerning the dose-effect relationships of intravenously administered amphetamine and cocaine on two widely used measures of psychomotor activation: locomotor crossover activity in neurologically intact rats, and rotational behavior in rats with a unilateral 6-hydroxydopamine lesion. There were marked differences in dose-effect relationships, both as a function of drug and of behavioral measure. Amphetamine produced a linear increase in rotational behavior over a wide range of doses (the highest effective dose was 76.8 times the lowest), but a linear increase in locomotor crossover activity over only a narrow dose range (the highest effective dose was only four times the lowest). In contrast, for cocaine, the dose-effect relationships for the two behaviors were very similar, but for both behaviors the effective dose range was quite narrow, the highest effective dose being only between two and four times the lowest. The data highlight the advantages and disadvantages of these measures as indices of the psychomotor activating effects of psychostimulant drugs.

The use of animal models is essential in carrying out research into clinical phenomena such as addiction. However, the complexity of the clinical condition inevitably means that even the best animal models are inadequate representations of the condition they seek to mimic. Such mismatches may account for apparent inconsistencies between discoveries in animal models, including the identification of potential novel therapies, and the translation of such discoveries to the clinic. We argue that it is overambitious to attempt to model human disorders such as addiction in animals, and especially in rodents, where "validity" of such models is often limited to superficial similarities, referred to as "face validity" that reflect quite different underlying phenomena and biological processes from the clinical situation. Instead, we suggest a more profitable approach may be to identify (a) well-defined intermediate human behavioral phenotypes that reflect defined, limited aspects of the human clinical disorder, and (b) to develop animal models that are homologous with those discrete human behavioral phenotypes in terms of psychological processes, and underlying neurobiological mechanisms. Examples of current weaknesses and suggestions for more limited approaches that may allow better homology between the test animal and human condition are made.

In appetitive Pavlovian associative learning, a stimulus (conditioned stimulus, CS) that has been associated with the delivery of a reinforcing event (unconditioned stimulus, US; e.g., food) can subsequently elicit or modulate goal-directed instrumental behaviors. For example, a Pavlovian CS can serve to reinforce (novel) instrumental behavior (conditioned reinforcement or CRf), or it can energize and potentiate ongoing instrumental responses when presented non-contingently (Pavlovian-instrumental transfer or PIT). Notably, these different effects of a Pavlovian CS on instrumental behavior are mediated by dissociable psychological and neurobiological mechanisms. Given the critical role that Pavlovian-instrumental interactions play in regulating motivated behavior and maladaptive manifestations of motivation such as eating disorders and addictions, understanding the underlying psychological and neurobiological mechanisms will be important. This unit describes behavioral protocols that produce robust and reliable PIT and CRf in mice and that open the door for future studies using transgenic approaches into the molecular mechanisms underlying associative learning and motivation.

In appetitive Pavlovian associative learning, a stimulus (conditioned stimulus, CS) that has been associated with the delivery of a reinforcing event (unconditioned stimulus, US; e.g., food) can subsequently elicit or modulate goal-directed instrumental behaviors. For example, a Pavlovian CS can serve to reinforce (novel) instrumental behavior (conditioned reinforcement or CRf), or it can energize and potentiate ongoing instrumental responses when presented non-contingently (Pavlovian-instrumental transfer or PIT). Notably, these different effects of a Pavlovian CS on instrumental behavior are mediated by dissociable psychological and neurobiological mechanisms. Given the critical role that Pavlovian-instrumental interactions play in regulating motivated behavior and maladaptive manifestations of motivation such as eating disorders and addictions, understanding the underlying psychological and neurobiological mechanisms will be important. This unit describes behavioral protocols that produce robust and reliable PIT and CRf in mice and that open the door for future studies using transgenic approaches into the molecular mechanisms underlying associative learning and motivation.

