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Cortico-striatal neurocircuits mediate goal-directed and habitual actions which are necessary for adaptive behaviour. It has recently been proposed that some of the core symptoms of autism spectrum disorder (ASD) and Gilles de la Tourette syndrome (GTS), such as tics and repetitive behaviours, may emerge because of imbalances in these neurocircuits. We have recently developed a model of ASD and GTS by knocking downImmp2l, a mitochondrial gene frequently associated with these disorders. The current study sought to determine whetherImmp2lknockdown (KD) in male mice alters flexible, goal- or cue-driven behaviour using procedures specifically designed to examine response-outcome and stimulus-response associations, which underlie goal-directed and habitual behaviour, respectively. WhetherImmp2lKD alters neuron density in cortico-striatal neurocircuits known to regulate these behaviours was also examined.Immp2lKD mice and wild type-like mice (WT) were trained on Pavlovian and instrumental learning procedures where auditory cues predicted food delivery and lever-press responses earned a food outcome. It was demonstrated that goal-directed learning was not changed forImmp2lKD mice compared to WT mice, as lever-press responses were sensitive to changes in the value of the food outcome, and to contingency reversal and degradation. There was also no difference in the capacity of KD mice to form habitual behaviours compared to WT mice following extending training of the instrumental action. However,Immp2lKD mice were more responsive to auditory stimuli paired with food as indicated by a non-specific increase in lever response rates during Pavlovian-to-instrumental transfer. Finally, there were no alterations to neuron density in striatum or any prefrontal cortex or limbic brain structures examined. Thus, the current study suggests that Immp2l is not necessary for learned maladaptive goal or stimulus driven behaviours in ASD or GTS, but that it may contribute to increased capacity for external stimuli to drive behaviour. Alterations to stimulus-driven behaviour could potentially influence the expression of tics and repetitive behaviours, suggesting that genetic alterations toImmp2lmay contribute to these core symptoms in ASD and GTS. Given that this is the first application of this battery of instrumental learning procedures to a mouse model of ASD or GTS, it is an important initial step in determining the contribution of known risk-genes to goal-directed versus habitual behaviours, which should be more broadly applied to other rodent models of ASD and GTS in the future.

Recognition memory provides the ability to distinguish familiar from novel objects and places, and is important for recording and updating events to guide appropriate behavior. The hippocampus (HPC) and medial prefrontal cortex (mPFC) have both been implicated in recognition memory, but the nature of HPC–mPFC interactions, and its impact on local circuits in mediating this process is not known. Here we show that novelty discrimination is accompanied with higher theta activity (4–10 Hz) and increased c-Fos expression in both these regions. Moreover, theta oscillations were highly coupled between the HPC and mPFC during recognition memory retrieval for novelty discrimination, with the HPC leading the mPFC, but not during initial learning. Principal neurons and interneurons in the mPFC responded more strongly during recognition memory retrieval compared with learning. Optogenetic silencing of HPC input to the mPFC disrupted coupled theta activity between these two structures, as well as the animals' (male Sprague Dawley rats) ability to differentiate novel from familiar objects. These results reveal a key role of monosynaptic connections between the HPC and mPFC in novelty discrimination via theta coupling and identify neural populations that underlie this recognition memory-guided behavior.SIGNIFICANCE STATEMENTMany memory processes are highly dependent on the interregional communication between the HPC and mPFC via neural oscillations. However, how these two brain regions coordinate their oscillatory activity to engage local neural populations to mediate recognition memory for novelty discrimination is poorly understood. This study revealed that the HPC and mPFC theta oscillations and their temporal coupling is correlated with recognition memory-guided behavior. During novel object recognition, the HPC drives mPFC interneurons to effectively reduce the activity of principal neurons. This study provides the first evidence for the requirement of the HPC–mPFC pathway to mediate recognition memory for novelty discrimination and describes a mechanism for how this memory is regulated.

Pathological forms of the microtubule-associated protein tau are involved in a large group of neurodegenerative diseases named tauopathies, including frontotemporal lobar degeneration (FTLD-tau). K369I mutant tau transgenic mice (K3 mice) recapitulate neural and behavioural symptoms of FTLD, including tau aggregates in the cortex, alterations to nigrostriatum, memory deficits and parkinsonism. The aim of this study was to further characterise the K3 mouse model by examining functional alterations to the striatum. Whole-cell patch-clamp electrophysiology was used to investigate the properties of striatal neurons in K3 mice and wildtype controls. Additionally, striatal-based instrumental learning tasks were conducted to assess goal-directed versus habitual behaviours (i.e., by examining sensitivity to outcome devaluation and progressive ratios). The K3 model demonstrated significant alterations in the discharge properties of striatal neurons relative to wildtype mice, which manifested as a shift in neuronal output towards a burst firing state. K3 mice acquired goal-directed responding faster than control mice and were goal-directed at test unlike wildtype mice, which is likely to indicate reduced capacity to develop habitual behaviour. The observed pattern of behaviour in K3 mice is suggestive of deficits in dorsal lateral striatal function and this was supported by our electrophysiological findings. Thus, both the electrophysiological and behavioural alterations indicate that K3 mice have early deficits in striatal function. This finding adds to the growing literature which indicate that the striatum is impacted in tau-related neuropathies such as FTLD, and further suggests that the K3 model is a unique mouse model for investigating FTLD especially with striatal involvement.

