CRF receptor systems in affective disorders

Neuroscience and Biobehavioral Reviews 29 (2005) 867–889 www.elsevier.com/locate/neubiorev

Review

Listening to mutant mice: a spotlight on the role of CRF/CRF receptor systems in affective disorders Martin E. Keck*, Frauke Ohl, Florian Holsboer, Marianne B. Mu¨ller Max Planck Institute of Psychiatry, Kraepelinstrasse 2-10, 80804 Munich, Germany

Abstract Genetically engineered mice were originally generated to delineate the role of a specific gene product in behavioral or neuroendocrine phenotypes, rather than to produce classic animal models of depression. To learn more about the neurobiological mechanisms underlying a clinical condition such as depression, it has proven worthwhile to investigate changes in behaviors characteristic of depressed humans, such as anxiety, regardless of whether or not these alterations may also occur in other disorders besides depression. The majority of patients with mood and anxiety disorders have measurable shifts in their stress hormone regulation as reflected by elevated secretion of central and peripheral stress hormones or by altered hormonal responses to neuroendocrine challenge tests. In recent years, these alterations have been increasingly translated into testable hypotheses addressing the pathogenesis of illness. Refined molecular technologies and the creation of genetically engineered mice have allowed to specifically target individual genes involved in regulation of corticotropin releasing factor (CRF) system elements (e.g. CRF and CRF-related peptides, their receptors, binding protein). Studies performed in such mice have complemented and extended our knowledge. The cumulative evidence makes a strong case implicating dysfunction of these systems in the pathogenesis of depression and leads us beyond the monoaminergic synapse in search of eagerly anticipated strategies to discover and develop better therapies for depression. q 2005 Elsevier Ltd. All rights reserved. Keywords: CRF; Depression; Anxiety; CRF receptor antagonist; R121919; NBI 30775; CRF receptor type 1; CRF receptor type 2; Transgenic mice; Conditional knockout

Contents 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

CRF neuronal circuitries and affective disorders-the clinical situation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . HPA system—a stress hormone system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Corticosteroid effects are mediated via two receptor subtypes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Dual action of CRF: activator of the HPA system and neurotransmitter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The CRF neuropeptide family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CRF receptors and CRF binding protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The role of CRF receptors in anxiety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genetically engineered mice to elucidate CRF neurocircuitry-related psychopathology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The use of rodents to investigate depression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genetically engineered mice-the ‘genotype-driven’ approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Is it reasonable to test for anxiety when investigating for depression? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Gene targeting methods: short overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.1. ‘Conventional’ transgenic mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.2. Conditional gene-silencing methods (‘conditional knock-out’) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13. Highlights from mutant mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . * Corresponding author. Tel.: C49 89 30622 233; fax: C49 89 30622 610. E-mail address: [email protected] (M.E. Keck).

0149-7634/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.neubiorev.2005.03.003

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13.1. CRF overexpression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 876 CRF knockout . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 877 Urocortin 1 (Ucn 1) knockout: conflicting behavioral data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 878 CRF-binding protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 878 16.1. CRF-binding protein overexpressing mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 878 16.2. CRF-binding protein knockout . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 879 17. Genetic targeting of CRF receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 879 17.1. Conventional inactivation of CRF1 decreases anxiety-related behavior . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 879 17.2. Conditional inactivation of CRF1 in the limbic system reduces anxiety-related behavior with normal basal HPA activity 879 17.3. CRF2 knockout: increased anxiety-related behavior? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 880 17.4. Life without CRF receptors: CRF1/CRF2 double knockout mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 881 18. First steps towards therapy: CRF1 antagonists for the treatment of affective disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 881 19. Clinical outlook and summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 883 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 884 14. 15. 16.

