Heidi M. B. Lesscher

Differential and age-dependent effects of maternal deprivation on the hypothalamic-pituitary-adrenal axis of brown norway rats from youth to senescence.

In this study, the hypothesis was tested that infants deprived from maternal care show persistent changes in hypothalamic‐pituitary‐adrenal activity. For this purpose, we studied the effect of maternal deprivation in one cohort of the healthy ageing Brown Norway rat strain showing still more than 80% survival rate at 32 months of age. Three‐day‐old male Brown Norway rats were either maternally deprived for 24 h or remained with the dam. In 3, 12 and 30–32 months (young, adult, senescent) deprived rats and their nondeprived littermates (controls), we determined basal resting and stress‐induced plasma adrenocorticotropic hormone (ACTH) and corticosterone as well as corticotropin releasing hormone (CRH) mRNA expression in the paraventricular nucleus (PVN) of the hypothalamus. Mineralocorticoid (MR) and glucocorticoid receptors (GR) in hippocampus and PVN were also assessed using in vitro cytosol binding and in situ hybridization. The effect of ageing per se showed that in the control nondeprived Brown Norway rats, basal corticosterone and ACTH concentrations did not change during life. However, with age, the corticosterone response to novelty stress became progressively attenuated, but prolonged, while there was an age‐related increase in the ACTH response. CRH mRNA expression in PVN decreased with age. Hippocampal MR binding and MR mRNA expression in the dentate gyrus were reduced at senescence, as were the GR binding capacities in hippocampus and hypothalamus. Maternal deprivation did not affect survival rate, body weight, nor adrenal weight of the ageing Brown Norway rats. Basal corticosterone and ACTH levels were not affected by deprivation, except for a rise in basal corticosterone concentrations at 3 months. At this age, the corticosterone output in response to novelty was attenuated in the deprived rats. In contrast, a striking surge in novelty stress‐induced corticosterone output occurred at midlife while, at senescence, the corticosterone and ACTH responses were attenuated again in the deprived animals, particularly after the more severe restraint stressor. CRH mRNA expression was reduced only during adulthood in the deprived animals. After maternal deprivation, the MR mRNA in dentate gyrus showed a transient midlife rise. GR binding in hypothalamus and hippocampus GR binding was reduced in young rats while, in the senescent deprived animals, a reduced GRmRNA expression was observed in PVN and hippocampal CA1. In conclusion, in the Brown Norway rat, ageing causes a progressive decline in corticosterone output after stress, which is paralleled at senescence by decreased MR and GR mRNA expression in hippocampus and hypothalamus. The long‐term effects of maternal deprivation become manifest differently at different ages and depend on test conditions. The deprivation effect culminates in a midlife corticosterone surge and results at senescence in a strongly reduced corticosterone output.

Endogenous cannabinoids are not involved in cocaine reinforcement and development of cocaine-induced behavioural sensitization

The endogenous cannabinoid system is a relatively novel discovered system consisting of cannabinoid CB1 receptors, which are expressed both in the periphery and in the central nervous system, peripheral cannabinoid CB2 receptors and endogenous cannabinoids, which are anandamide and 2-arachidonyl glycerol. The cannabinoid CB1 receptors have recently been implicated in rewarding aspects of not only the cannabinoid drug Delta9-tetrahydrocannabinol (Delta9-THC), but also of other drugs of abuse, including cocaine. The present study was designed to further investigate the role of CB1 receptors in reward-related effects of cocaine. Using the CB1 receptor selective antagonist SR141716A, the involvement of CB1 receptors in cocaine reinforcement was determined by intravenous cocaine self-administration. In addition, the effects of the CB1 receptor selective antagonist SR141716A upon the development of cocaine-induced behavioural sensitization were investigated. SR141716A did not affect cocaine reinforcement nor did it affect the development of behavioural sensitization to the locomotor stimulant effects of cocaine. These findings suggest that CB1 receptors are not involved in acute cocaine reinforcement nor in cocaine-induced behavioural sensitization.

