Behavioral studies on anxiety and depression in a drug discovery environment: keys to a successful future.

The review describes a personal journey through 25 years of animal research with a focus on the contribution of rodent models for anxiety and depression to the development of new medicines in a drug discovery environment. Several classic acute models for mood disorders are briefly described as well as chronic stress and disease-induction models. The paper highlights a variety of factors that influence the quality and consistency of behavioral data in a laboratory setting. The importance of meta-analysis techniques for study validation (tolerance interval) and assay sensitivity (Monte Carlo modeling) are demonstrated by examples that use historic data. It is essential for successful discovery of new potential drugs to maintain a high level of control in animal research and to bridge knowledge across in silico modeling, and in vitro and in vivo assays. Today, drug discovery is a highly dynamic environment in search of new types of treatments and new animal models which should be guided by enhanced two-way translation between bench and bed. Although productivity has been disappointing in the search of new and better medicines in psychiatry over the past decades, there has been and will always be an important role for in vivo models in-between preclinical discovery and clinical development. The right balance between good science and proper judgment versus a decent level of innovation, assay development and two-way translation will open the doors to a very bright future.

5-HT(1A) receptor sensitivity in 5-HT(1B) receptor KO mice is unaffected by chronic fluvoxamine treatment.

The 5-HT(1B) receptor has been implicated in disorders such as depression, anxiety and obsessive-compulsive disorder. In mice lacking the 5-HT(1B) receptor (5-HT(1B) knockout mice), important changes in physiology and behavior exist. In the absence of presynaptic 5-HT(1B) receptor inhibition, chronic SSRI treatment may differentially affect 5-HT(1A) receptor functionality. The present studies tested the hypothesis that chronically reducing 5-HT transporter (5-HTT) function with selective serotonin reuptake inhibitor (SSRI) treatment would accelerate 5-HT(1A) receptor desensitization in 5-HT(1B) knockout mice. Moreover, as 5-HT(1B) knockout mice have been found to display exaggerated autonomic and locomotor responses to environmental stressors, the effects of chronic SSRI treatment on the hyperreactive phenotype of 5-HT(1B) knockout mice were investigated. The stress-reducing effect of the 5-HT(1A) receptor agonist flesinoxan on increases in body temperature, heart rate and locomotor activity was similar in wild type and 5-HT(1B) knockout mice before and after chronic 21-day treatment with the SSRI fluvoxamine, indicating no apparent alteration of 5-HT(1A) receptor sensitivity in 5-HT(1B) knockout mice. Also, chronic SSRI treatment did not alter the increased stress reactivity to mild environmental stressors in 5-HT(1B) knockout mice. We demonstrate that no apparent differences in 5-HT(1A) receptor sensitivity occur between 5-HT(1B) knockout and wild type mice after chronic fluvoxamine treatment. Also, the hyperreactive phenotype of 5-HT(1B) knockout mice is unresponsive to chronic SSRI treatment. Taken together, these results indicate that constitutive absence of 5-HT(1B) receptors does not result in adaptive changes in 5-HT(1A) receptor functionality and that chronic SSRI treatment does not modify stress reactivity in 5-HT(1B) knockout mice.

IMPA1 is essential for embryonic development and lithium-like pilocarpine sensitivity.

Lithium has been the standard pharmacological treatment for bipolar disorder over the last 50 years; however, the molecular targets through which lithium exerts its therapeutic effects are still not defined. We characterized the phenotype of mice with a dysfunctional IMPA1 gene (IMPA1-/-) to study the in vivo physiological functions of IMPA1, in general, and more specifically its potential role as a molecular target in mediating lithium-dependent physiological effects. Homozygote IMPA1-/- mice died in utero between days 9.5 and 10.5 post coitum (p.c.) demonstrating the importance of IMPA1 in early embryonic development. Intriguingly, the embryonic lethality could be reversed by myo-inositol supplementation via the pregnant mothers. In brains of adult IMPA1-/- mice, IMPase activity levels were found to be reduced (up to 65% in hippocampus); however, inositol levels were not found to be altered. Behavioral analysis of the IMPA1-/- mice indicated an increased motor activity in both the open-field test and the forced-swim test as well as a strongly increased sensitivity to pilocarpine-induced seizures, the latter supporting the idea that IMPA1 represents a physiologically relevant target for lithium. In conclusion the IMPA1-/- mouse represents a novel model to study inositol homeostasis, and indicates that genetic inactivation of IMPA1 can mimic some actions of lithium.