Instrumental actions are initially goal-directed but with repeated performance can become habitual. Habitual actions are adaptive, learned behaviours that are automated in order to reduce cognitive load and to allow for efficient interaction with the environment. Goal-directed and habitual actions are mediated by distinct neurocircuits which centre on the dorsal striatum and involve different cortical and limbic inputs. The lateral hypothalamus (LH) has yet to be considered in this neurocircuitry despite its anatomical connections with these neurocircuits and its established role in motivated behaviour. The aim of the current study was to determine whether the LH has a role in the development of habitual actions in rats by knocking down protein expression in the LH with short hairpin RNAs (shRNA). Two shRNAs were utilised, both of which were shown to reduce the expression of two neuropeptides within the LH, orexin and melanin-concentrating hormone, compared to a saline-vehicle control. This was unexpected given that one shRNA was a control vector (i.e, scrambled sequence), and the other shRNA was supposed to selectively target orexin's precursor protein. Given this lack of specificity and that shRNA's are known to be neurotoxic, the current study examined the impact of non-selective dysfunction of the LH on habitual actions. Adult male Long-Evans rats were trained to press a lever for a food outcome and were tested for goal directed and habitual behaviour following devaluation of the food. The shRNA groups displayed goal-directed actions following moderate instrumental training, but did not develop habitual actions following extended training. That is, control rats developed the expected habitual behaviour where lever-response rates were insensitive to outcome value when tested, whilst the shRNA groups reduced rates of responding on the lever under devalued conditioned and hence remained goal-directed. This failure to demonstrate habitual actions was unlikely to be secondary to changes in motivation or arousal as the shRNA groups did not show altered food consumption, body weight, lever response rates, or motor performance on a rota rod or tapered balance beam. However, locomotor activity was reduced in an open field test, consistent with the proposed role of the LH in spontaneous locomotor activity. Therefore, this study implicates the LH in habitual learning, and adds to the emerging evidence that the LH has a role in associative learning processes. This finding has implications for human conditions where there is dysfunction or neurodegeneration in the LH, as well as altered habitual actions, such as in Parkinson's disease and drug addiction.

The neuropeptide orexin-A (OX-A) has diverse functions, including maintaining arousal, autonomic control, motor activity and stress responses. These functions are regulated at different terminal regions where OX-A is released. The current study examined the physiological and behavioural effects of OX-A microinjections into the central amygdala (CeA) under basal and stressed conditions in rats. When OX-A was microinjected into the CeA and the animals returned to the home-cage, heart rate and mean arterial pressure were increased compared to vehicle-injected controls. General activity of the animal was also increased, indicating that OX-A activity in CeA contributes to increased arousal. This outcome is similar to the effects of central intracerebroventricular infusions of OX-A, as well as the cardiovascular effects previously demonstrated at many of OX’s efferent hypothalamic and brainstem structures. In a second study, animals were fear-conditioned to a context by delivery of electric footshocks and then animals were re-exposed to the conditioned context at test. When OX-A was microinjected at test, freezing behaviour was reduced and there was a corresponding increase in the animal’s activity but no impact on the pressor and cardiac responses (i.e, blood pressure and heart rate were unchanged). This reduction in freezing suggests that OX-A activates amygdala neurons that inhibit freezing, which is similar to the actions of other neuropeptides in the CeA that modulate the appropriate defence response to fearful stimuli. Overall, these data indicate that the CeA is an important site of OX-A modulation of cardiovascular and motor activity, as well as conditioned freezing responses.

Goldberg-Shprintzen syndrome is a poorly understood condition characterized by learning difficulties, facial dysmorphism, microcephaly, and Hirschsprung disease. GOSHS is due to recessive mutations in KIAA1279, which encodes kinesin family member 1 binding protein (KIF1BP, also known as KBP). We examined the effects of inactivation of Kif1bp in mice. Mice lacking Kif1bp died shortly after birth, and exhibited smaller brains, olfactory bulbs and anterior commissures, and defects in the vagal and sympathetic innervation of the gut. Kif1bp was found to interact with Ret to regulate the development of the vagal innervation of the stomach. Although newborn Kif1bp−/− mice had neurons along the entire bowel, the colonization of the gut by neural crest-derived cells was delayed. The data show an essential in vivo role for KIF1BP in axon extension from some neurons, and the reduced size of the olfactory bulb also suggests additional roles for KIF1BP. Our mouse model provides a valuable resource to understand GOSHS.