Serum response factor (SRF) is a ubiquitously expressed stimulus-dependent transcription factor that regulates gene expression by binding to serum response element in the promoter region of target genes. Recent studies in mice have shown that SRF is important for activity-dependent gene expression and synaptic plasticity in the adult brain but is dispensable for neuronal survival. Given these important functions of SRF in the CNS, it is expected to play a critical role in several aspects of learning and memory. Here we evaluated the role of SRF in conditioned reinforcement using two lines of conditional SRF mutant mice. These SRF mutant mice exhibited different spatial patterns of SRF deletion in the post-natal forebrain and notably within the hippocampus. SRF deletion was more widespread in SRF-CKCre mutants than in SRF-SynCre mutants, particularly in areas of the cortex and striatum. Mutant and wild-type mice were trained to associate one auditory cue (CS+) with reward, whereas a second cue remained relatively neutral (CS−). All mice readily acquired this discrimination, entering the food cup during CS+ but not during CS−. In a subsequent test of conditioned reinforcement, in the absence of food, wild-type control mice and SRF-SynCre mice learned to selectively perform an instrumental response that yielded CS+ presentation rather than another response that produced CS−. SRF-CKCre mutants failed to show this preferential responding for CS+. These results suggest a role for SRF in conditioned reinforcement, a manifestation of incentive learning that has been implicated in many aspects of adaptive and maladaptive behavior, such as substance abuse and eating disorders.

The mouse has emerged as a uniquely valuable species for studying the molecular and genetic basis of complex behaviors and modeling neuropsychiatric disease states. While valid and reliable preclinical assays for reward-related behaviors are critical to understanding addiction-related processes, and various behavioral procedures have been developed and characterized in rats and primates, there have been relatively few studies using operant-based addiction-relevant behavioral paradigms in the mouse. Here we describe the performance of the C57BL/6J inbred mouse strain on three major reward-related paradigms, and replicate the same procedures in two other commonly used inbred strains (DBA/2J, BALB/cJ). We examined Pavlovian-instrumental transfer (PIT) by measuring the ability of an auditory cue associated with food reward to promote an instrumental (lever press) response. In a separate experiment, we assessed the acquisition and extinction of a simple stimulus-reward instrumental behavior on a touch screen based task. Reinstatement of this behavior was then examined following either continuous exposure to cues (conditioned reinforcers, CRs) associated with reward, brief reward and CR exposure, or brief reward exposure followed by continuous CR exposure. The third paradigm examined sensitivity of an instrumental (lever press) response to devaluation of food reward (a probe for outcome insensitive, habitual behavior) by repeated pairing with malaise. Results showed that C57BL/6J mice displayed robust PIT, as well as clear extinction and reinstatement, but were insensitive to reinforcer devaluation. DBA/2J mice showed good PIT and (rewarded) reinstatement, but were slow to extinguish and did not show reinforcer devaluation or significant CR-reinstatement. BALB/cJ mice also displayed good PIT, extinction and reinstatement, and retained instrumental responding following devaluation, but, unlike the other strains, demonstrated reduced Pavlovian approach behavior (food magazine head entries). Overall, these assays provide robust paradigms for future studies using the mouse to elucidate the neural, molecular and genetic factors underpinning reward-related behaviors relevant to addiction research.

Reward is a concept fundamental to discussions of drug abuse and addiction. The idea that altered sensitivity to either drug-reward, or to rewards in general, contributes to, or results from, drug-taking is a common theme in several theories of addiction. However, the concept of reward is problematic in that it is used to refer to apparently different behavioural phenomena, and even to diverse neurobiological processes (reward pathways). Whether these different phenomena are different behavioural expressions of a common underlying process is not established, and much research suggests that there may be only loose relationships among different aspects of reward. Measures of rewarding effects of drugs in humans often depend upon subjective reports. In animal studies, such insights are not available, and behavioural measures must be relied upon to infer rewarding effects of drugs or other events. In such animal studies, but also in many human methods established to objectify measures of reward, many other factors contribute to the behaviour being studied. For that reason, studying the biological (including genetic) bases of performance of tasks that ostensibly measure reward cannot provide unequivocal answers. The current overview outlines the strengths and weaknesses of current approaches that hinder the conciliation of cross-species studies of the genetics of reward sensitivity and the dysregulation of reward processes by drugs of abuse. Some suggestions are made as to how human and animal studies may be made to address more closely homologous behaviours, even if those processes are only partly able to isolate 'reward' from other factors contributing to behavioural output.