Blocking melanin-concentrating hormone MCH1 receptor affects rat sleep-wake architecture.

Melanin-concentrating hormone (MCH) is a hypothalamic peptide that centrally regulates food intake, energy balance and emotion. Interestingly, MCH and melanin-concentrating hormone MCH(1) receptors are distributed in brain areas known to regulate vigilance states. Effects of subcutaneous administration of two selective melanin-concentrating hormone MCH(1) receptor antagonists, labeled A and B were examined over a broad dose range (1, 3, 10, 20, 40 mg/kg) on rat sleep-wake architecture. Both compounds have a nanomolar antagonist activity at recombinant human melanin-concentrating hormone MCH(1) receptor (IC(50)=44.1+/-6.1 nM and 26.6+/-5.4 nM, respectively) and potently inhibited the MCH-induced mobilization of [Ca(2+)] (IC(50) 29.1+/-8.1 nM and 10.5+/-4.1 nM, respectively). The selectivity of both compounds was further confirmed on a panel of receptors, transporters and channels. In vivo, both compounds dose-dependently decreased deep sleep primarily by decreasing the mean duration of episodes during the first 4 h post-administration. In parallel, REM sleep and intermediate stage sleep were decreased while active and passive waking increased. Deep sleep and REM sleep onset latencies were significantly prolonged at higher doses. No homeostatic rebound of deep sleep was observed, while a tendency for recovery of REM sleep was found during subsequent dark phase. Together, the results support a role of the melanin-concentrating hormone MCH(1) receptor in the regulation of deep slow-wave sleep-REM sleep cycle. Therapeutic application of melanin-concentrating hormone MCH(1) receptor-inhibiting agents should take into account the significant decreases in deep sleep without recovery as these may interfere with sleep dependent memory consolidation.

Differential effects of exposure to low-light or high-light open-field on anxiety-related behaviors: relationship to c-Fos expression in serotonergic and non-serotonergic neurons in the dorsal raphe nucleus.

Serotonergic systems arising from the mid-rostrocaudal and caudal dorsal raphe nucleus (DR) have been implicated in the facilitation of anxiety-related behavioral responses to anxiogenic drugs or aversive stimuli. In this study we attempted to determine a threshold to engage serotonergic neurons in the DR following exposure to aversive conditions in an anxiety-related behavioral test. We manipulated the intensity of anxiogenic stimuli in studies of male Wistar rats by leaving them undisturbed (CO), briefly handling them (HA), or exposing them to an open-field arena for 15-min under low-light (LL: 8-13 lx) or high-light (HL: 400-500 lx) conditions. Rats exposed to HL conditions responded with reduced locomotor activity, reduced time spent exploring the center of the arena, a lower frequency of rearing and grooming, and an increased frequency of facing the corner of the arena compared to LL rats. Rats exposed to HL conditions had small but significant increases in c-Fos expression within serotonergic neurons in subdivisions of the rostral DR. Exposure to HL conditions did not alter c-Fos responses in serotonergic neurons in any other DR subdivision. In contrast, rats exposed to the open-field arena had increased c-Fos expression in non-serotonergic cells throughout the DR compared to CO rats, and this effect was particularly apparent in the dorsolateral part of the DR. We conclude that exposure to HL conditions, compared to LL conditions, increased anxiety-related behavioral responses in an open-field arena but this stimulus was at or below the threshold required to increase c-Fos expression in serotonergic neurons.

Exposure to high- and low-light conditions in an open-field test of anxiety increases c-Fos expression in specific subdivisions of the rat basolateral amygdaloid complex.

Anxiety states and anxiety-related behaviors appear to be regulated by a distributed and highly interconnected system of forebrain structures including the basolateral amygdaloid complex (basolateral amygdala). Despite a wealth of research examining the role of the basolateral amygdala in anxiety-related behaviors and anxiety states, the specific subdivisions of the basolateral amygdala that are involved in responses to anxiogenic stimuli have not been examined. In this study, we investigated the effects of exposure to a novel open-field environment, with either low- or high-levels of illumination, on expression of the protein product of the immediate-early gene c-Fos in subdivisions of the rat basolateral amygdala. The subdivisions studied included the lateral, ventrolateral and ventromedial parts of the lateral amygdaloid nucleus, the anterior, posterior and ventral parts of the basolateral amygdaloid nucleus and the anterior and posterior part of the basomedial amygdaloid nucleus. Small increases in the number of c-Fos-immunoreactive cells were observed in several, but not all, of the subdivisions of the basolateral amygdala studied following exposure of rats to either the high- or low-light conditions, compared to home cage or handled control groups. Open-field exposure in both the high- and low-light conditions resulted in a marked increase in c-Fos expression in the anterior part of the basolateral amygdaloid nucleus compared to either home cage or handled control groups. These findings point toward anatomical and functional heterogeneity within the basolateral amygdaloid complex and an important role of the anterior part of the basolateral amygdaloid nucleus in the neural mechanisms underlying physiological or behavioral responses to this anxiety-related stimulus.

