Mice overexpressing CRH show reduced responsiveness in plasma corticosterone after a 5-HT1A receptor challenge
M. M. Van Gaalen,
Max Planck Institute of Psychiatry, Kraepelinstraße 2–10, D-80804 Munich, Germany, and
Corresponding author (present address): Research Institute Neurosciences VU, Department of Medical Pharmacology, Vumc, Van der Boechorststraat 7, 1081 BT Amsterdam, the Netherlands. E-mail: email@example.com
§ Present address: Johnson & Johnson Pharmaceutical Research & Development, a divison of Janssen Pharmaceutica N.V., Turnhoutseweg 30, 2340 Beerse, Belgium.
Corticotropin-releasing hormone (CRH) overproduction and serotonergic dysfunction have both been implicated in a range of psychiatric disorders, such as anxiety and depression, and several studies have shown interactions between these two neurotransmitter systems. In this study, we investigated the effects of CRH challenge on hypothalamo-pituitary-adrenal (HPA) axis activity in female transgenic mice overproducing CRH. Furthermore, the effects of mild stress on HPA axis activity and body temperature were investigated in these mice. Pre- and post-synaptic 5-HT1A receptor function were studied by monitoring body temperature and plasma corticosterone levels after challenge with the 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propyl-amino)-tetralin (8-OH-DPAT). Hypothermia in response to 8-OH-DPAT treatment did not differ between transgenic and wild type mice, indicating unaltered somatodendritic 5-HT1A autoreceptor function in mice overproducing CRH. In wild type mice 8-OH-DPAT increased plasma corticosterone levels, but not in transgenic animals. CRH injection, however, increased corticosterone levels in both groups. These data suggest desensitization of post-synaptic, but not pre-synaptic, 5-HT1A receptors in mice overproducing CRH. These findings resemble those seen in depressed patients following 5-HT1A challenge, which is in accord with the hypothesized role of CRH in the pathogenesis of depression.
Both Corticotropin-releasing hormone (CRH) and serotonin (5-HT), in particular the 5-HT1A receptor, have been implicated in anxiety and depression (Deakin 1998; Holsboer 1999). CRH, a 41-amino acid mammalian neuropeptide, is a potent mediator of endocrine, autonomic and behavioral responses to stress (De Souza 1995). It has been shown that CRH can affect serotonergic function via actions at the level of the cell body region of the dorsal raphe serotonergic system (Price et al. 1998; Kirby et al. 2000), and serotonergic activity is altered after long-term intracereboventricular infusion of CRH (Linthorst et al. 1997). In the present experiment, the long-term consequences of CRH overproduction on 5-HT1A receptor function were studied in transgenic mice overexpressing CRH. Basal plasma corticosterone levels are increased in these mice (Stenzel-Poore et al. 1992). Behaviorally, transgenic animals overexpressing CRH display hypoactivity in novel environments (Stenzel-Poore et al. 1994) and enhanced anxiety-like behavior (Stenzel-Poore et al. 1994; Heinrichs et al. 1997), both of which are believed to be independent of hypothalamo-pituitary-adrenal (HPA) axis activity (Heinrichs et al. 1997). However, it remains unclear whether these effects could be mediated through alterations in serotonergic activity secondary to CRH overexpression. Therefore, we studied pre- and post-synaptic 5-HT1A receptor function, using 8-OH-DPAT-induced hypothermia and corticosterone release as markers.
5-HT1A receptors serve both somatodendritic autoreceptor- and post-synaptic heteroreceptor function. In mice, 8-OH-DPAT-induced hypothermia has been attributed to activation of somatodendritic 5-HT1A autoreceptors (Bill et al. 1991), while there is strong evidence to suggest that increased secretion of corticotropin (ACTH) and corticosterone following 8-OH-DPAT administration results, at least in part, from stimulation of post-synaptic 5-HT1A receptors at the level of the hypothalamic paraventricular nucleus (PVN) (Pan & Gilbert 1992).
