Thomas Steckler

Impaired stress response and reduced anxiety in mice lacking a functional corticotropin-releasing hormone receptor 1

Corticotropin-releasing hormone (CRH) is a potent mediator of endocrine, autonomic, behavioural and immune responses to stress, and has been implicated in the stress-like and other aversive consequences of drug abuse, such as withdrawal from alcohol. Two CRH receptors, Crhr1 and Crhr2, have been identified in the mouse. Crhr1 is highly expressed in the anterior pituitary, neocortex, hippocampus, amygdala and cerebellum, and activation of this receptor stimulates adenylate cyclase. Here we show that in mice lacking Crhr1, the medulla of the adrenal gland is atrophied and stress-induced release of adrenocorticotropic hormone (ACTH) and corticosterone is reduced. The homozygous mutants exhibit increased exploratory activity and reduced anxiety-related behaviour under both basal conditions and following alcohol withdrawal. Our results demonstrate a key role of the Crhr1 receptor in mediating the stress response and anxiety-related behaviour.

Genome-wide, large-scale production of mutant mice by ENU mutagenesis

In the post-genome era, the mouse will have a major role as a model system for functional genome analysis. This requires a large number of mutants similar to the collections available from other model organisms such as Drosophila melanogaster and Caenorhabditis elegans. Here we report on a systematic, genome-wide, mutagenesis screen in mice. As part of the German Human Genome Project, we have undertaken a large-scale ENU-mutagenesis screen for dominant mutations and a limited screen for recessive mutations. In screening over 14,000 mice for a large number of clinically relevant parameters, we recovered 182 mouse mutants for a variety of phenotypes. In addition, 247 variant mouse mutants are currently in genetic confirmation testing and will result in additional new mutant lines. This mutagenesis screen, along with the screen described in the accompanying paper, leads to a significant increase in the number of mouse models available to the scientific community. Our mutant lines are freely accessible to non-commercial users (for information, see http://www.gsf.de/ieg/groups/enu-mouse.html).

Corticotropin-releasing hormone receptor subtypes and emotion

Preclinical data indicate that corticotropin-releasing hormone (CRH) has anxiogenic properties and a dysregulation in CRH systems has been suggested to play a role in a variety of stress-related psychiatric disorders, such as anxiety, depression, and eating disorders. Two CRH receptor subtypes have been identified, termed CRH1 receptor (CRH1) and CRH2 receptor (CRH2), with its splice variants CRH2 alpha and CRH2 beta. These receptor subtypes differ in their pharmacology and expression pattern in the brain. Mouse mutants in which the CRH1 receptor subtype has been deleted show an impaired stress response, reduced anxiety-related behavior, and cognitive deficits. Studies using antisense oligodeoxynucleotides directed against CRH1 or CRH2 alpha identified the CRH1 receptor as the main target for CRH in mediating anxiogenesis, although recent data also suggest a possible role for CRH2 alpha. More clearly, CRH2 alpha is involved in the CRH effects on food intake. Moreover, local injection of CRH into areas rich in CRH2 alpha also result in altered sexual female behavior. Therefore, it is suggested that the CRH2 alpha may primarily influence a system concerned with implicit processes necessary for survival, i.e., with motivational types of behavior including feeding, reproduction, and possibly defense, whereas the CRH1 may be more concerned with explicit processes, including attention, executive functions, the conscious experience of emotions, and possibly learning and memory related to these emotions. This also suggests that patients suffering from anxiety and depression may benefit from treatment with CRH1 antagonistic drugs, while drugs targeting CRH2 alpha may be of particular benefit for patients with eating disorders.

Transgenic mice overexpressing glycogen synthase kinase 3beta: a putative model of hyperactivity and mania.

Lithium is used as treatment for bipolar disorder with particular efficacy in the treatment of mania. Lithium inhibits glycogen synthase kinase 3β (GSK-3β) directly or indirectly via stimulation of the kinase Akt-1. We therefore investigated the possibility that transgenic mice overexpressing GSK-3β could be of relevance to model bipolar disorder. Transgenic mice showed hypophagia, an increased general locomotor activity, and decreased habituation as assessed in an open field, an increased acoustic startle response, and again decreased habituation. The forced swim test revealed a reduced immobility in transgenic mice, but this is probably related to the hyperactivity of the animals. There were no differences in baseline and stress-induced increases of plasma adrenocorticotrophic hormone and corticosterone levels. Molecular analysis suggests compensatory mechanisms in the striatum of these transgenic mice for the overload of active GSK-3β by dimming the endogenous GSK-3β signaling pathway via upregulation of Akt-1 expression. Brain-derived neurotrophic factor protein levels were increased in the hippocampus of the transgenic mice. This suggests some kind of compensatory mechanism to the observed reduction in brain weight, which has been related previously to a reduced size of the somatodendritic compartment. Together, in mice overexpressing GSK-3β, specific intracellular signaling pathways are affected, which is accompanied by altered plasticity processes and increased activity and reactivity, whereas habituation processes seem to be decreased. The behavioral observations led to the suggestion that the model at hand recapitulates hyperactivity as observed in the manic phase of bipolar disorder.