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 3beta (GSK-3beta) directly or indirectly via stimulation of the kinase Akt-1. We therefore investigated the possibility that transgenic mice overexpressing GSK-3beta 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-3beta by dimming the endogenous GSK-3beta 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-3beta, 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 anxiogenic drug FG-7142 increases serotonin metabolism in the rat medial prefrontal cortex.

The neural mechanisms underlying anxiety states are believed to involve interactions among forebrain limbic circuits and brainstem serotonergic systems. Consistent with this hypothesis, FG-7142, a partial inverse agonist at the benzodiazepine allosteric site of the GABAA receptor, increases c-Fos expression within a subpopulation of brainstem serotonergic neurons. Paradoxically, FG-7142 has no effect on extracellular serotonin concentrations, as measured using in vivo microdialysis, in certain anxiety-related brain structures. This study tested the hypothesis that FG-7142 alters serotonin metabolism within one or more nodes of a defined anxiety-related forebrain circuit. Rats received one of four treatments (vehicle, 1.9, 3.8, or 7.5 mg/kg FG-7142, i.p.) and brains were collected 1 h following treatment. Thirteen forebrain regions were microdissected and analyzed for l-tryptophan, serotonin, and 5-hydroxyindoleacetic acid concentrations using high pressure liquid chromatography with electrochemical detection. FG-7142 (7.5 mg/kg) increased l-tryptophan, serotonin, and 5-hydroxyindoleacetic acid concentrations in the prelimbic cortex but not in several other regions studied including subdivisions of the amygdala and bed nucleus of the stria terminalis. These data demonstrate that FG-7142 alters brain tryptophan concentrations and serotonin metabolism in specific components of an anxiety-related forebrain circuit including the medial prefrontal cortex, an important structure involved in executive function and the regulation of emotional behavior.

Abnormalities of social interactions and home-cage behavior in a mouse model of Rett syndrome.

Rett syndrome (RTT) is an autistic spectrum disorder with a known genetic basis. RTT is caused by loss of function mutations in the X-linked gene MECP2 and is characterized by loss of acquired motor, social and language skills in females beginning at 6-18 months of age. MECP2 mutations also cause non-syndromic mental retardation in males and females, and abnormalities of MeCP2 expression in the brain have been found in autistic spectrum disorders. We studied home-cage behavior and social interactions in a mouse model of RTT (Mecp2(308/Y)) carrying a mutation similar to common RTT causing alleles. Young adult mutant mice showed abnormal home-cage diurnal activity in the absence of motor skill deficits. Nesting, a phenotype related to social behavior, and social interactions were both impaired in these animals. Mecp2(308/Y) mice showed deficits in nest building and decreased nest use. Although there were no differences in aggression or exploration of novel inanimate stimuli, mutant mice took less initiative and were less decisive approaching unfamiliar males and spent less time in close vicinity to them in several social interaction paradigms. The abnormalities of diurnal activity and social behavior in Mecp2(308/Y) mice are reminiscent of the sleep/wake dysfunction and autistic features of RTT. These data suggest that MECP2 regulates the expression and/or function of genes involved in social behavior. The study of Mecp2(308/Y) mice will allow the identification of the molecular basis of social impairment in RTT and related autistic spectrum disorders.

Stress-induced hyperthermia in the mouse: c-fos expression, corticosterone and temperature changes.

In mammals, stress exposure is frequently associated with an elevated body temperature ['emotional fever', stress-induced hyperthermia (SIH)]. Rectal measurement of body core temperature of the mouse induces a rise of 1-1.5 degrees C over a 10- to 15-min time interval. This phenomenon has been exploited to design a specific test for measuring stress-induced hyperthermia: the singly-housed SIH paradigm in mice. In the present experiments, changes in body temperature and corticosterone levels were studied 10, 30, 60, 90 and 120 min after the first insertion of the rectal probe. In addition, changes in patterns of neural activation, as observed after immunostaining for Fos-immunoreactivity (Fos-IR), were studied in the brains of animals perfused at times 0, 60 or 120 min. Our results show that SIH and corticosterone levels have their peak values between 10 and 30 min and are no longer different from control values after 60 min. Patterns of Fos-IR have been studied in 11 brain areas, of which 2 brain areas (anterodorsal preoptic and periolivary nuclei) showed a continuing rise in Fos-IR after 60 and 120 min, while six nuclei, mostly hypothalamic and septal, showed a peak induction of Fos-IR after 60 min. In three brain areas, no consistent changes in Fos-IR could be observed. The authors conclude that the changes observed in the patterns of Fos-IR, after application of the singly-housed SIH-test in mice, reflect the effects of both the stressor application and the ensuing thermoregulatory responses. The role of each activated brain area in either one of these effects is discussed in view of data available from the literature.