1. CRF neuronal circuitries and affective disorders-the clinical situation There is increasing evidence for a hyperactivity of the hypothalamic-pituitary-adrenocortical (HPA) system in human affective disorders, and it has been proposed that hypercortisolemia is in some way integral to the pathogenesis and maintenance of psychopathology and cognitive deficits in these diseases (Holsboer, 1999b, 2000b; Nemeroff, 1996; Nemeroff and Owens, 2002). Accordingly, hyperactivity of central neuropeptidergic circuits such as corticotropin releasing factor (CRF; also termed corticotropin releasing hormone, CRH) and vasopressin (AVP) neuronal systems is thought to play a causal role in the etiology and symptomatology of mood and anxiety disorders (Gold et al., 1984; Ho¨kfelt et al., 2000; Holsboer, 2000b). The most prominent and well-documented neuroendocrine change in affective disorders, in particular major depression, is overactivity of the HPA system. This is reflected by increased numbers of CRF- and AVP-secreting neurons in the hypothalamus and locus coeruleus of depressed patients (Bissette et al., 2003; Purba et al., 1996; Raadsheer et al., 1994), increased levels of CRF in the cerebrospinal fluid, and characteristic changes in neuroendocrine challenge tests, such as the combined dexamethasone/CRF challenge test (Heuser et al., 1994a; for a detailed review see: Holsboer, 1999c, 2000a). In major depression, the combined dexamethasone/CRF test, in which dexamethasone-pretreated subjects receive a single dose of CRF, has proven to be the most sensitive tool for the detection of altered HPA system regulation. Depending on age and gender, up to 90% of patients with depression show this neuroendocrine phenomenon (Heuser et al., 1994b). The more severe the patient’s depression, i.e. patients with psychotic features and melancholia, the more robust the HPA hyperactivity (Nemeroff, 1996). Moreover, decreased CRF binding in the prefrontal cortex of depressed suicide victims has been reported, probably reflecting an adaptive down-regulation due to central CRF hypersecretion (Nemeroff, 1996). In recent years, a considerable amount of evidence has been

accumulated suggesting that normalization of the HPA system might be the final step necessary for stable remission of the disease (Barden et al., 1995; Holsboer, 2000b; Zobel et al., 1999). Antidepressant drugs and electroconvulsive therapy, in turn, have been shown to attenuate and normalize HPA system abnormalities (De Bellis et al., 1993; Heuser et al., 1996; Kling et al., 1994; Nemeroff et al., 1991). Although these findings derived from peripheral HPA assessments in depressed patients led to the concept of CRF hyperactivity, it is now clear that central CRF neuropeptidergic circuits other than those driving the peripherally accessible HPA system may well be overactive and act on behaviorally relevant extrapituitary brain sites. The scope of this review focuses on genetically engineered mice which were helpful to substantiate this hypothesis. Depression is a heterogeneous illness and it is, therefore, of interest to note that while the weight of available evidence suggests that a significant proportion of depressed patients display CRF hypersecretion, another subgroup of patients suffering from depression appears to have a hypoactive HPA system and decreased central CRF concentrations (Geriacoti et al., 1997, 1992). These patients often have special clinical characteristics such as chronicity or suffer from ‘atypical’ depression, a syndrome characterized by hypersomnia, hyperphagia and mood reactivity (Geriacoti et al., 1997; Gold and Chrousos, 2002). Abnormalities in CRF secretion or signal conduction, therefore, might be a part of the pathophysiology of depression regardless of whether they result in either the under- or overamplification of the CRF signal.

