Targeted mutations of the corticotropin-releasing factor system: effects on physiology and behavior.

Genetic modifications of the genes that encode proteins integral to the corticotropin-releasing factor (CRF) system have been employed in the creation of mutant mice that serve as tools for studying the role of this neuropeptide in regulated and dysregulated behaviors and physiology. Overexpression of the CRF peptide and CRF binding protein as well as deletion of the peptide, binding protein, and both known receptors has been achieved and these mouse models have been characterized for anatomical, neuroendocrine, and behavioral sequelae. The profile of results, consistent with current knowledge of CRF function from more traditional assays, indicates that enhancement of CRF function is associated with an activation of the hypothalamic-pituitary-adrenal axis, an anxious phenotype, alterations in cognitive performance and reductions in feeding. In general, blockade of CRF function produces the opposite effects. Genetic mouse models allow further analysis of specific elements in the CRF circuitry for which more traditional tools have not existed. These animal models are valuable for increasing our understanding of the underlying pathology associated with a variety of psychiatric and neuroendocrine disorders and for the development and testing of novel treatment agents.

Increased ethanol self-administration in delta-opioid receptor knockout mice.

BACKGROUND: The role of the delta-opioid receptor in ethanol drinking has remained unclear despite the use of traditional pharmacological and correlational approaches. The results of several studies suggest that pharmacological blockade of these receptors results in decreases in ethanol drinking behavior, but an approximately equal number of reports have failed to observe an effect of delta-receptor antagonism on ethanol drinking. It is clear that alternative approaches to understanding opioid-receptor involvement in ethanol drinking are needed. METHODS: In this study, ethanol drinking was examined in delta-opioid receptor knockout (KO) mice by using first a two-bottle-choice test, then an operant self-administration paradigm and a second two-bottle-choice test, in that order. In addition, because KO mice were previously shown to display enhanced anxiety-like behavior relative to wild-type (WT) mice, the effect of ethanol self-administration on anxiety-like responses was determined. RESULTS: delta KO mice initially showed no evidence of a preference for ethanol in the first two-bottle-choice drinking test; however, after an experience of operant self-administration of ethanol, a preference for ethanol developed in the second two-bottle-choice test. KO mice also showed a preference for ethanol over water and self-administered more ethanol than WT mice in the operant self-administration paradigm. The ethanol self-administered in this procedure was sufficient to reverse the innate anxiety-like response observed in this strain. CONCLUSIONS: delta KO mice showed a greater preference for ethanol and consumed more ethanol than their WT counterparts, suggesting that a decrease in delta-receptor activity is associated with increased ethanol-drinking behavior. It is hypothesized that delta receptors may influence ethanol self-administration at least partly through an effect of these receptors on anxiety-like behavior.

Intravenous self-administration of heroin/cocaine combinations (speedball) using nose-poke or lever-press operant responding in mice.

Acquisition and dose-related self-administration of heroin (H)/cocaine (C) combinations in C57BL/6 x SJL mice were studied in nose-poke or lever-press operant responding procedures. C57BL/6 x SJL mice readily acquired self-administration in both operant procedures with a combination of doses known to be ineffective when each drug was used alone (H: 15 microg/kg and C: 150 microg/kg per injection). Similar numbers of infusions were obtained under conditions of fixed-ratio (FR) 3 versus 1 for the nose-poke and lever-press responses, respectively. Dose-effect curves for heroin:cocaine combinations revealed a pattern corresponding to a leftward shift of the dose-response for intravenous cocaine self-administration. Curves were similar whether generated with 1 or 3 days of availability per dose, or including subjects that did not respond preferentially (> 70% responses) to the hole or lever associated with drug delivery, along with those that did. Motor activity induced by a combination of low doses for each drug was examined (H: 0.375 mg/kg and C: 3.75 mg/kg, i.p.). Under these conditions, the combination of both drugs induced an initial cocaine-like stimulation of horizontal activity, in contrast to the tendency of heroin to decrease activity. It is concluded that heroin:cocaine combinations used in the present study had reinforcing effects in C57BL/6 x SJL mice, and produced a cocaine-like effect in the early part of drug-induced activity sessions, followed by a locomotor profile corresponding to the average of both drugs.