Behavioral and physiological mouse models for anxiety: effects of flesinoxan in 129S6/SvEvTac and C57BL/6J mice.

Serotonin(1A) (5-HT(1A)) receptors are involved in anxiety. This study focuses on the role of genetic factors on the anxiety-related effects of 5-HT(1A) receptor stimulation using both a within subject design. The effects of 5-HT(1A) receptor activation were studied in high- and low-anxiety mice (129S6/SvEvTac (S6) and C57BL/6J (B6), respectively) in behavioral and physiological anxiety-related assays. These two strains were also selected because they are frequently used in gene-targeting studies. Mice were treated with the selective 5-HT(1A) receptor agonist flesinoxan (0-0.3-1.0-3.0 mg/kg s.c.) and tested in either the open-field activity test, the light-dark exploration test, or the stress-induced hyperthermia paradigm. Flesinoxan unexpectedly increased anxiety, but also decreased activity on several behavioral measures in B6 mice. Flesinoxan produced only minimal effects in the behavioral tests in the high-anxiety S6 strain. In contrast, the physiological hyperthermia response showed anxiolytic-like effects of flesinoxan in both strains. Our data indicate that the role of 5-HT(1A) receptor activation on anxiety-related responses is dependent on genetic background and selected paradigm used to assess anxiety. These findings indicate that it is critical to use a multi-level approach to develop mouse models for human diseases. In addition, the implication of such findings for studies on genetically modified mice is discussed.

Effects of repeated testing in two inbred strains on flesinoxan dose-response curves in three mouse models for anxiety.

Over the last decade, many genetically modified mice have been developed as models for psychiatric diseases such as anxiety. Limited availability of such mutant mice highlights the importance of studying the possibility of repeatedly testing the same individuals. We tested mice four times with 1-week intervals with the same dose of the 5-HT(1A) receptor agonist flesinoxan (0-0.3-1.0-3.0 mg/kg s.c.) in three anxiety-related paradigms: light-dark exploration, open-field activity and stress-induced hyperthermia. The two inbred strains studied were the highly anxious 129S6/SvEvTac (S6) and low-anxiety C57BL/6J (B6) mice. The results indicate that the effects of repeated testing were relatively mild. B6 mice showed some mild habituation in the open-field test when treated with vehicle, whereas S6 mice developed reduced initial activity in the light-dark box after drug treatment. In contrast, responses to flesinoxan treatment were strong and highly consistent for most parameters. In the open-field and light-dark tests, B6 mice showed reduced activity and anxiogenic-like behavioral responses, whereas S6 mice were minimally affected. Anxiolytic-like responses were found in both strains in the stress-induced hyperthermia paradigm. We conclude that B6 and S6 mice can be tested repeatedly with agents such as 5-HT(1A) receptor agonists with 1-week intervals in the three paradigms tested.

Overexpression of corticotropin-releasing hormone in transgenic mice and chronic stress-like autonomic and physiological alterations.

To gain a greater insight into the relationship between hyperactivity of the corticotropin-releasing hormone (CRH) system and autonomic and physiological changes associated with chronic stress, we developed a transgenic mouse model of central CRH overproduction. The extent of central and peripheral CRH overexpression, and the amount of bioactive CRH in the hypothalamus were determined in two lines of CRH-overexpressing (CRH-OE) mice. Furthermore, 24 h patterns of body temperature, heart rate, and activity were assessed using radiotelemetry, as well as cumulative water and food consumption and body weight gain over a 7-day period. CRH-OE mice showed increased amounts of CRH peptide and mRNA only in the central nervous system. Despite the presence of the same CRH transgene in their genome, only in one of the two established lines of CRH-OE mice (line 2122, but not 2123) was overexpression of CRH associated with increased levels of bioactive CRH in the hypothalamus, increased body temperature and heart rate (predominantly during the light (inactive) phase of the diurnal cycle), decreased heart rate variability during the dark (active) phase, and increased food and water consumption, when compared with littermate wildtype mice. Because line 2122 of the CRH transgenic mice showed chronic stress-like neuroendocrine and autonomic changes, these mice appear to represent a valid animal model for chronic stress and might be valuable in the research on the consequences of CRH excess in situations of chronic stress.