2. HPA system—a stress hormone system Stress has repeatedly been shown to precipitate major depression and to influence its incidence, severity and course (Kessler et al., 1994). In this context the question arises as to how the technical term ‘stress’ is defined. In neurobiology, stress is an internal or external cue that disrupts the homeostatic status of a subject. In order to

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survive, the organism must be able to adapt to stress, which can be manifested in a variety of forms. This stress response involves a large number of mechanisms and processes, which, altogether, serve to control the body’s defense reactions to stress, so as to restore homeostasis and to facilitate adaptation. One major neuroendocrine system involved in mediating the stress response is the HPA axis. CRF is the primary hypothalamic hypophysiotropic hormone, which regulates both basal and stress-induced release of pituitary corticotropin (ACTH) and is the major constituent of the HPA system (Vale et al., 1981b). At the pituitary level, the effects of CRF are amplified by AVP, which, after prolonged stress, is increasingly co-expressed and co-secreted from hypothalamic CRF neurons (Antoni, 1993; Keck et al., 2000, 2002). CRF triggers the immediate release of ACTH from the anterior pituitary, subsequently leading to release of glucocorticoid hormones (GC, cortisol in humans and corticosterone in rats and mice) from the adrenal cortex (Fig. 1).

Fig. 1. Regulation of the HPA system under physiological conditions. Diagram illustrating the regulation of the hypothalamic-pituitary-adrenocortical system under basal conditions: hypothalamic corticotropinreleasing factor (CRF), together with vasopressin (AVP), is released into the hypophyseal portal system and triggers the release of corticotropin (ACTH) from the anterior pituitary via stimulation of CRF1. ACTH, in turn, stimulates the secretion of glucocorticoid hormones (cortisol or corticosterone) from the adrenal cortex. Increasing glucocorticoid levels suppress hypothalamic CRF expression via negative feed-back through hippocampal and hypothalamic corticosteroid receptors. Neural inputs include excitatory afferents from the amygdala.

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During the last years, several research groups surmised a hypothesis relating aberrant stress hormone regulation to causality of depression (Holsboer, 1999c, 2000a). This ‘corticosteroid receptor hypothesis’ submits that corticosteroid receptor signaling is impaired in depression (Holsboer, 2000a). Specifically, based on the finding of increased CRF and AVP levels which account for a large number or the signs and symptoms prevalent in depression and anxiety disorders, it can be hypothesized that corticosteroid receptor signaling, exerting a negative feedback on AVP and CRF gene expression, is defunct.

3. Corticosteroid effects are mediated via two receptor subtypes GC exert their regulatory effects on the HPA system via two types of corticosteroid receptors: the glucocorticoid receptor (GR) and the mineralocorticoid receptor (MR) (Reul and deKloet, 1985). GRs occur everywhere in the brain but are most abundant in hypothalamic CRF neurons and pituitary corticotropes. MRs, in contrast, are highly expressed in the hippocampus, and, at lower expression levels, in hypothalamic sites. The MR binds GC with a tenfold higher affinity than does the GR (Reul and deKloet, 1985). These findings on corticosteroid receptor diversity led to the working hypothesis that the tonic influences of corticosterone are exerted via hippocampal MRs, while the additional occupancy of GRs with higher levels of corticosterone mediates feedback actions aimed to restore disturbances in homeostasis. Recently, a new mechanism of cross-talk between the CRF neuropeptidergic systems and hippocampal MRs was described: acute stressors act via a CRF receptor mediated action to cause an elevation in MR levels in the hippocampus, which is associated with an augmented MR-mediated inhibition of HPA activity (Gesing et al., 2001). Thus, CRF receptors are involved in central feedback control of the HPA system and there is increasing evidence that this phenomenon may be mediated via CRF1 (Gesing et al., 2001; Mu¨ller et al., 2003). Activation of GRs at the level of the PVN reduces CRF and AVP activity (Erkut et al., 1998). This negative feedback is a fundamental way in which the HPA system is restrained during stress and activity, and this restraint of HPA activation by glucocorticoids is rapid and profound. In contrast, induction of CRF expression by increasing glucocorticoid levels has been described to occur at the level of the central amygdala and the bed nucleus of the stria terminalis (BNST) (for review: Schulkin et al., 1998; Watts, 1996). The latter is derived embryologically from the amygdala, and plays a fundamental role in the regulation of the HPA system during stress (for review: Holsboer, 2000). In brief, stress initially activates the hypothalamic CRF and AVP system, resulting in the hypersecretion of glucocorticoids from the adrenal gland. In addition, the presumed psychological component of the stressor