Individually housed female transgenic mice overproducing CRH (Stenzel-Poore et al. 1992; Stenzel-Poore et al. 1994) (2–4 months of age, originally generated on a SJL × C57BL/6 background and backcrossed seven generations onto C57BL/6) were compared to sex- and age-matched wild-type litter mates (n = 5–9 per group). In the first experiment, animals were anaesthetized with isofluran and decapitated, with as short delay as possible (< 15 s), either under basal conditions (killed between 8 and 9 am) or 20 min after subcutaneous (sc) CRH injection (30 microgram/kg). Trunk blood was collected in ice-chilled EDTA-coated tubes containing 140 µg aprotinin (Trasylol; Bayer, Cologne, Germany). Plasma was separated and stored at −80 °C until corticosterone measurement by radioimmunoassay (RIA) (ICN Biomedicals, Costa Mesa, CA, USA). In the second experiment, rectal temperature was recorded over 4 s (Thermo-electra, Pijnacker, the Netherlands), after which animals received a sc injection of either 8-OH-DPAT (0.5 mg/kg) or saline and were returned to their homecage for 20 min, followed by another measurement of rectal temperature. Subsequently, trunkblood was collected and plasma corticosterone levels were estimated (see above). Data were analyzed by multiple analyses of variance (manova), followed by post hoc Tukey test, when appropriate. The experiments were performed in compliance with the guidelines of the European Communities Council Directive of 24 November 1986 (86/609/EEC).
Plasma corticosterone levels were overall increased in transgenic mice (F1,24 = 48.25, P < 0.001; Fig. 1). Furthermore, CRH injection resulted in an increase in corticosterone plasma levels (F1,24 = 50.10, P < 0.001), whereas no genotype–treatment interaction was found (F1,24 = 0.36, P > 0.050).
Body temperature was increased after injection stress, independently of genotype (Fig. 2). There was a significant time × treatment effect (F1,24 = 94.7, P < 0.001) on body temperature after 8-OH-DPAT administration, but no significant time × genotype (F1,24 = 0.12, P > 0.050) or time × genotype × treatment (F1,24 = 0.60, P > 0.050) interactions were observed (Fig. 2). Post hoc testing revealed that saline injection increased body temperature (P < 0.001), whereas 8-OH-DPAT reduced body temperature (P < 0.001).
Both genotype (F1,24 = 22.6, P < 0.001) and treatment (F1,24 = 7.92, P < 0.010) affected plasma corticosterone levels, and ANOVA revealed a significant genotype–treatment interaction (F1,24 = 94.7, P < 0.010; Fig. 3). Additional post hoc testing indicated that plasma corticosterone levels were higher in wild-type animals treated with 8-OH-DPAT than in all other groups, while 8-OH-DPAT had no effect in CRH overexpressing mice.
Central CRH administrations are known to induce hyperthermic responses, and both stress and CRH-induced hyperthermia can be blocked by α-helical CRH(9–41), a non-selective CRH antagonist (Morimoto et al. 1993), suggesting a key role for CRH in stress-induced hyperthermia. The fact that stress-induced hyperthermia did not differ between transgenic and wild-type mice suggests that CRH overproduction did not affect this measure, or that compensatory mechanisms are operating to maintain hyperthermic responses induced by stress in the face of CRH hypersecretion. 8-OH-DPAT induced hypothermia was not different between groups, suggesting that pre-synaptic 5-HT1A receptor function remained intact in transgenic mice.