Behavioral and physiological mouse assays for anxiety: a survey in nine mouse strains.

In order to find better and new treatments for anxiety in humans, a variety of paradigms are used to study anxiety-related processes in rodents. We studied mice in two different anxiety-related assays: the physiological stress-induced hyperthermia (SIH) paradigm and the behavioral light-dark exploration (LD) test. Eight inbred strains (129S6/SvEvTac, 129S1/SvImJ, A/J, BALB/cByJ, C3H/HeJ, C57BL/6J, DBA/2J, and FVB/NJ) and one outbred strain (CD1-ICR) were tested in both assays repeatedly. This study describes the first strain survey for the SIH paradigm. All strains showed an SIH response, but the magnitude of the response varied between lines. The inbred strain distribution pattern for the behavioral responses in the LD assay was not correlated with the SIH response. The lack of a significant correlation suggests that there is no genetic relation between such responses. Mice could be tested repeatedly in both assays without affecting the results. A new paradigm, in which both assays were combined, elucidated that behavioral responses were not altered by segments of the SIH paradigm. In contrast, exposure to the light-dark box instead of the home-cage showed a strain-dependent effect on the physiological response. We conclude that a combination of behavioral and physiological responses might lead to a better understanding in anxiety-related processes.

GABAA-benzodiazepine receptor complex ligands and stress-induced hyperthermia in singly housed mice.

Stress-induced hyperthermia (SIH) in singly housed mice, in which the rectal temperature of a mouse is measured twice with a 10-min interval, enables to study the effects of a drug on the basal (T1) and on the stress-enhanced temperature (T2), 10 min later, using the rectal procedure as stressor. SIH (T2-T1) reflects a stress-induced phenomenon sensitive to stress- or anxiety-modifying effects of drugs. Several benzodiazepine agonists (diazepam, chlordiazepoxide, oxazepam and alprazolam) dose-dependently antagonized SIH either in NMRI mice from two different breeders or in BALB/c mice. No major differences in the sensitivity for any of the drugs tested were found between strains or between substrains from different breeders. The selective BZ1 receptor agonists alpidem and zolpidem only at relatively high doses antagonized SIH, whereas flumazenil, FG7142, pentylenetetrazol and phenobarbital did not affect SIH. Alcohol antagonized SIH, and the effects of diazepam could be antagonized by flumazenil. The findings that full BZ receptor agonists have anxiolytic-like effects in the singly housed SIH paradigm are comparable to those previously found in the group-housed version. The singly housed SIH is proposed as a simple and reliable screen for detecting anxiety-like properties of drugs that is valid in every mouse strain tested so far.

5-HT(1B) receptor knockout mice show no adaptive changes in 5-HT(1A) receptor function as measured telemetrically on body temperature and heart rate responses.

Two presynaptic receptors play an important role in the regulation of serotonergic neurotransmission, i.e., the 5-HT(1A) and 5-HT(1B) receptor. The present study focuses on putative adaptive changes in the 5-HT(1A) receptor system in mice that lack 5-HT(1B) receptors (5-HT(1B) KO). 5-HT(1A) receptor sensitivity was assessed in vivo in two models of presynaptic 5-HT(1A) receptor activity: agonist-induced hypothermia and prevention of stress-induced hyperthermia. The effects of 5-HT(1A) receptor activation by flesinoxan (0.1-3.0 mg/kg s.c.) were determined telemetrically on body temperature and heart rate in 5-HT(1B) KO and wild-type (WT) mice. Flesinoxan induced hypothermia dose-dependently without affecting heart rate and prevented stress-induced hyperthermia and tachycardia equipotently in both genotypes. Specificity of these responses was confirmed by blockade with the selective 5-HT(1A) receptor antagonist WAY100635 (1.0 mg/kg s.c.). The importance of continuous sampling in freely moving subjects to improve appropriate characterization of mutants is discussed. 5-HT(1B) KO mice showed no shift in 5-HT(1A) receptor sensitivity compared to WT mice. This study found no indications for adaptive changes in presynaptic 5-HT(1A) receptor function in 5-HT(1B) KO mice as measured telemetrically on body temperature and heart rate responses.