Functional consequences of repeated (+/-)3,4-methylenedioxymethamphetamine (MDMA) treatment in rhesus monkeys.

Six rhesus monkeys were trained to stable performance on neuropsychological tests of memory, reinforcer efficacy, reaction time and bimanual motor coordination. Three monkeys were then exposed to a high-dose, short course regimen of (+/-)3,4-Methylenedioxymethamphetamine (MDMA, "Ecstasy") (4 days, 10 mg/kg i.m., b.i.d.). Following treatment, concentrations of 5-hydroxyindoleacetic acid (5-HIAA) in cerebrospinal fluid (CSF) were reduced by approximately 50% in the treated animals, and this effect persisted for approximately three months post-MDMA. Behavioral performance was disrupted during acute MDMA treatment but returned to baseline within one week following treatment. MDMA also produced persistent alterations in late peak latencies of brainstem auditory evoked potentials (BSAEP), lasting three months post-MDMA. Both CSF 5-HIAA concentrations and evoked potential latencies were normalized four months after treatment. These findings indicate that serotonergic alterations associated with MDMA use may result in persisting changes in brain function.

Anxiogenic-like effects limit rewarding effects of cocaine in balb/cbyj mice.

Previous studies have reported intravenous cocaine self-administration behavior in several strains of mice with the exception of BALB/cByJ, a strain considered a mouse model of high emotional reactivity. The present experiments further investigated acquisition of self-administration in BALB/cByJ mice using a low dose and a habituation session. Following evidence of an initial drug-seeking behavior, we observed a progressive decline of intravenous self-administration. Pretreatment with diazepam (0.5 mg/kg, IP), reinstated cocaine-maintained responding. To test the hypothesis that injections directly into a reward-relevant brain region might support consistent cocaine-seeking behavior, BALB/cByJ mice implanted in the nucleus accumbens (NAc) or the caudate-putamen nucleus (CPu) were trained to discriminate between the arm enabling a microinjection of cocaine (30 pmol/50 nl or 150 pmol/50 nl) and the neutral arm of a Y-maze. Only NAc subjects exhibited a spatial discrimination toward the cocaine-reinforced arm and the D2 antagonist, sulpiride (50 mg/kg, IP) eliminated intra-NAc cocaine self-administration. However, after several days of cocaine self-injection, animals developed an approach/avoidance-like behavior between the start box and the reinforced arm. This behavior was suppressed by systemic diazepam (1 mg/kg, IP) pretreatment. We conclude that: (1) medio-ventral NAc is involved both in the rewarding (via a D2 dopaminergic mechanism) and aversive effects of cocaine in mice; and (2) anxiolytic pretreatment (diazepam) indirectly enhanced the reinforcing properties of cocaine in BALB/cByJ, suggesting that emotionality can act as a protective mechanism against stimulant abuse.

DARPP-32 knockout mice exhibit impaired reversal learning in a discriminated operant task.

The current study was conducted to examine the performance of mice with a targeted deletion of the gene for DARPP-32 in a discriminated operant task using food reinforcement. DARPP-32 plays a central role in regulating the efficacy of dopaminergic neurotransmission. Initially, wild-type and DARPP-32 knockout mice were trained to nose-poke for food on a continuous reinforcement schedule. The minimum response requirement was increased every 5 days until the animals were responding on an FR-15 schedule of reinforcement. At the completion of extensive operant training, reversal learning was assessed. Wild-type and DARPP-32 knockout mice exhibited equivalent performance during acquisition of this task, with both groups increasing operant responding as the schedule of reinforcement was raised. However, significant differences in discrimination learning were observed during the reversal phase, with DARPP-32 knockout mice requiring significantly more trials to reach criterion than wild-type controls. These results provide evidence for a functional role of DARPP-32 in the mediation of processes underlying learning and memory.

mu-Opioid receptor knockout mice do not self-administer alcohol.