Protein Kinase Cδ Regulates Ethanol Intoxication and Enhancement of GABA-Stimulated Tonic Current

Ethanol alters the distribution and abundance of PKCδ in neural cell lines. Here we investigated whether PKCδ also regulates behavioral responses to ethanol. PKCδ−/− mice showed reduced intoxication when administered ethanol and reduced ataxia when administered the nonselective GABAA receptor agonists pentobarbital and pregnanolone. However, their response to flunitrazepam was not altered, suggesting that PKCδ regulates benzodiazepine-insensitive GABAA receptors, most of which contain δ subunits and mediate tonic inhibitory currents in neurons. Indeed, the distribution of PKCδ overlapped with GABAA δ subunits in thalamus and hippocampus, and ethanol failed to enhance tonic GABA currents in PKCδ−/− thalamic and hippocampal neurons. Moreover, using an ATP analog-sensitive PKCδ mutant in mouse L(tk−) fibroblasts that express α4β3δ GABAA receptors, we found that ethanol enhancement of GABA currents was PKCδ-dependent. Thus, PKCδ enhances ethanol intoxication partly through regulation of GABAA receptors that contain δ subunits and mediate tonic inhibitory currents. These findings indicate that PKCδ contributes to a high level of behavioral response to ethanol, which is negatively associated with risk of developing an alcohol use disorder in humans.

Mu-opioid receptor knockout mice show diminished food-anticipatory activity

We have previously suggested that during or prior to activation of anticipatory behaviour to a coming reward, µ‐opioid receptors are activated. To test this hypothesis schedule induced food‐anticipatory activity in µ‐opioid receptor knockout mice was measured using running wheels. We hypothesized that µ‐knockout mice show little food‐anticipatory activity. In wildtype mice we observed that food‐anticipatory activity increased proportional to reduced food intake levels during daily scheduled food access, and thus reflects the animals physiological need for food. µ‐Knockout mice do not adjust their schedule induced running wheel behaviour prior to and during feeding time in the same way as wildtype mice; rather than showing more running wheel activity before than during feeding, they showed an equal amount of activity before and during feeding. As food‐anticipatory activity is dependent on the mesolimbic dopamine system and µ‐opioid receptors regulate dopaminergic activity, these data suggest a change in the dopamine systems activity in µ‐knockout mice. As we observed that µ‐knockout mice tended to show a stronger locomotor activity response than wildtype mice to the indirect dopamine agonist d‐amphetamine, it appears that the dopaminergic system per se is intact and sensitive to activation. We found no differences in the expression of pro‐opiomelanocortin, a precursor of endogenous endorphin, in the arcuate nucleus between µ‐knockout mice and wildtype mice during restricted feeding, showing that the µ‐opioid receptor does not regulate endogenous endorphin levels. These data overall suggest a role for µ‐opioid receptors in adapting reward related behaviour to the requirements of the environment.

Increased GABAergic input to ventral tegmental area dopaminergic neurons associated with decreased cocaine reinforcement in μ-opioid receptor knockout mice

There is general agreement that dopaminergic neurons projecting from the ventral tegmental area (VTA) to the nucleus accumbens and prefrontal cortex play a key role in drug reinforcement. The activity of these neurons is strongly modulated by the inhibitory and excitatory input they receive. Activation of mu-opioid receptors, located on GABAergic neurons in the VTA, causes hyperpolarization of these GABAergic neurons, thereby causing a disinhibition of VTA dopaminergic neurons. This effect of mu-opioid receptors upon GABA neurotransmission is a likely mechanism for mu-opioid receptor modulation of drug reinforcement. We studied mu-opioid receptor signaling in relation to cocaine reinforcement in wild-type and mu-opioid receptor knockout mice using a cocaine self-administration paradigm and in vitro electrophysiology. Cocaine self-administration was reduced in mu-opioid receptor knockout mice, suggesting a critical role of mu-opioid receptors in cocaine reinforcement. The frequency of spontaneous inhibitory post-synaptic currents onto dopaminergic neurons in the ventral tegmental area was increased in mu-opioid receptor knockout mice compared with wild-type controls, while the frequency of spontaneous excitatory post-synaptic currents was unaltered. The reduced cocaine self-administration and increased GABAergic input to VTA dopaminergic neurons in mu-opioid receptor knockout mice supports the notion that suppression of GABAergic input onto dopaminergic neurons in the VTA contributes to mu-opioid receptor modulation of cocaine reinforcement.

Inflexible and Indifferent Alcohol Drinking in Male Mice

BACKGROUND Alcoholism is characterized by compulsive alcohol intake, but this critical feature of alcoholism is seldom captured in preclinical studies. Here, we evaluated whether alcohol-preferring C57BL/6J mice develop compulsive alcohol drinking patterns, using adulteration of the alcohol solution with quinine, in a limited access choice paradigm. We assessed 2 independent aspects of compulsive drinking: (i) inflexible alcohol intake by testing whether mice would drink bitter alcohol solutions if this was their only source of alcohol and (ii) indifferent drinking by comparing intake of aversive and nonaversive alcohol solutions. METHODS Male C57BL/6J mice consumed alcohol for 2 or 8 consecutive weeks. The alcohol solution was then adulterated with graded quinine concentrations, and the effect on alcohol intake was determined. RESULTS C57BL/6J mice rapidly developed compulsive alcohol drinking patterns. Adulteration of the alcohol solution with an aversive quinine concentration failed to reduce intake, indicative of inflexible drinking behavior, after only 2 weeks of alcohol experience, although quinine adulteration did suppress the acquisition of alcohol drinking in naïve mice. After 8 weeks of alcohol consumption, the mice also became indifferent to quinine. They consumed an aversive, quinine-containing alcohol solution, despite the simultaneous availability of an unadulterated alcohol solution. Prolonged alcohol ingestion did not alter the sensitivity to the bitter taste of quinine itself. CONCLUSION These findings demonstrate the staged occurrence in mice of 2 distinct behavioral characteristics of alcoholism, i.e., inflexible and indifferent alcohol drinking.

