Anneloes Dirks

HPA axis dysregulation in mice overexpressing corticotropin releasing hormone

BACKGROUND Hypersecretion of corticotropin-releasing hormone (CRH) in the brain has been implicated in stress-related human pathologies. We developed a transgenic mouse line overexpressing CRH (CRH-OE) exclusively in neural tissues to assess the effect of long-term CRH overproduction on regulation of the hypothalamic-pituitary-adrenal (HPA) axis. METHODS Male transgenic CRH-OE(2122) mice on a C57BL/6J background were used. Littermate wildtype mice served as control animals. Basal plasma corticotropin and corticosterone concentrations were measured, and adrenal gland weight was determined. A dexamethasone suppression test measured the effects of long-term CRH hypersecretion on negative feedback control. Additionally, we measured plasma corticosterone concentrations in reaction to stress. RESULTS CRH-OE(2122) mice showed elevated basal plasma corticosterone concentrations, hypertrophy of the adrenal gland, and dexamethasone nonsuppression. Basal plasma ACTH concentrations of wildtype and CRH-OE(2122) mice did not differ significantly. In reaction to stress, CRH-OE(2122) mice showed a normal corticosterone response. CONCLUSIONS The HPA axis abnormalities observed in CRH-OE(2122) mice suggest that long-term hypersecretion of CRH in the brain can be a main cause of HPA axis dysregulation. The alterations in HPA axis regulation are reminiscent of changes reported in major depressive disorder. As such, these CRH -OE(2122) mice may model the neuroendocrine changes observed in major depressive disorder.

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.

5-HT1A receptor knockout mice and mice overexpressing corticotropin-releasing hormone in models of anxiety

Pharmacological experiments have implicated a role for serotonin (5-HT)(1A) receptors in the modulation of anxiety. More recent is the interest in corticotropin-releasing hormone (CRH) system as a potential target for the treatment of anxiety disorders. However, selective pharmacological tools for the CRH system are limited, hampering research in this field. Gene targeting is a relatively new approach to study mechanisms underlying anxiety disorders. 5-HT(1A) receptor knockout (1AKO) mice have been created on three different background strains, and two different lines of mice, overexpressing CRH (CRH-OE), have been generated. In the present review, behavioural and physiological findings reported for 1AKO mice and CRH-OE mice will be reviewed. As behavioural phenotyping is often limited to one or two approach avoidance paradigms, we extended these observations and also tested 1AKO and CRH-OE mice in a conditioned fear paradigm. This paradigm reflects essentially different aspect of anxiety than approach avoidance paradigms. 1AKO mice on a 129/Sv background strain showed similar freezing as wild-type (WT) mice. In CRH-OE mice, less freezing was observed than in the corresponding wild-type mice. The fact that the anxious phenotype of these genetically altered mice seems less clear than initially reported will be discussed. Rather than studying the direct consequences of alterations in the targeted gene, 1AKO and CRH-OE mice seem very valuable to study compensatory processes that seem to have taken place in reaction to life-long changes in gene expression.

Reduced startle reactivity and plasticity in transgenic mice overexpressing corticotropin-releasing hormone

BACKGROUND Corticotropin-releasing hormone (CRH) hyperactivity in transgenic mice overexpressing CRH in the brain (CRH-OE(2122)) appears to be associated with chronic stress-like alterations, including increased CRH content in the hypothalamus, changes in hypothalamus-pituitary-adrenal axis regulation, and increased heart rate and body temperature. In the present study, we investigated if sensory information processing of startling auditory stimuli was affected in CRH-OE(2122) mice. METHODS CRH-OE(2122) mice (on C57BL/6J background) were subjected to a number of procedures probing sensory information processing mechanisms, including the acoustic startle response, habituation, and prepulse inhibition of startle. RESULTS CRH-OE(2122) mice displayed reduced acoustic startle reactivity and increased motor activity during startle testing compared to wild-type mice. Furthermore, transgenic mice did not show habituation of the startle response after repeated exposure to the auditory stimulus, or habituation across procedures. CRH-OE(2122) mice exhibited robust impairments of prepulse inhibition in two different paradigms. CONCLUSIONS The results in CRH-OE(2122) mice indicate that chronic CRH hyperactivity is associated with reductions in startle reactivity, habituation, and prepulse inhibition. The latter two abnormalities are also observed in schizophrenia patients. We conclude that chronic CRH excess may reduce behavioral reactivity to environmental stimuli and impair information processing mechanisms.

