Differential behavioural effects of chronic infusion of CRH 1 and CRH 2 receptor antisense oligonucleotides into the rat brain

Abstract

The purpose of this study was to investigate the functional role the two corticotropin-releasing hormone (CRH) receptor subtypes play in regulating the behavioural performance of rats in various well-defined test situations. Antisense oligodeoxynucleotides (ODNs) corresponding to either the rat CRH1 or CRH2 receptor mRNA were infused chronically into the lateral ventricle of male rats via osmotic minipumps (5 μg/0.5 μl/h over 6 days). Control groups received infusions of either a scrambled sequence ODN or mixed bases ODN or vehicle. On day 4 after surgery, the rats were subjected to 10 min of social defeat and immediately afterwards tested on the elevated plus-maze. Compared to a scrambled sequence control ODN, CRH1 receptor antisense ODN infusion was found to exert an anxiolytic-like effect whereas CRH2 receptor antisense ODN infusion had no effect on defeat-induced anxiety-related behaviour. In contrast, the CRH2 receptor antisense ODN increased immobility in a forced swim test whereas CRH1 receptor ODN-treated rats did not differ from controls. No influence of either ODN was found on general locomotor activity in an open field or on short-term memory performance in a social discrimination test. Furthermore, the CRH2 receptor antisense ODN did not affect spatial learning in a Morris water maze task. An additional experiment comparing a mixture of both missense ODNs and a vehicle control group confirmed that the former failed to induce non-specific (toxic) side effects, further substantiating the specificity of the respective antisense effects measured in this study. The results support the hypothesis that the two CRH receptor subtypes selectively mediate differential effects of endogenous CRH or CRH-related peptides at the brain level with the CRH1 receptor contributing predominantly to emotional behaviour and the CRH2 receptor being involved in the regulation of stress coping behaviour.

Introduction

Over the past few years, experimental evidence has accumulated indicating a central role for the neuropeptide corticotropin-releasing hormone (CRH) and its brain systems in coordinating the organisms endocrine, immune, autonomic and behavioral responses to stress (Dunn and Berridge, 1990; Owens and Nemeroff, 1991; Koob et al., 1994; Turnbull and Rivier, 1997). Dysregulation in CRH systems has been implicated in a variety of stress-related human psychiatric disorders such as anxiety and depression (Behan et al., 1996; Holsboer and Barden, 1996; Heit et al., 1997).

In the rat brain, as in the human brain, two different receptor subtypes (termed CRH1 and CRH2 receptors) have been identified (Perrin et al., 1993; Lovenberg et al., 1995a) that mediate the effects of CRH and related peptides. The observations that the two receptor subtypes differ in their pharmacologies, their affinities for CRH and other endogenous CRH-related peptides and in their discrete anatomical distribution (Chalmers et al., 1995; Primus et al., 1997; Turnbull and Rivier, 1997; Vale et al., 1997) suggest that they probably also mediate distinct CRH functions. The CRH1 receptor is widely distributed throughout the rat brain, being found in the cortex, brain stem and anterior pituitary, but also in limbic structures such as the amygdala (Potter et al., 1994; Chalmers et al., 1995; Primus et al., 1997). It is functionally well characterised, its local expression in the brain is changed as part of an adaptive response and it seems to play an important role in the regulation of emotional behavioural reactions to stressful stimuli (Heinrichs et al., 1992, Heinrichs et al., 1997a, Heinrichs et al., 1997b; Liebsch et al., 1995; Iredale et al., 1996; Gilligan et al., 1997). In contrast, the CRH2 receptor (especially the 2α splice variant) is found in different rat brain areas, predominantly the septum, hypothalamus, nucleus of the solitary tract and raphe nucleus (Lovenberg et al., 1995b; Primus et al., 1997). To date, due to the lack of highly specific CRH2 receptor agonists or antagonists, there is little information on the functional significance of this second receptor subtype. An alternative approach to reveal its functional significance despite the lack of a specific antagonist is antisense targeting, an extensively used method for selectively and transiently manipulating specific receptors or receptor subtypes in distinct brain areas of adult animals (Landgraf, 1996; Landgraf et al., 1997; Nicot and Pfaff, 1997). Recently, this technique has been successfully applied to study the functional difference between the two CRH receptor subtypes. Preliminary findings from our laboratory indicate that intracerebroventricular (i.c.v.) infusion of CRH1, but not CRH2, receptor antisense ODNs has an anxiolytic-like effect in rats on the elevated plus-maze (Liebsch et al., 1996; Montkowski et al., 1996) and, indeed, Heinrichs et al., 1997afound similar differential behavioural effects in the defensive withdrawal paradigm. The present study was designed to investigate the effect of chronic i.c.v. administration of CRH1 and CRH2 receptor antisense ODNs on the performance of rats in various behavioural test situations, some of which are known to involve CRH neurotransmission (Heinrichs et al., 1992; Koob et al., 1994; Liebsch et al., 1995). Social defeat, i.e. confrontation with an aggressive male rat in its home territory, was used to increase anxiety-related behaviour in the experimental subjects which could then be assessed in an elevated plus-maze test (Heinrichs et al., 1994; Pich et al., 1993). A forced swim test was used to examine stress coping behaviour (Porsolt et al., 1977; Marti and Armario, 1993), with general locomotor activity being determined in an open field test (Harro, 1993). Additionally, two tests of learning and memory, the social discrimination paradigm (Engelmann et al., 1995) and the Morris water maze (Morris, 1981), were included in the experimental design to study the involvement of CRH receptors in cognitive functions.