The role of serotonergic-cholinergic interactions in the mediation of cognitive behaviour.

Cholinergic systems have been linked to cognitive processes such as attention, learning and mnemonic function. However, other neurotransmitter systems, such as the serotonergic one, which may have only minor effects on cognitive function on their own, interact with cholinergic function and their combined effects may have marked behavioural actions. Some studies have dealt with serotonergic-cholinergic interactions, but it is unclear whether both systems affect cognition directly or whether interactions at a behavioural level result from additional alterations in non-cognitive factors. This distinction is difficult, since it is possible that the diverse cholinergic and serotonergic systems serve different roles in the mediation of cognitive processes, both at the neuroanatomical and neurochemical level. Nevertheless, it is possible that cholinergic systems primarily alter accuracy in cognitive tasks, whereas serotonergic neurotransmission modulates behaviour by altering bias (motivation, motor processes). Whether serotonin alters accuracy or bias, however, may also depend on the cognitive process under investigation: it is suggested that attention, stimulus processing and/or arousal can be influenced by both cholinergic and serotonergic systems independently from each other. Cholinergic and serotonergic projections to cortex and thalamus may be of importance in the mediation of these cognitive processes. Serotonergic-cholinergic interactions could also be of importance in the mediation of learning processes and trial-by-trial working memory. The data available do not allow an unambiguous conclusion about the role of these interactive processes in the mediation of long-term reference memory. These processes may rely on serotonergic-cholinergic interactions at the hippocampal level. It is concluded that serotonergic-cholinergic interactions play an important role in the mediation of behavioural, including cognitive, performance, but that further studies are necessary in order to elucidate the exact nature of these interactions.

Removing Obstacles in Neuroscience Drug Discovery : The Future Path for Animal Models

Despite great advances in basic neuroscience knowledge, the improved understanding of brain functioning has not yet led to the introduction of truly novel pharmacological approaches to the treatment of central nervous system (CNS) disorders. This situation has been partly attributed to the difficulty of predicting efficacy in patients based on results from preclinical studies. To address these issues, this review critically discusses the traditional role of animal models in drug discovery, the difficulties encountered, and the reasons why this approach has led to suboptimal utilization of the information animal models provide. The discussion focuses on how animal models can contribute most effectively to translational medicine and drug discovery and the changes needed to increase the probability of achieving clinical benefit. Emphasis is placed on the need to improve the flow of information from the clinical/human domain to the preclinical domain and the benefits of using truly translational measures in both preclinical and clinical testing. Few would dispute the need to move away from the concept of modeling CNS diseases in their entirety using animals. However, the current emphasis on specific dimensions of psychopathology that can be objectively assessed in both clinical populations and animal models has not yet provided concrete examples of successful preclinical–clinical translation in CNS drug discovery. The purpose of this review is to strongly encourage ever more intensive clinical and preclinical interactions to ensure that basic science knowledge gained from improved animal models with good predictive and construct validity readily becomes available to the pharmaceutical industry and clinical researchers to benefit patients as quickly as possible.

Behavioural analysis of four mouse strains in an anxiety test battery

Differences in locomotor activity, exploratory activity and anxiety-like behaviour of C57BL/6ChR,C57BL/6J, Swiss Webster/J and A/J strain were investigated in an anxiety battery. The battery consisted of paradigms studying spontaneous behaviour after a mild stressor, tasks of innate anxiety (light-dark box, elevated plus maze, novel object exploration), response to a conflict situation (Vogel conflict), conditioned fear and response to inescapable swim stress. Locomotor activity was studied in an open field and compared with locomotion in the other tests. Exploratory behaviour was studied in a 16-hole board task. The data confirm previous studies suggesting that A/J mice are a relatively anxious strain. Also, the data indicated that locomotor activity was independent of the paradigm employed, while the rank order of strain-dependent effects on anxiety-related behaviour changed as a function of the task under study. Our data provide further support for the notion that choice of strain is essential in studies of anxiety-related behaviour. Influence of strain should be considered in pharmacological and lesion studies, as well as in studies with mutant mice. In addition, the data indicate that different anxiety paradigms tax different aspects of anxiety, suggesting that a battery of different tests should be used in studies of anxiety-related behaviour.