Distress vocalizations in maternally separated mouse pups: modulation via 5-HT1A, 5-HT1B and GABAA receptors

Abstract Rationale: Young rodents emit ultrasonic vocalizations (USVs) when separated from their dams and littermates. Pharmacological agents that act on GABAA and/or 5-HT receptors and that alleviate anxiety in humans reduce the emission of these calls. Objectives: 1) to investigate specific 5-HT1 receptor subtypes that modulate maternal separation-induced USVs in mice; 2) to assess the behavioral specificity of these effects; and 3) to compare 5-HT1 agonists with a positive neurosteroid modulator of the GABAA receptor complex. Methods: Seven-day old CFW mouse pups were isolated from their littermates and placed onto a 20°C surface for 4 min. USVs between 30 and 80 kHz, grid crossing, and rectal temperature were measured in separate groups of mouse pups following subcutaneous administration of 5-HT1A and 5-HT1B receptor agonists and antagonists, the neurosteroid allopregnanolone, or the benzodiazepine midazolam. Results: The 5-HT1A agonists (+)8-OH-DPAT (0.01–0.1 mg/kg) and flesinoxan (0.3–1.0 mg/kg), the selective 5-HT1B agonist CP-94,253 (0.03–30.0 mg/kg), and the mixed 5-HT1B/2C receptor agonist TFMPP (0.1–10.0 mg/kg) dose-dependently reduced USVs. These effects were reversed by the 5-HT1A receptor antagonist WAY 100,635 (0.1 mg/kg) or the 5-HT1B/D receptor antagonist GR 127935 (0.1 mg/kg). The effects of TFMPP were biphasic; low doses (i.e. 0.01 and 0.03 mg/kg) increased the rate of vocalization. Midazolam and allopregnanolone also reduced USVs. The highest doses of flesinoxan, (+)8-OH-DPAT, and allopregnanolone suppressed locomotion, whereas CP-94,253, TFMPP, and midazolam stimulated motor activity. Conclusions: These experiments confirm that agonists at the 5-HT1 receptors and a positive allosteric modulator of the GABAA receptor complex decrease maternal separation-induced USVs in mice, with 5-HT1B manipulations dissociating the effects on vocalizations from sedative effects.

Reversal of startle gating deficits in transgenic mice overexpressing corticotropin-releasing factor by antipsychotic drugs.

Chronically elevated levels of corticotropin-releasing factor (CRF) in transgenic mice overexpressing CRF in the brain (CRF-OE) appear to be associated with alterations commonly associated with major depressive disorder, as well as with sensorimotor gating deficits commonly associated with schizophrenia. In the present study, we tested the hypothesis that antipsychotics may be effective in normalizing prepulse inhibition (PPI) of acoustic startle in CRF-OE mice, which display impaired sensorimotor gating compared to wild-type (WT) mice. The typical antipsychotic haloperidol and atypical antipsychotic risperidone improved PPI in the CRF-OE mice, but were ineffective in WT mice. The atypical antipsychotic clozapine did not influence PPI in CRF-OE mice, but reduced gating in WT mice. This effect of clozapine in the CRF-OE mice may thus be regarded as a relative improvement, consistent with the observed effect of haloperidol and risperidone. As expected, the anxiolytic, nonantipsychotic chlordiazepoxide was devoid of any effect. All four compounds dose-dependently reduced the acoustic startle response irrespective of genotype. These results indicate that antipsychotic drugs are effective in improving startle gating deficits in the CRF-OE mice. Hence, the CRF-OE mouse model may represent an animal model for certain aspects of psychotic depression, and could be a valuable tool for research addressing the impact of chronically elevated levels of CRF on information processing.

5-HT1B receptor knockout, but not 5-HT1A receptor knockout mice, show reduced startle reactivity and footshock-induced sensitization, as measured with the acoustic startle response

To investigate whether the hyperreactivity to mild environmental and novel stimuli in 5-HT1B receptor knockout (1BKO) mice, as suggested by measures of exploratory, aggressive, and impulsive behaviors, can be extended to phasic stimuli, 1BKO and wildtype mice were tested in acoustic startle reactivity and plasticity paradigms, including habituation, prepulse inhibition, and footshock-induced sensitization of the startle response. Furthermore, we compared 5-HT1A receptor knockout (1AKO) and 1BKO mice to further test the suggested opposite behavioral profiles in these two genotypes. Results show that startle reactivity and footshock-induced sensitization was reduced in 1BKO mice, with no changes in habituation or PPI. In contrast, 1AKO mice did not differ from WT mice in any of the measures. These results indicate that an absence of 5-HT1B receptors, but not of 5-HT1A receptors, affects the modulation of startle reactivity and footshock-induced sensitization, without influencing startle plasticity. Moreover, this study suggests that 1AKO mice display a distinct, but not opposite behavioral profile from 1BKO mice. Furthermore, it is concluded that the hyperreactivity in 1BKO mice cannot be generalized to all stimuli, including the startling stimuli used in this study, but is probably restricted to mild environmental stimuli only.