Goal-directed actions are controlled by the value of the consequences they produce and so increase when what they produce is valuable and decrease when it is not. With continued invariant practice, however, goal-directed actions can become habits, controlled not by their consequences but by antecedent, reward-related states and stimuli. Here, we show that pre-exposure to methamphetamine (METH) caused abnormally rapid development of habitual control. Furthermore, these drug-induced habits differed strikingly from conventional habits; we found that they were insensitive both to changes in reward value and to the effects of negative feedback. In addition to these behavioral changes, METH exposure produced bidirectional changes to synaptic proteins in the dorsal striatum. In the dorsomedial striatum, a structure critical for goal-directed action, METH exposure was associated with a reduction in glutamate receptor and glutamate vesicular proteins, whereas in the dorsolateral striatum, a region that has previously been implicated in habit learning, there was an increase in these proteins. Together, these results indicate that METH exposure promotes habitual control of action that appears to be the result of bidirectional changes in glutamatergic transmission in the circuits underlying goal-directed and habit-based learning.

The organization of the mouse spinal dorsal horn has been delineated in 2D for the six Rexed laminae in our publication Atlas of the Spinal Cord: Mouse, Rat, Rhesus, Marmoset, and Human. in the present study, the tissue clearing technique CLARITY was used to observe the cyto- and chemoarchitecture of the mouse spinal cord in 3D, using a variety of immunohistochemical markers. We confirm prior observations regarding the location of glycine and serotonin immunoreactivities. Novel observations include the demonstration of numerous calcitonin gene-related peptide (CGRP) perikarya, as well as CGRP fibers and terminals in all laminae of the dorsal horn. We also observed sparse choline acetyltransferase (ChAT) immunoreactivity in small perikarya and fibers and terminals in all dorsal horn laminae, while gamma aminobutyric acid (GABA) and glutamate decarboxylase-67 (GAD67) immunoreactivities were found only in small perikarya and fibers. Finally, numerous serotonergic fibers were observed in all laminae of the dorsal horn. in conclusion, CLARITY confirmed the 2D immunohistochemical properties of the spinal cord. Furthermore, we observed novel anatomical characteristics of the spinal cord and demonstrated that CLARITY can be used on spinal cord tissue to examine many proteins of interest., National Health and Medical Research CouncilNational Health and Medical Research Council of Australia [APP1140295, APP1188744]; Scientific and Technological Research Council of TurkeyTurkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [SBAG-114S405]; Rebecca L. Cooper Medical Research Foundation; Centre of Excellence for Integrative Brain Function, Australian Research Council [CE140100007], We thank Dr. Emma Schofield for her technical support. This research was funded by the National Health and Medical Research Council (APP1140295 and APP1188744, awarded to George Paxinos), Scientific and Technological Research Council of Turkey (awarded to Gulgun Sengul, SBAG-114S405), Rebecca L. Cooper Medical Research Foundation (no grant number available, awarded to Huazheng Liang), and Centre of Excellence for Integrative Brain Function, Australian Research Council (CE140100007, awarded to George Paxinos), and the APC was funded by the Rebecca L. Cooper Medical Research Foundation.

Rationale Ethanol has rewarding and aversive properties, and the balance of these properties influences voluntary ethanol consumption. Preclinical and clinical evidence show that the aversive properties of ethanol limit intake. The neural circuits underlying ethanol-induced aversion learning are not fully understood. We have previously shown that the lateral habenula (LHb), a region critical for aversive conditioning, plays an important role in ethanol-directed behaviors. However, the neurocircuitry through which LHb exerts its actions is unknown. Objective In the present study, we investigate a role for the rostromedial tegmental nucleus (RMTg), a major LHb projection target, in regulating ethanol-directed behaviors. Methods Rats received either sham or RMTg lesions and were studied during voluntary ethanol consumption; operant ethanol self-administration, extinction, and yohimbine-induced reinstatement of ethanol-seeking; and ethanol-induced conditioned taste aversion (CTA). Results RMTg lesions increased voluntary ethanol consumption and accelerated extinction of ethanol-induced CTA. Conclusions The RMTg plays an important role in regulating voluntary ethanol consumption, possibly by mediating ethanol-induced aversive conditioning.