Opioid peptides long have been hypothesized to play a role in ethanol reinforcement. Neuropharmacological studies have shown that opioid receptor antagonists decrease ethanol self-administration in rodents and prevent relapse in humans. However, the exact mechanism for such powerful effects has remained elusive. The availability of mu-opioid receptor knockout mice has made possible the direct examination of the role of the mu-opioid receptor in mediating ethanol self-administration. In the present experiments, both nosepoke and lever operant ethanol self-administration and several tests of two bottle-choice ethanol drinking were studied in these genetically engineered mice. In no case did knockout mice show evidence of ethanol self-administration, and, in fact, these mice showed evidence of an aversion to ethanol under several experimental conditions. These data provide new evidence for a critical role for mu-opioid receptors in ethanol self-administration assessed with a variety of behavioral paradigms and new insights into the neuropharmacological basis for ethanol reinforcement.

Dissociation of locomotor activation and suppression of food intake induced by CRF in CRFR1-deficient mice.

Corticotropin-releasing factor (CRF) systems are involved in locomotor and feeding behaviors. Two distinct CRF receptor subtypes, CRFR1 and CRFR2, are thought to mediate CRF actions in the central nervous system. However, the role for each receptor in locomotor activity and feeding remains to be determined. Using CRFR1 null mutant mice, the present study examined the functional significance of this receptor in ambulation and feeding. CRF treatment of wild-type mice resulted in increased levels of locomotion whereas no change was observed in CRFR1-deficient mice as compared to vehicle-treated mutant mice. In contrast, CRF decreased food-water intake in both wild type and CRFR1-deficient mice equally. These results support an important role for CRFR1 in mediating CRF-induced locomotor activation, whereas other receptor subtypes, likely CRFR2, may mediate the appetite-suppressing effects of CRF-like peptides.

Mice deficient for corticotropin-releasing hormone receptor-2 display anxiety-like behaviour and are hypersensitive to stress.

Corticotropin-releasing hormone (Crh) is a critical coordinator of the hypothalamic-pituitary-adrenal (HPA) axis. In response to stress, Crh released from the paraventricular nucleus (PVN) of the hypothalamus activates Crh receptors on anterior pituitary corticotropes, resulting in release of adrenocorticotropic hormone (Acth) into the bloodstream. Acth in turn activates Acth receptors in the adrenal cortex to increase synthesis and release of glucocorticoids. The receptors for Crh, Crhr1 and Crhr2, are found throughout the central nervous system and periphery. Crh has a higher affinity for Crhr1 than for Crhr2, and urocortin (Ucn), a Crh-related peptide, is thought to be the endogenous ligand for Crhr2 because it binds with almost 40-fold higher affinity than does Crh. Crhr1 and Crhr2 share approximately 71% amino acid sequence similarity and are distinct in their localization within the brain and peripheral tissues. We generated mice deficient for Crhr2 to determine the physiological role of this receptor. Crhr2-mutant mice are hypersensitive to stress and display increased anxiety-like behaviour. Mutant mice have normal basal feeding and weight gain, but decreased food intake following food deprivation. Intravenous Ucn produces no effect on mean arterial pressure in the mutant mice.

Genetic differences in response to novelty and spatial memory using a two-trial recognition task in mice.

A two-trial memory task, based on a free-choice exploration paradigm in a Y-maze, was previously developed to study recognition processes in Sprague-Dawley rats. Because this paradigm avoids the use of electric shock or deprivation that may have nonspecific effects and does not require learning of a rule, it may be particularly useful for studying memory in mice. Four inbred strains (Balb/cByJ, DBA/2J, C57BL/6J, and SJL/J), an F1 hybrid (C57BL/6 x SJL/J), and one outbred strain (CD1) were used to validate this task in mice and to characterize a strain distribution in response to novelty and working memory. Exploration was measured with a short (2 min) intertrial interval (ITI) between acquisition and retrieval, while memory was examined with longer intervals (30 min, 1 h, and 2 h). A study of the time course of the response to novelty revealed varying degrees of preference and/or habituation to novelty among the different strains, with CD1 exhibiting a very high response to novelty and others showing lower (C57 x SJL hybrids) to complete absence (SJL) of exploration of novelty. Memory span, assessed with increasing ITIs, varied widely among strains from 30 min (C57 x SJL hybrids) to at least 2 h (C57 and BALB). Such demonstrated sensitivity to a wide range of behavioral phenotypes supports the use of this spatial memory task as an effective tool for the study of genetic influences on the response to novelty and recognition processes in mice.