Penetration of Amitriptyline, but Not of Fluoxetine, into Brain is Enhanced in Mice with Blood-Brain Barrier Deficiency Due to Mdr1a P-Glycoprotein Gene Disruption

Mice with a genetic disruption (knockout) of the multiple drug resistance (Mdr1a) gene were used to examine the effect of the absence of the drug-transporting P-glycoprotein at the blood-brain barrier on the uptake of amitriptyline (AMI) and fluoxetine (FLU) and their metabolites into the brain. One hour after intraperitoneal injection of AMI or FLU, knockout (−/−) and wild-type (+/+) mice were sacrificed and drug concentrations of brain, kidney, liver, testis, and plasma were measured. The plasma concentrations of the AMI metabolites and the brain:spleen ratios of AMI, nortriptyline (NOR), 10-OH-AMI and 10-OH-NOR were significantly higher in the −/− mice, demonstrating that AMI and its metabolites are substrates of the P-glycoprotein and that mdr1a activity at the level of the blood-brain barrier reduces the penetration of these substances into the brain. In contrast, tissue distributions of FLU and its metabolites among the various tissues tested were indistinguishable between groups. The herein reported differences in brain penetration of antidepressant drugs depending on the presence of the mdr1a gene may offer an explanation for differences in the treatment response at a given plasma concentration. Moreover, individual differences in mdr1 gene activity may account for variable response patterns at different episodes and development of therapy resistance.

Effects of transgenic overproduction of CRH on anxiety-like behaviour

Central administration of corticotropin‐releasing hormone increases anxiety‐like behaviour and arousal in rodents, and increased anxiety‐like behaviour has been shown in mice overproducing corticotropin‐releasing hormone on an elevated plus maze and in a dark–light emergence task. However, evidence is accumulating that measures obtained from different anxiety tasks may reflect different aspects of anxiety‐like behaviour in animals. We therefore tested mice overproducing corticotropin‐releasing hormone in a battery of paradigms, studying spontaneous behaviour after a mild stressor, tasks of innate anxiety‐like behaviour (light–dark box), lick suppression (Vogel conflict), conditioned fear, and forced swimming. Exploratory behaviour was studied in a 16‐hole board task. Furthermore, pain threshold, water intake, locomotor activity and sensorimotor learning/co‐ordination were tested to control for confounding factors. In line with previous findings, increased anxiety‐like behaviour of transgenic mice was observed in the light–dark box paradigm. However, no differences were seen in the conflict paradigm. Conditioned fear was decreased 1 h but not 24 h after conditioning in transgenic mice, and immobility was increased in forced swimming in corticotropin‐releasing hormone overexpressors. Locomotor activity in a novel open field and on the hole board was reduced in transgenics. Exploratory behaviour (hole pokes) was reduced during initial exploration of an unfamiliar hole board. Moreover, sensorimotor performance on a rotorod was impaired, and water intake was reduced in corticotropin‐releasing hormone overproducing mice, while no changes were seen in nociception. No differences in locomotor activity were seen in a second group of mice, tested in a familiar open field. When these animals were challenged with diazepam, transgenic mice were less susceptible to the sedative effects of the drug on locomotor activity. These data suggest that corticotropin‐releasing hormone overproduction leads to specific effects in a subset of anxiety paradigms, and that these transgenic mice suffer from a motor deficit in addition to altered anxiety‐like behaviour/arousal.

Synaptic plasticity in the basolateral amygdala in transgenic mice expressing dominant‐negative cAMP response element‐binding protein (CREB) in forebrain

Electrophysiological and behavioural experiments were performed in transgenic mice expressing a dominant‐negative form of cAMP response element‐binding protein (CREBA133) in the limbic system. In control littermate in vitro slice preparation, tetanizing the lateral amygdala–basolateral amygdala (BLA) pathway with a single train (100 Hz for 1 s) produced short‐term potentiation (STP) in the BLA. Five trains (10‐s interstimulus interval) induced long‐term potentiation (LTP), which was completely blocked by the N‐methyl‐d‐aspartate (NMDA) receptor antagonist d(–)‐2‐amino‐5‐phosphonopentanoic acid (AP5; 50 μm). When GABAergic (γ‐aminobutyric acid) inhibition was blocked by picrotoxin (10 μm), LTP became more pronounced. Low‐frequency stimulation (1 Hz for 15 min) induced either long‐term depression (LTD) or depotentiation. LTD remained unaffected by AP5 (50 μm) or by the L‐ and T‐type Ca2+‐channel blockers nifedipine (20 μm) and Ni2+ (50 μm), but was prevented by picrotoxin (10 μm), indicating a GABAergic link in the expression of LTD in the BLA. When conditioned fear was tested, a mild impairment was seen in one of three transgenic lines only. Although high levels of mRNA encoding CREBA133 lead to downregulation of endogenous CREB, expression of LTP and depotentiation were unaltered in BLA of these transgenic animals. These results could suggest that residual CREB activity was still present or that CREB per se is dispensable. Alternatively, other CREB‐like proteins were able to compensate for impaired CREB function.