Instrumental actions are initially goal-directed and driven by their associated outcome. However, with repeated experience habitual actions develop which are automated and efficient, as they are instead driven by antecedent stimuli. Dopamine is thought to facilitate the transition from goal-directed to habitual actions. This idea has been largely derived from evidence that psychostimulants accelerate the development of habitual actions. In the current study, we examined the impact of L-dopa (levodopa or L-dihydroxyphenylalanine), which also potentiates dopamine activity, on habitual learning. L-dopa was systemically administered prior to training rats to press a lever for a food outcome. When tested, L-dopa exposed animals were insensitive to changes in the value of the food outcome, and hence demonstrated accelerated habitual behavioral control compared to control animals that remained goal directed. We also showed that when N-acetylcysteine (NAC), an antioxidant and regulator of glutamate activity, was co-administered with L-dopa, it prevented the transition to habitual behavior; an effect demonstrated previously for cocaine. Therefore, this study establishes similarities between L-dopa and psychostimulants in both the development and prevention of habitual actions, and supports the notion that excess dopamine potentiates habitual learning. This finding extends the limited existing knowledge of the impact of L-dopa on learning and behavior, and has implications for neurological disorders where L-dopa is the primary treatment.

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Establishing the neurocircuitry involved in inhibiting fear is important for understanding and treating anxiety disorders. To date, extinction procedures have been predominately used to examine the inhibition of learned fear, where fear is reduced to a conditioned stimulus (CS) by presenting it in the absence of the unconditioned stimulus (US). However, learned fear can also be reduced by habituation procedures where the US is presented in the absence of the CS. Here we used expression of the activity marker c-Fos in rats to compare the recruitment of several forebrain structures following fear habituation and extinction. Following fear conditioning where a tone CS was paired with a loud noise US, fear was then reduced the following day by either presentation of the CS or US alone (i.e. CS extinction or US habituation, respectively). This extinction and habituation training recruited several common structures, including infralimbic cortex, basolateral amygdala, midline thalamus and medial hypothalamus (orexin neurons). Moreover, this overlap was shared when examining the neural correlates of the expression of habituation and extinction, with common recruitment of infralimbic cortex and midline thalamus. However, there were also important differences. Specifically, acquisition of habituation was associated with greater recruitment of prelimbic cortex whereas expression of habituation was associated with greater recruitment of paraventricular thalamus. There was also less recruitment of central amygdala for habituation compared to extinction in the retention phase. These findings indicate that largely overlapping neurocircuitries underlie habituation and fear extinction and imply common mechanisms for reducing fear across different inhibitory treatments.

Addiction is characterized by a persistent loss of behavioral control resulting in insensitivity to negative feedback and abnormal decision-making. Here, we investigated the influence of methamphetamine (METH)-paired contextual cues on decision-making in rats. Choice between goal-directed actions was sensitive to outcome devaluation in a saline-paired context but was impaired in the METH-paired context, a deficit that was also found when negative feedback was provided. Reductions in c-Fos-related immunoreactivity were found in dorsomedial striatum (DMS) but not dorsolateral striatum after exposure to the METH context suggesting this effect reflected a loss specifically in goal-directed control in the METH context. This reduction in c-Fos was localized to non-enkephalin-expressing neurons in the DMS, likely dopamine D1-expressing direct pathway neurons, suggesting a relative change in control by the D1-direct versus D2-indirect pathways originating in the DMS may have been induced by METH-context exposure. To test this suggestion, we infused the adenosine 2A receptor antagonist ZM241385 into the DMS prior to test to reduce activity in D2 neurons relative to D1 neurons in the hope of reducing the inhibitory output from this region of the striatum. We found that this treatment fully restored sensitivity to negative feedback in a test conducted in the METH-paired context. These results suggest that drug exposure alters decision-making by downregulation of the circuitry mediating goal-directed action, an effect that can be ameliorated by acute A2A receptor inhibition in this circuit.

Drug addiction is characterized in part by a lack of flexible control over drug-related behaviors. This could be the result of strengthening drug-related habits or diminished goal-directed control. The present chapter reviews research on the effects of drugs of abuse on the medial and lateral subregions of the dorsal striatum known to mediate goal-directed and habitual behaviors. Evidence is presented, which demonstrates that drugs of abuse can cause persistent neuroplastic changes to both the goal-directed and habit systems. Importantly, it is also demonstrated that goal-directed control can be rescued by interventions that either decrease activity of the dorsolateral striatal habit system or promote or normalize activity in the dorsomedial striatal goal-directed system. Identifying when habits dominate behavioral control and localizing changes to underlying circuits will be essential for developing the most effective treatments to restore behavioral control in drug addiction.