Basal corticosterone plasma levels were elevated in transgenic animals, which is in line with a previous report from Stenzel-Poore and colleagues (Stenzel-Poore et al. 1992). However, no differences between groups were seen on this measure following mild stress (saline administration, and body temperature measurements) in the present study. This is in agreement with a recent study, reporting a blunted response of HPA axis activity after swim stress in transgenic mice overexpressing CRH (Heinrichs et al. 1997), despite the fact that anxiety-like behavior is increased in these animals (Stenzel-Poore et al. 1994; Heinrichs et al. 1997). Thus, although the hyperthermic response remained unaffected in transgenic mice, HPA axis reactivity after mild stress was blunted in these animals, suggesting distinct mechanisms involved in adjusting to increased CRH activity.
CRH injection elevated corticosterone plasma levels in both wildtype and transgenic mice, demonstrating that HPA axis activity is sensitive to CRH stimulation in both groups. Administration of 8-OH DPAT, however, increased plasma corticosterone levels in wild type animals, but not in transgenics. As elevation of plasma ACTH levels induced by 5-HT1A receptor activation may be mediated by 5-HT1A receptors affecting the release of CRH from the PVN (Pan & Gilbert 1992), it may be speculated that the blunted HPA response seen after 8-OH-DPAT administration in transgenic mice is due to desensitization of 5-HT1A receptors at the level of the PVN. Such phenomenon may also explain the lack of mild stress-induced HPA-axis activation in transgenic mice. Activation of PVN neurones is affected by multiple sources, such as brainstem aminergic and peptidergic afferents, blood-borne information, indirect input from limbic system-associated regions, and local interaction with the preoptic-hypothalamic region (Herman et al. 1996). The pathways involved in stress-induced HPA-axis activation seem to depend on the nature of the stressor (Herman et al. 1996; Jorgensen et al. 1998). Restrained stress-induced ACTH release, in contrast to ether stress-, cold swim stress-, or endotoxin-induced ACTH release, has been reported to be inhibited by the 5-HT1A antagonist WAY 100635 (Jorgensen et al. 1998). This suggests that the effects of certain stressors are under the control of serotonergic tone. It could be speculated that this mechanism also plays a role in the current study, and the lack of injection stress-induced HPA-axis activation may be due to desensitisation of post-synaptic 5-HT1A receptors. Of course, other mechanisms cannot be excluded.
Several clinical studies have demonstrated that administration of 5-HT1A agonists results in a decrease in body temperature and an increase in cortisol release in humans. Different profiles were found when the responses to 5-HT1A receptor challenge were studied in affective disorders. Thus, patients diagnosed as suffering from unipolar or major depression, exhibit significantly decreased cortisol responses to the partial 5-HT1A agonist ipsapirone, while basal cortisol secretion is increased when compared to controls (Lesch et al. 1990; Meltzer & Maes 1995). Despite altered HPA responsivity to 5-HT1A challenge, no significant differences were observed in ipsapirone-induced hypothermia (Meltzer & Maes 1995). This contrasts with data from patients with bipolar depression, who did not differ in cortisol release (Shiah et al. 1998), and patients with mania, who showed increased cortisol release after ipsapirone challenge (Yatham et al. 1999). Hypothermic responses were not different from controls in both patients groups. Patients suffering from obsessive compulsive disorder, on the other hand, showed no differences after 5-HT1A challenge in either parameter (Lesch et al. 1991), whereas attenuation of both thermoregulation and cortisol release was seen in patients with panic disorder (Lesch et al. 1992). Thus, the responses seen in mice overproducing CRH strikingly resemble those observed in depression.
In summary, the data indicate that 5-HT1A receptor challenging results in reduced responsiveness in plasma corticosterone levels in mice overproducing CRH, whereas body temperature changes after stress or following 5-HT1A receptor challenge is unaltered. This suggests that 5-HT1A receptor function at the post-synaptic level is diminished in CRH overexpressing mice, whereas 5-HT1A receptor function is unaltered at the pre-synaptic level. Extrapolating this situation to the human, these data open the possibility that the desensitization of 5-HT1A receptors seen in the brains of depressed subjects may result from hyperactivity of the CRH system frequently seen in these patients (Steckler & Holsboer 1999).