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stimulates the amygdaloid CRF system in a manner distinguishable from presumed physiological stressors such as, e.g. hemorrhage (Dayas and Day, 2001). Chronic stressful life events could result in a loss of capacity of CRF or CRF-related peptides to upregulate hippocampal MR levels, leading to a loosening of the tonic inhibitory influence on parvocellular neurons in the PVN. Consequently, levels of CRF and AVP will increase in these neurons, providing an enhanced drive on HPA activity. Subsequently, the elevated circulating glucocorticoid levels will raise CRF expression in the central nucleus of the amygdala, resulting in an enhanced stimulatory influence on the PVN. In addition, in the chronic phase of stress, down-regulation of GR in the PVN and other brain structures such as the locus coeruleus fails to restrain hyperfunction of the HPA system, and persistent activation of the HPA axis further up-regulates the amygdaloid CRF system. In this manner, a feed-forward loop develops, accelerating the establishment of a state of sustained HPA hyperactivity (Reul and Holsboer, 2002). Thus, the hypothalamic and the amygdaloid CRF systems cooperatively constitute stress-responsive, anxiety-producing neurocircuitry during chronic stress, which may be responsible for the clinical manifestation of stress-associated disorders such as major depression and anxiety disorders.

4. Dual action of CRF: activator of the HPA system and neurotransmitter Parvocellular neurons of the hypothalamic PVN are the major source of CRF within the central nervous system. These parvocellular neurons project via the external zone of the median eminence to the anterior pituitary where CRF is released into hypophyseal portal blood vessels to activate the HPA system by triggering ACTH release from pituitary corticotropes through activation of CRF 1 receptors (CRF1). Furthermore, independently of its action on ACTH release, CRF acts as a neurotransmitter in several brain areas. High densities of CRF-like immunoreactivity have been observed throughout the neocortex (particularly in the prefrontal and cingulate cortices), the central nucleus of the amygdala (Van Bockstaele et al., 1998), the BNST, the hippocampus, the nucleus accumbens, some thalamic nuclei, substantia nigra, raphe´ nuclei, locus coeruleus, periaqueductal gray and cerebellum (Swanson et al., 1983).

5. The CRF neuropeptide family With the discovery of more endogenous ligands-besides CRF-that bind to CRF receptors, the concept is dawning that CRF, its congeners and their receptors form an intricate network in the brain that potentially provides a variety of targets for drug interventions (Dautzenberg and Hauger, 2002; Reul and Holsboer, 2002; Rex et al., 1998). By now,