Performance norms for a rhesus monkey neuropsychological testing battery: acquisition and long-term performance.

A computerized behavioral battery based upon human neuropsychological tests (CANTAB, CeNeS, Cambridge, UK) has been developed to assess cognitive behaviors of rhesus monkeys. Monkeys reliably performed multiple tasks, providing long-term assessment of changes in a number of behaviors for a given animal. The overall goal of the test battery is to characterize changes in cognitive behaviors following central nervous system (CNS) manipulations. The battery addresses memory (delayed non-matching to sample, DNMS; spatial working memory, using a self-ordered spatial search task, SOSS), attention (intra-/extra-dimensional shift, ID/ED), motivation (progressive-ratio, PR), reaction time (RT) and motor coordination (bimanual task). As with human neuropsychological batteries, different tasks are thought to involve different neural substrates, and therefore performance profiles should assess function in particular brain regions. Monkeys were tested in transport cages, and responding on a touch sensitive computer monitor was maintained by food reinforcement. Parametric manipulations of several tasks demonstrated the sensitivity of performance to increases in task difficulty. Furthermore, the factors influencing difficulty for rhesus monkeys were the same as those shown to affect human performance. Data from this study represent performance of a population of healthy normal monkeys that will be used for comparison in subsequent studies of performance following CNS manipulations such as infection with simian immunodeficiency virus (NeuroAIDS) or drug administration.

Scopolamine alters rhesus monkey performance on a novel neuropsychological test battery.

Rhesus monkeys (6) were trained on a test battery including cognitive tests adapted from a human neuropsychological assessment battery (CANTAB; CeNeS, Cambridge, UK) as well as a bimanual motor skill task. The complete battery included tests of memory (delayed non-match to sample, DNMS; self-ordered spatial search, SOSS), reaction time (RT), motivation (progressive ratio; PR) and fine motor coordination (bimanual). The animals were trained to asymptotic performance in all tasks and then were administered two of the four CANTAB tasks on alternate weekdays (PR/SWM; DNMS/RT) with the bimanual task being administered on each weekday. The effect of acute administration of scopolamine (3-24 microg/kg, i.m.) on performance was then determined. Although performance on DNMS was impaired there was no interaction of drug treatment with retention interval, suggesting that scopolamine does not increase the rate of forgetting in this task. Scopolamine administration produced a decrement in SOSS performance that was dependent on task difficulty as well as dose. Scopolamine also impaired motor responses, resulting in increased time required to complete the bimanual motor task and increased movement time in the RT task. Performance in the PR task was decreased in a dose-dependent fashion by scopolamine. The results suggest that scopolamine interferes with memory storage and motor responses but not memory retention/retrieval or vigilance. The findings demonstrate that the test battery is useful for distinguishing the effects of neuropharmacological manipulation on various aspects of cognitive performance in monkeys.

Reduced anxiety-like and cognitive performance in mice lacking the corticotropin-releasing factor receptor 1.

Corticotropin-releasing factor (CRF) has been hypothesized to be involved in the pathophysiology of anxiety, depression, cognitive and feeding disorders. Two distinct CRF receptor subtypes, CRFR1 and CRFR2, are thought to mediate CRF actions in the CNS. However, the role for each receptor subtype in animal models of neuropsychiatric disorders remains to be determined. Using CRFR1 deficient mice, the present study investigated the functional significance of this CRF receptor subtype in anxiety-like and memory processes. CRFR1 knockout mice displayed an increased exploratory behavior in both the Elevated Plus-maze (EPM) and the Black and White (B-W) test box models of anxiety, indicating an anxiolytic-like effect of the CRFR1 gene deletion. In contrast, during the retrieval trial of a two-trial spatial memory task wild type mice made more visits to and spent more time in the novel arm as opposed to the two familiar ones of a Y-maze apparatus. No increase in the level of exploration of the novel arm by the CRFR1 deficient mice was observed. This indicates that CRFR1 knockout mice are impaired in spatial recognition memory. These results demonstrate that genetic deletion of the CRFR1 receptor can lead to impairments in anxiety-like and cognitive behaviors, supporting a critical role for this receptor in anxiety and cognitive biological processes.

Structural and functional neuropathology in transgenic mice with CNS expression of IFN-alpha.