Access to highly palatable and calorically dense foods contributes to increasing rates of obesity worldwide. Some have made the controversial argument that consumption of such foods can lead to “food addiction,” yet little is known about how long-term access to highly palatable foods might alter goal-directed learning and decision making. In the following experiments, rats were given 5 weeks of continuous or restricted daily access to sweetened condensed milk (SCM) before instrumental training for food reward. Subsequently we examined whether goal-directed performance was impaired in these groups using the outcome-devaluation task. Control rats reduced responding following devaluation of the earned outcome as did those with previous continuous access to SCM. Of interest, rats with previous restricted access to SCM responded similarly under the devalued and nondevalued conditions, indicating loss of goal-directed control of responding. To identify whether the loss of goal-directed control was accompanied by differences in neuronal activity, we used c-Fos immunohistochemistry to examine the patterns of activation during devaluation testing. We observed greater c-Fos immunoreactivity in the dorsolateral striatum (DLS) and associated cortical regions in the group that received previous restricted access to SCM and demonstrated a lack of sensitivity to outcome devaluation. Infusion of the AMPA-receptor antagonist CNQX or dopamine D1-receptor antagonist SCH-23390 into the DLS before testing restored goal-directed performance in the restricted SCM group, confirming that this region is essential for habit-based performance. These results indicate that previous diet can alter subsequent learning and activity in the neural circuits that support performance.

The lateral habenula (LHb) is an epithalamic brain region implicated in aversive processing via negative modulation of midbrain dopamine (DA) and serotonin (5-HT) systems. Given the role of the LHb in inhibiting DA and 5-HT systems, it is thought to be involved in various psychiatric pathologies, including drug addiction. In support, it has been shown that LHb plays a critical role in cocaine- and ethanol-related behaviors, most likely by mediating drug-induced aversive conditioning. In our previous work, we showed that LHb lesions increased voluntary ethanol consumption and operant ethanol self-administration and blocked yohimbine-induced reinstatement of ethanol self-administration. LHb lesions also attenuated ethanol-induced conditioned taste aversion suggesting that a mechanism for the increased intake of ethanol may be reduced aversion learning. However, whether afferents to the LHb are required for mediating effects of the LHb on these behaviors remained to be investigated. Our present results show that lesioning the fiber bundle carrying afferent inputs to the LHb, the stria medullaris (SM), increases voluntary ethanol consumption, suggesting that afferent structures projecting to the LHb are important for mediating ethanol-directed behaviors. We then chose two afferent structures as the focus of our investigation. We specifically studied the role of the inputs from the lateral hypothalamus (LH) and ventral pallidum (VP) to the LHb in ethanol-directed behaviors. Our results show that the LH-LHb projection is necessary for regulating voluntary ethanol consumption. These results are an important first step towards understanding the functional role of afferents to LHb with regard to ethanol consumption.

Toluene is a commonly abused inhalant that is easily accessible to adolescents. Despite the increasing incidence of use, our understanding of its long-term impact remains limited. Here, we used a range of techniques to examine the acute and chronic effects of toluene exposure on glutameteric and GABAergic function, and on indices of psychological function in adult rats after adolescent exposure. Metabolomics conducted on cortical tissue established that acute exposure to toluene produces alterations in cellular metabolism indicative of a glutamatergic and GABAergic profile. Similarly, in vitro electrophysiology in Xenopus oocytes found that acute toluene exposure reduced NMDA receptor signalling. Finally, in an adolescent rodent model of chronic intermittent exposure to toluene (10 000 ppm), we found that, while toluene exposure did not affect initial learning, it induced a deficit in updating that learning when response-outcome relationships were reversed or degraded in an instrumental conditioning paradigm. There were also group differences when more effort was required to obtain the reward; toluene-exposed animals were less sensitive to progressive ratio schedules and to delayed discounting. These behavioural deficits were accompanied by changes in subunit expression of both NMDA and GABA receptors in adulthood, up to 10 weeks after the final exposure to toluene in the hippocampus, prefrontal cortex and ventromedial striatum; regions with recognized roles in behavioural flexibility and decision-making. Collectively, our data suggest that exposure to toluene is sufficient to induce adaptive changes in glutamatergic and GABAergic systems and in adaptive behaviour that may underlie the deficits observed following adolescent inhalant abuse, including susceptibility to further drug-use.

Extinction promotes abstinence from drug seeking. Extinction expression is an active process, dependent on infralimbic prefrontal cortex (ilPFC). However, the neurocircuitry mediating extinction expression is unknown. Here we studied the neural mechanisms for expression of extinction of alcoholic beer seeking in rats. We first examined the pattern of activation in prefrontal cortex projections to medial dorsal hypothalamus (MDH) (i.e., perifornical and dorsomedial nuclei) during extinction expression. Double labeling for retrograde tracer cholera toxin B subunit (CTb) and the neuronal activity marker c-Fos revealed significant recruitment of MDH projecting ilPFC neurons during extinction expression. We then studied the causal role of MDH in inhibiting alcoholic beer seeking during extinction expression. MDH infusion of the inhibitory neuropeptide cocaine- and amphetamine-regulated transcript prevented extinction expression, showing that MDH is necessary for extinction expression. Next we examined the pattern of activation in MDH projections to paraventricular thalamus (PVT) during extinction expression. Double labeling for CTb and c-Fos revealed significant recruitment of PVT projecting MDH neurons during extinction expression. We also showed, using triple-label immunofluorescence, that the majority of PVT projecting extinction neurons express prodynorphin, suggesting that actions at κ opioid receptors (KORs) in PVT may be critical for inhibiting alcoholic beer seeking. Consistent with this, infusions of a KOR agonist into PVT prevented reinstatement of alcoholic beer seeking showing that PVT KOR activation is sufficient to inhibit alcoholic beer seeking. Together, these findings identify a role for MDH and its ilPFC afferents and PVT efferents in inhibiting alcoholic beer seeking during extinction expression.