Effects of corticotropin-releasing hormone on distress vocalizations and locomotion in maternally separated mouse pups

The behavioral effects of corticotropin-releasing hormone (CRH) appear to depend on the baseline state of arousal of the animal. In this study, this hypothesis was tested using a 4-min maternal separation procedure in 7-day-old male and female mouse pups (outbred CFW strain). Two intensities of stress were used to assess the effects of intracerebroventricularly administered r/hCRH: a mild stress condition where the ambient temperature was close to nest temperature (30 degrees C) and rates of maternal separation-induced ultrasonic vocalizations (USVs) were relatively low (ca. 25/4 min), and a more stressful condition where the temperature was 19 degrees C and the rates of USVs were high (ca. 250/4 min). Differential effects of CRH on vocalization rate and locomotor behavior were observed to be dependent on the level of stress. In the more stressful 19 degrees C condition, r/hCRH dose-dependently reduced the number of USVs without affecting motor behavior, as indexed by grid crossings. In contrast, in the 30 degrees C condition, only the highest dose of r/hCRH reduced calling while r/hCRH activated motor behavior over a wider range of doses. These effects were independent of hypothalamus-pituitary-adrenal (HPA) axis activity, as measured by plasma corticosterone levels. The present study indicates that in mouse pups, the effects of CRH administration depend on baseline levels of arousal and that the behavioral effects of CRH administration can be dissociated under mild and more stressful conditions.

CRF1 Not Glucocorticoid Receptors Mediate Prepulse Inhibition Deficits in Mice Overexpressing CRF

BACKGROUND Both corticotropin-releasing factor (CRF) and glucocorticoid receptors (GR) are implicated in the psychotic symptoms of psychiatric disorders. Correspondingly, it is of interest to determine their respective involvement in the sensorimotor gating deficits displayed by transgenic mice overexpressing CRF. These mice reveal lifelong elevations of CRF and corticosterone levels. METHODS Effects of the GR antagonists ORG34517 (5-45 mg/kg by mouth [PO]) and mifepristone (5-45 mg/kg PO) and the CRF(1) receptor antagonists CP154,526 (20-80 mg/kg intraperitoneally [IP]) and DMP695 (2.5-40.0 mg/kg IP) on prepulse inhibition (PPI) of the acoustic startle response were studied in mice overexpressing CRF and in their wild-type littermates. In addition, PPI was measured in both genotypes 2 weeks after adrenalectomy with or without exogenous corticosterone administration via subcutaneous pellet implant (20 mg corticosterone). RESULTS ORG34517 and mifepristone did not influence perturbation of PPI in mice overexpressing CRF; reducing corticosterone levels by adrenalectomy likewise did not improve PPI. Further, elevation in corticosterone levels by pellet implantation did not disrupt PPI in wild-type mice. Conversely, both CRF(1) receptor antagonists, CP154,526 (40-80 mg/kg IP) and DMP695 (40 mg/kg IP), significantly restored PPI in CRF-overexpressing mice. CONCLUSIONS Sustained overactivation of CRF(1) receptors rather than excessive GR receptor stimulation underlies impaired sensorimotor gating in CRF-overexpressing mice. CRF(1) receptors thus may play a role in the expression of psychotic features in stress-related psychiatric disorders.

Infusion of flesinoxan into the amygdala blocks the fear-potentiated startle.

In a previous study it was demonstrated that flesinoxan, a selective serotonin (5-HT)1A receptor agonist, had anxiolytic properties in the fear-potentiated startle paradigm. The present study investigated the putative site of action of flesinoxan in this paradigm. Flesinoxan infused either into the dorsal raphe nucleus or the median raphe nucleus did not affect startle potentiation. Bilateral infusion of flesinoxan into the central nucleus of the amygdala on the other hand, dose-dependently blocked the fear-potentiated startle response. These data indicate that flesinoxan exerts it anxiolytic effects in the fear-potentiated startle paradigm via the central nucleus of the amygdala, whereas the dorsal and median raphe nuclei are not directly involved in this process.