Amygdala protein kinase C epsilon controls alcohol consumption

Alcoholism is a progressive disorder that involves the amygdala. Mice lacking protein kinase C epsilon (PKCɛ) show reduced ethanol consumption, sensitivity and reward. We therefore investigated whether PKCɛ signaling in the amygdala is involved in ethanol consumption. Local knockdown of PKCɛ in the amygdala reduced ethanol consumption and preference in a limited‐access paradigm. Further, mice that are heterozygous for the PKCɛ allele consume less ethanol compared with wild‐type mice in this paradigm. These mice have a >50% reduction in the abundance of PKCɛ in the amygdala compared with wild‐type mice. We conclude that amygdala PKCɛ is important for ethanol consumption in mice.

Rodent models for compulsive alcohol intake

Continued seeking and drinking of alcohol despite adverse legal, health, economic, and societal consequences is a central hallmark of human alcohol use disorders. This compulsive drive for alcohol, defined by resistance to adverse and deleterious consequences, represents a major challenge when attempting to treat alcoholism clinically. Thus, there has long been interest in developing pre-clinical rodent models for the compulsive drug use that characterizes drug addiction. Here, we review recent studies that have attempted to model compulsive aspects of alcohol and cocaine intake in rodents, and consider technical and conceptual issues that need to be addressed when trying to recapitulate compulsive aspects of human addiction. Aversion-resistant alcohol intake has been examined by pairing intake or seeking with the bitter tastant quinine or with footshock, and exciting recent work has used these models to identify neuroadaptations in the amygdala, cortex, and striatal regions that promote compulsive intake. Thus, rodent models do seem to reflect important aspects of compulsive drives that sustain human addiction, and will likely provide critical insights into the molecular and circuit underpinnings of aversion-resistant intake as well as novel therapeutic interventions for compulsive aspects of addiction.

Compulsive drug use and its neural substrates

Abstract Drug addiction is a chronic relapsing brain disease, characterized by compulsive drug use. Despite the fact that drug addiction affects millions of people worldwide, treatments for this disorder are limited in number and efficacy. In our opinion, understanding the neural underpinnings of drug addiction would open new avenues for the development of innovative treatments for this disorder. Based on an awareness that drug use and drug reward do not equal drug addiction, there has been increasing interest in developing animal models of addiction that mimick the loss of control over drug use more closely than existing models aimed at studying drug reward. The present review provides an overview of animal studies of compulsive drug use and the neural mechanisms involved. First, the employed models are summarized, with a particular emphasis on models of escalation of drug use and resistance to punishment. Next, we discuss mechanisms within the (ventral and dorsal) striatum and (central) amygdala that have recently been implicated in the compulsive seeking and taking of alcohol and cocaine. The studies discussed here provide a promising line of research that will advance our knowledge of the neural circuits involved in the self-destructive behavior that characterizes drug addiction.

Amygdala protein kinase C epsilon regulates corticotropin-releasing factor and anxiety-like behavior.

Corticotropin‐releasing factor (CRF), its receptors, and signaling pathways that regulate CRF expression and responses are areas of intense investigation for new drugs to treat affective disorders. Here, we report that protein kinase C epsilon (PKCɛ) null mutant mice, which show reduced anxiety‐like behavior, have reduced levels of CRF messenger RNA and peptide in the amygdala. In primary amygdala neurons, a selective PKCɛ activator, ψɛRACK, increased levels of pro‐CRF, whereas reducing PKCɛ levels through RNA interference blocked phorbol ester‐stimulated increases in CRF. Local knockdown of amygdala PKCɛ by RNA interference reduced anxiety‐like behavior in wild‐type mice. Furthermore, local infusion of CRF into the amygdala of PKCɛ−/− mice increased their anxiety‐like behavior. These results are consistent with a novel mechanism of PKCɛ control over anxiety‐like behavior through regulation of CRF in the amygdala.