We studied the role of cocaine and amphetamine related transcript (CART) in regulating context induced reinstatement (renewal) of reward seeking. Rats were trained to respond for alcoholic beer in context A before extinction in context B. Rats were tested for responding in context A (ABA) and context B (ABB). Intracerebroventricular (ICV) infusions of the active fragment CART55-102 but not the inactive fragment CART1-27 before test prevented ABA renewal of extinguished responding. ICV CART55-102 had no effect on responding in the extinction context (ABB). ICV CART55-102 also altered the profile of behavioral responses observed on test in the training (ABA) but not extinction (ABB) context. These results identify a novel role for CART in preventing reinstatement of reward seeking.

The aim of this study was to test the role of the perifornical hypothalamus and adjacent areas in the behavioral and cardiovascular responses to two forms of stress, conditioned fear to context and restraint. Of particular interest was the role of the hypocretin (orexin) containing neurons in these responses. Rats implanted with radio-telemetric probes and fear conditioned to a context received bilateral injections of the neurotoxin hypocretin-2-saporin centered on the perifornical area. One week later, the animals were tested for conditioned fear to context and restraint while recording freezing, 22 kHz ultrasonic vocalizations, activity, mean arterial pressure and heart rate. Histological verification revealed that the lesions were not specific since virtually all the neurons within the injection area were lost. Nevertheless, these lesions, which were centered on the perifornical area, markedly reduced all recorded components of the contextual fear response (by 70%) but had no effect on the response to restraint. The lesions also caused a reduction in body weight and reduced the circadian rhythm of the recorded parameters. The results show (i) that hypocretin-2-saporin was not specific enough to produce lesions restricted to the hypocretin system, (ii) that neurons of the perifornical area are necessary for the expression of both the cardiovascular and behavioral components of conditioned fear to context, and (iii) that the same neurons are not necessary for the cardiovascular response to restraint. Thus, the perifornical hypothalamus is critical for some forms of stress but not others. We propose that it is a necessary relay for emotional responses in which the psychological component is stronger than the sensory component.

Abused amphetamines, such as d-amphetamine (AMPH) and methamphetamine (METH), are highly addictive and destructive to health and productive lifestyles. The abuse of these drugs is associated with impulsive behavior, which is likely to contribute to addiction. The amphetamines also differentially damage dopamine (DA) and serotonin (5-HT) systems, which regulate impulsive behavior; therefore, exposure to these drugs may differentially alter impulsive behavior to effect the progression of addiction. We examined the impact of neurotoxicity induced by three amphetamines on impulsive action using a stop-signal task in rats. Animals were rewarded with a food pellet after lever pressing (i.e., a go trial), unless an auditory cue was presented and withholding lever press gained reward (i.e., a stop trial). Animals were trained on the task and then exposed to a neurotoxic regimen of either AMPH, p-chloroamphetamine (PCA), or METH. These regimens preferentially reduced DA transporter levels in striatum, 5-HT transporter levels in prefrontal cortex, or both, respectively. Assessment of performance on the stop-signal task beginning 1 week after the treatment revealed that AMPH produced a deficit in go-trial performance, whereas PCA did not alter performance on either trial type. In contrast, METH produced a deficit in stop-trial performance (i.e., impulsive action) but not go-trial performance. These findings suggest that the different neurotoxic consequences of substituted amphetamines are associated with different effects on inhibitory control over behavior. Thus, the course of addiction and maladaptive behavior resulting from exposure to these substances is likely to differ.

Alcohol-related stimuli can trigger relapse of alcohol-seeking behaviors even after extended periods of abstinence. Extinction of such stimuli can reduce their impact on relapse; however, the expression of extinction can be disrupted when testing occurs outside the context where extinction learning took place, an effect termed renewal. Behavioral and pharmacological methods have recently been shown to augment extinction learning; yet, it is not known whether the improved expression of extinction following these treatments remains context-dependent. Here we examined whether two methods, compound–stimulus extinction and treatment with the noradrenaline reuptake inhibitor atomoxetine, would reduce the vulnerability of extinction to a change in context. Following alcohol self-administration, responding was extinguished in a distinct context. After initial extinction, further extinction was given to a target stimulus presented in compound with another alcohol-predictive stimulus intended to augment prediction error (Experiment 1) or after a systemic injection of atomoxetine (1.0 mg kg−1; Experiment 2). A stimulus extinguished as part of a compound elicited less responding than a stimulus receiving equal extinction alone regardless of whether animals were tested in the training or extinction context; however, reliable renewal was not observed in this paradigm. Importantly, atomoxetine enhanced extinction relative to controls even in the presence of a reliable renewal effect. Thus, extinction of alcohol-seeking behavior can be improved by extinguishing multiple alcohol-predictive stimuli or enhancing noradrenaline neurotransmission during extinction training. Importantly, both methods improve extinction even when the context is changed between extinction training and test, and thus could be utilized to enhance the outcome of extinction-based treatments for alcohol-use disorders.