Cytokines belonging to the type I interferon (e.g. interferon-alpha) family are important in the host response to infection and may have complex and broad ranging actions in the central nervous system (CNS) that may be beneficial or harmful. To better understand the impact of the CNS expression of the type I interferons (IFN), transgenic mice were developed that produce IFN-alpha(1) chronically from astrocytes. In two independent transgenic lines with moderate and low levels of astrocyte IFN-alpha mRNA expression respectively, a spectrum of transgene dose- and age-dependent structural and functional neurological alterations are induced. Structural changes include neurodegeneration with loss of cholinergic neurons, gliosis, angiopathy with mononuclear cell cuffing, progressive calcification affecting basal ganglia and cerebellum and the up-regulation of a number of IFN-alpha-regulated genes. At a functional level, in vivo and in vitro electrophysiological studies revealed impaired neuronal function and disturbed synaptic plasticity with pronounced hippocampal hyperexcitability. Severe behavioral alterations were also evident in higher expressor GFAP-IFNalpha mice which developed fatal seizures around 13 weeks of age precluding their further behavioral assessment. Modest impairments in discrimination learning were measured in lower expressor GFAP-IFNalpha mice at various ages (7-42 weeks). The behavioral and electrophysiological findings suggest regional changes in hippocampal excitability which may be linked to abnormal calcium metabolism and loss of cholinergic neurons in the GIFN mice. Thus, these transgenic mice provide a novel animal model in which to further evaluate the mechanisms that underlie the diverse actions of type I interferons in the intact CNS and to link specific structural changes with functional impairments.

Strain distribution of mice in discriminated Y-maze avoidance learning: genetic and procedural differences.

The current study was conducted to characterize discriminated avoidance learning in mice by using a Y-maze task. In Experiment 1, the task parameters were manipulated, including the amount of time spent in the start arm, the amount of time to make the avoidance response, and the intertrial interval (ITI) using C57 x SJL F1 hybrid mice. Avoidance performance was significantly improved with longer times to avoid the shock and longer ITIs. In Experiment 2, mice from 4 inbred strains (BALB/cByJ, DBA/2J, C57BL/6J, and SJL/J), an F1 hybrid (C57 x SJL), and 1 outbred strain (CD1) were tested with various ITIs. Strain differences were observed in avoidance learning, with BALB, DBA, C57 x SJL and CD1 mice showing significantly better avoidance learning than C57 mice, which were better than SJL mice. These data demonstrate that Y-maze performance is significantly influenced by the genetic background of the mouse and the parameters of the task.

Understanding corticotropin releasing factor neurobiology: contributions from mutant mice.

Mice with transgenic expression or deletion of the CRF peptide, transgenic expression of the CRF-BP or deletion of specific CRF receptor subtypes exist and will be valuable for examining candidate mediators in animal model systems recapitulating a variety of normal function. In particular, results described in this review implicate CRF in acute emotional responses studied in animal models of anxiety and drug abstinence. CRF also appears to play a role in behavioral and physiological plasticity judging by alterations in HPA reactivity to stress, information processing and energy balance regulation in CRF mutant models. Accordingly, the creation of genetically engineered mice now permits the evaluation of contributory roles for several CRF-related gene products in the pathophysiology of a variety of complex behavioral disorders. For example, the postulated causal linkage between overactivation of CRF systems and the hyper-emotionality which characterizes human affective disorders can now be more thoroughly evaluated by examining the phenotype of CRF mutant mice in animal models of depression, dementia and substance abuse.

Treatment of depression during pregnancy.

Increasing numbers of patients are being treated for mood disorders. The majority of these patients, particularly with the diagnosis of major depression, are women of childbearing years. Concerns about fetal exposure to medication, both planned and unplanned, are becoming more pressing in the clinical practices of both psychiatrists and primary care physicians. There are relatively few study data available to guide clinicians in the use of psychotropic medications during pregnancy because of obvious problems in designing studies of the effects of medication on pregnant women, fetuses, and infants. Clinicians in all specialties receive little or no formal training in this area of psychopharmacology. This article gathers clinically relevant studies and practice information and provides suggestions regarding the approach to treatment of mood disorders during pregnancy, based on a risk assessment model.