three other mammalian neuropeptides of the CRF family have been discovered, the so-called urocortin (Ucn 1; Vaughan et al., 1995), urocortin 2 (Ucn 2; Reyes et al., 2001b), and urocortin 3 (Ucn 3; Lewis et al., 2001a) the latter of which is an N-terminally shortened sequence of stresscopin (Hsu and Hsueh, 2001). CRF peptides only share four amino acids with each other and secondary structure, rather than linear sequence, appears to determine biological activity (Dautzenberg and Hauger, 2002). Because Ucn 2 and Ucn 3 have been identified by molecular cloning strategies (Reyes et al., 2001a, Lewis et al., 2001b), their exact size has not been established yet and this issue will not be conclusively resolved until endogenous forms of these two peptides are isolated in various species. It is of interest to note that human Ucn 2 lacks the standard consensus site required for proteolytic cleavage and C-terminated amidation, which is a prerequisite for biological potency (Dautzenberg and Hauger, 2002). The possibility, therefore, exists that human Ucn 2 may not be processed into a biologically active peptide in vivo and only the isolation of Ucn 2 peptide from native human tissue would resolve this intriguing issue (Hauger et al., 2003). The CRF and Ucn 1 precursor genes both contain two exons with the second exon encoding the entire precursor protein (Zhao et al., 1998). Two non-mammalian CRF-like peptides, the 40-amino-acid amphibian peptide sauvagine and the 41-amino-acid fish peptide urotensin I share about 50% sequence identity with human CRF. CRF 1 receptor (CRF1) and CRF 2 receptor (CRF2) differ in their ligand affinities for CRF, Ucn 1, Ucn 2 and Ucn 3 (CRF1: Ucn 1OCRF; CRF2: Ucn 1OUcn 2OUcn 3[CRF) (Chalmers et al., 1996; Donaldson et al., 1996; Lovenberg et al., 1995b). Compared to CRF, Ucn 1 has an approximately 100-fold higher affinity for the CRF2 and a roughly 6-fold higher affinity for CRF1 (Dautzenberg et al., 2001; Vaughan et al., 1995). Ucn 2 and Ucn 3 display specific affinity for the CRF1 at very high local concentrations only. Of known agonists, Ucn 3 displays the highest degree of selectivity in binding to the CRHR2. It is, therefore, likely that CRF and Ucn 1 represent the natural agonists for the CRF1, whereas Ucn 1, Ucn 2 and Ucn 3 are likely to be the natural ligands for the CRF2 with CRF remaining a candidate natural ligand for the CRF2 at sufficiently high local concentrations. It cannot be ruled out, however, that yet undiscovered receptors exist. Ucn 1 has many of the effects of CRF, such as a high ACTH secretagogue potency (Asaba et al., 1998). Ucn 1 immunoreactivity has been shown to be widely expressed in the brain (Bittencourt et al., 1999), with high levels in various neocortical areas and the Edinger-Westphal nucleus (cell bodies and fibers), and with moderate levels in the basal ganglia (mostly fibers), lateral and medial septum (mostly fibers), the hypothalamus (cell bodies and fibers), the superior colliculus (fibers), the raphe´ nuclei (cell bodies and fibers) and cerebellum (fibers) (Iino et al., 1999; Kozicz et al., 1998; Wong et al., 1998; Yamamoto et al., 1998). By use of well characterized antisera Ucn 1 was found

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to be absent or relatively scarce in the hippocampus and amygdaloid nuclei (Bittencourt et al., 1999). Neurons that display both Ucn 1 mRNA and peptide expression were found to be centered in the Edinger-Westphal, lateral superior olivary, and supraoptic nuclei. Lower levels of expression are seen in small populations of neurons in the forebrain. Additional sites of Ucn 1 mRNA and peptide expression detected only in colchicine-treated rats are considered to be minor ones (Bittencourt et al., 1999). In peripheral tissues, Ucn 1 is more broadly expressed, especially in the pituitary, thymus, kidney, spleen, testis, and gastrointestinal tract (Kageyama et al., 1999). In general, there seems to be only limited overlap between the distribution of CRF and Ucn 1 (Morin et al., 1999). Ucn 2 mRNA displays a limited subcortical distribution in the rodent brain that is unique, although ostensibly overlapping in part with those of CRF (paraventricular nucleus) and Ucn 1 (brainstem and spinal motor nuclei). Of particular interest is the fact that Ucn 2 is expressed in cell groups involved in stress-related physiological and behavioral functions. This includes the locus coeruleus, the hypothalamic paraventricular nucleus and the arcuate nucleus (Reyes et al., 2001b). Mouse Ucn 3 mRNA expression is relatively restricted to stress-related brain regions such as the dorsal and lateral aspects of the hypothalamic PVN, the basomedial and cortical (but not the central) nuclei of the amygdala, the BNST, the anterior thalamic nucleus, the medial preoptic nucleus and the lateral hypothalamic area (Lewis et al., 2001b; Li et al., 2002; Venihaki et al., 2004). Using RT-PCR, blot hybridization and DNA sequence analysis Ucn 3 mRNA was also detected to be robustly expressed in the pituitary. Major Ucn 3 terminal fields include the lateral septum and the ventromedial hypothalamus, which are known to express high levels of CRF2 (Li et al., 2002). In brief, the central distribution of Ucn 1, Ucn 2, and CRF2 expressing neurons suggests that Ucn 1 may serve as the major CRF2 ligand in the hindbrain whereas Ucn 3 may serve as the major CRF2 ligand in the forebrain. Ucn 2 or a novel endogenous ligand may signal at CRF2 expressed in brain regions lacking Ucn 1 or Ucn 3 innervation, e.g. the hippocampus (Hauger et al., 2003).