Rats were shocked in a context and then exposed to that context in the absence of shock. Shorter intervals between these extinction trials produced more long-term freezing than did longer ones, and shorter intervals between the final extinction trial and test produced more freezing than did longer ones. A short interval between a context extinction trial and test with an extinguished conditioned stimulus (CS) produced more freezing than did a longer one, and a short interval between a nonreinforced context exposure and an extinguished CS reinstated freezing when the CS was tested 24 hr later. The results suggest that recent fear acts to favor subsequent retrieval of the memory formed at conditioning rather than extinction and to render the retrieved memory more salient.

Ventral tegmental area (VTA) dopamine neurons are crucial for appetitive responses to Pavlovian cues, including cue-induced reinstatement of drug seeking. However, it is unknown which VTA inputs help activate these neurons, transducing stimuli into salient cues that drive drug-seeking behavior. Here we examined 56 VTA afferents from forebrain and midbrain that are Fos activated during cue-induced reinstatement. We injected the retrograde tracer cholera toxin β subunit (CTb) unilaterally into rostral or caudal VTA of male rats. All animals were trained to self-administer cocaine, then extinguished of this behavior. On a final test day, animals were exposed to response-contingent cocaine-associated cues, extinction conditions, a non-cocaine-predictive CS-, or a novel environment, and brains were processed to visualize CTb and Fos immunoreactivity to identify VTA afferents activated in relation to behaviors. VTA-projecting neurons in subregions of medial accumbens shell, ventral pallidum, elements of extended amygdala, and lateral septum (but not prefrontal cortex) were activated specifically during cue-induced cocaine seeking, and some of these were also activated proportionately to the degree of cocaine seeking. Surprisingly, though efferents from the lateral hypothalamic orexin field were also Fos activated during reinstatement, these were largely non-orexinergic. Also, VTA afferents from the rostromedial tegmental nucleus and lateral habenula were specifically activated during extinction and CS- tests, when cocaine was not expected. These findings point to a select set of subcortical nuclei which provide reinstatement-related inputs to VTA, translating conditioned stimuli into cocaine-seeking behavior.

Psychological stress evokes increases in sympathetic activity and blood pressure, which are due at least in part to an upward resetting of the baroreceptor-sympathetic reflex. In this study we determined whether sympathetic premotor neurons in the rostral ventrolateral medulla (RVLM), which have a critical role in the reflex control of sympathetic activity, are activated during air puff stress, a moderate psychological stressor. Secondly, we identified neurons that are activated by air puff stress and that also project to the nucleus tractus solitarius (NTS), a key site for modulation of the baroreceptor reflex. Air puff stress resulted in increased c-Fos expression in several hypothalamic and brainstem nuclei, including the paraventricular nucleus (PVN), dorsomedial hypothalamus, perifornical area (PeF), periaqueductal gray (PAG), NTS and rostral ventromedial medulla, but not in the RVLM region that contains sympathetic premotor neurons. In contrast, neurons in this RVLM region, including catecholamine-synthesizing neurons, did express c-Fos following induced hypotension, which reflexly activates RVLM sympathetic premotor neurons. The highest proportion of NTS-projecting neurons that were double-labelled with c-Fos after air puff stress was in the ventrolateral PAG (29.3 ± 5.5%), with smaller but still significant proportions of double-labelled NTS-projecting neurons in the PVN and PeF (6.5 ± 1.8 and 6.4 ± 1.7%, respectively). The results suggest that the increased sympathetic activity during psychological stress is not driven primarily by RVLM sympathetic premotor neurons, and that neurons in the PVN, PeF and ventrolateral PAG may contribute to the resetting of the baroreceptor-sympathetic reflex that is associated with psychological stress.

The midbrain periaqueductal grey (PAG) contains four longitudinal columns, referred to as the dorsomedial (dmPAG), dorsolateral (dlPAG), lateral (lPAG) and ventrolateral (vlPAG) subdivisions, which collectively have a pivotal role in integrating behavioural and physiological responses to external stressors as well as other functions. This review is focussed on the dlPAG, which is believed to be an important component of the central mechanisms that generate the defensive response to acute psychological stressors, such as the presence of a predator or other immediate threat. The anatomical connections of the dlPAG are highly specific and distinctly different from those of the other PAG subregions. The chemical properties of the dlPAG are also distinctly different from the other PAG subregions (e.g. there is a very high density of neurons that synthesize nitric oxide in the dlPAG but very few such neurons in the other PAG subregions). Recent functional studies have demonstrated that neurons in the dlPAG exert a powerful control over both sympathetic and respiratory activity, and that the pattern of the evoked respiratory changes is also distinctly different from those evoked from other PAG subregions. These studies also showed that the sympathetic and respiratory changes evoked from the dlPAG are highly correlated, suggesting the possibility that a common population of "command neurons" within this region may generate the sympathetic and respiratory changes that accompany defensive behavioural responses to acute psychological stressors. Finally, although the anatomical connections and functional properties of the dlPAG are distinctly different from the other PAG subregions, they have many similarities with adjacent parts of the superior colliculus, suggesting that the dlPAG and deep layers of the superior colliculus may be part of a common defence system in the midbrain.

Pavlovian fear conditioning depends on prediction error, or the discrepancy between actual and expected outcomes. We used immunohistochemistry, neuronal tract tracing, and reversible inactivation to study the role of prefrontal cortex and thalamocortical pathways in predictive fear learning. Unexpected, but not expected, conditioned stimulus (CS)-unconditioned stimulus (US) presentations caused increased c-Fos expression in the prefrontal cortex (PFC), midline thalamus, lateral amygdala, as well as retrograde labeled midline thalamic afferents to PFC. Reversible inactivation of dorsomedial PFC, but not infralimbic PFC, prevented the associative blocking of fear learning. These results suggest a role for dorsomedial PFC (dmPFC), and a thalamic → dmPFC pathway, in signaling whether or not aversive events are expected or unexpected and so whether they are to be learned about.

The nucleus accumbens shell (AcbSh) is required to inhibit drug seeking after extinction training. Conversely, the lateral hypothalamus (LH), which receives projections from AcbSh, mediates reinstatement of previously extinguished drug seeking. We hypothesized that reversible inactivation of AcbSh using GABA agonists (baclofen/muscimol) would reinstate extinguished alcohol seeking and increase neuronal activation in LH. Rats underwent self-administration training for 4% (v/v) alcoholic beer followed by extinction. AcbSh inactivation reinstated extinguished alcohol seeking when infusions were made after, but not before, extinction training. We then used immunohistochemical detection of c-Fos as a marker of neuronal activity, combined with immunohistochemical detection of the orexin and cocaine- and amphetamine-related transcript (CART) peptides, to study the profile and phenotype of neural activation during reinstatement produced by AcbSh inactivation. AcbSh inactivation increased c-Fos expression in hypothalamus, as well as in paraventricular thalamus and amygdala. Within hypothalamus, there was an increase in the number of orexin and CART cells expressing c-Fos. Finally, we hypothesized that concurrent inactivation of LH would prevent reinstatement produced by inactivation of AcbSh alone. Our results confirmed this. Together, these findings suggest that AcbSh mediates extinction of reward seeking by inhibiting hypothalamic neuropeptide neurons. Reversible inactivation of the AcbSh removes this influence, thereby releasing hypothalamus from AcbSh inhibition and enabling reinstatement of reward seeking. These ventral striatal–hypothalamic circuits for extinction overlap with those that mediate satiety, and we suggest that extinction training inhibits drug seeking because it co-opts neural circuits originally selected to produce satiety.

Hypocretin/orexin has a well-established role in wakefulness and in the maintenance of arousal. Because stress is associated with arousal, it has been proposed that hypocretin is also involved in stress. However, it is not clear if this is true for all forms of stress. To clarify this issue, we compared four conditions combining high arousal with no or low stress (wakefulness and exploration) or high stress (contextual fear and restraint) in the rat. We looked at Fos expression in hypocretin neurons, hypocretin-1 levels in cerebrospinal fluid and cardiovascular and behavioural changes after pharmacological blockade with the dual hypocretin receptor antagonist, almorexant. Fos expression in hypocretin neurons was highest with wakefulness and exploration, also high with fear but not significant with restraint. Hypocretin-1 levels were consistent with this pattern, although the differences were not as marked. Hypocretin receptor blockade with almorexant reduced the pressor, tachycardic and locomotor responses of wakefulness and exploration as well as the pressor and sympathetic component of the tachycardic response of fear. In contrast, almorexant did not reduce the pressor and tachycardic responses of restraint and nor did it reduce the pressor, tachycardic and locomotor responses of another stressor, i.e. cold exposure. Thus, hypocretin is not involved in all forms of stress. Comparison of the different conditions suggests that, regardless of stress, hypocretin involvement occurs when the arousal associated with the response includes increased attention to environmental cues. When it does, hypocretin will at least contribute to the cardiovascular response. The findings are of clinical relevance to some forms of psychological stress.