6. CRF receptors and CRF binding protein Specific, high-affinity, G protein-coupled seven-transmembrane membrane receptors mediate the biological actions of CRF, Ucn 1, Ucn 2 and Ucn 3. To date, two distinct mammalian receptor subtypes have been characterized: CRF1 and CRF2 display a markedly different tissue distribution and pharmacological specificity (Chalmers et al., 1995; Steckler and Holsboer, 1999). In general, CRF1 has been proposed to mediate the effects of CRF on HPA system function and anxiety-related behavior (Liebsch et al., 1999; Liebsch et al., 1995; Skutella et al., 1998),

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whereas CRF2 might be predominantly involved in the regulation of feeding behavior (Spina et al., 1996), cardiovascular function and the recovery phase of the HPA response (Coste et al., 2000). A complex role for CRF2 in modulating anxiety-related behavior, however, is also very likely (see below). Highest densities of CRF1 mRNA have been described in the anterior pituitary, cerebral cortex, cerebellum, amygdala, hippocampus and olfactory bulb (Chalmers et al., 1995; Sanchez et al., 1999). Although there is some controversy, in non-human primates CRF1 mRNA could also be localized in the locus coeruleus (Sanchez et al., 1999). Basal expression of CRF1 mRNA within the PVN is very low. However, in the rat, but not in the mouse, PVN CRF1 mRNA could be induced via stress exposure or intraPVN CRF microinfusion (Imaki et al., 2003, 2001; Konishi et al., 2003). In the periphery, low levels of CRF1 mRNA occur in the testis, ovary, retina and adrenal gland. The CRF2 receptor family has additional diversity in that two isoforms have been described: CRF 2(a) and CRF2(b) (Chalmers et al., 1995; Lovenberg et al., 1995a). The CRF2(a) receptor is expressed primarily in rat subcortical neuronal populations (lateral septum, amygdala, hippocampus, parvoventricular nucleus of the hypothalamus), whereas the CRF2(b) isoform is expressed in non-neuronal cells in the central nervous system (e.g. cerebral arterioles and choroid plexus). Peripherally, CRF2(b) mRNA is found in cardiac myocytes, lung, ovary and skeletal muscle (Chalmers et al., 1995; Lovenberg et al., 1995a). Taken together, with respect to species-specific differences, it is of importance that in the monkey brain both CRF1 and CRF2 were found in the pituitary and throughout the neocortex (i.e. in prefrontal, cingulate, striate, and insular cortices), amygdala, and hippocampal formation (Sanchez et al., 1999). In the rat and mouse brain only the CRF1 is found in the anterior pituitary and the CRF2 is present only at low expression levels in the neocortex. These results suggest that, in primates, both CRF1 and CRF2 may be involved in mediating the effects of CRF on cognition, behavior, and HPA system function. The presence of CRF1 (but not CRF2) within the amygdala, cerebellar cortex, nucleus of the solitary tract, thalamus, and striatum and of CRF2 (but not CRF1) receptors in the choroid plexus, certain hypothalamic nuclei, the nucleus prepositus, and the nucleus of the stria terminalis suggests that each receptor subtype also may have distinct functional roles within the primate central nervous system. In humans only, a third functional splice variant, CRF2(c), has been identified which is expressed in selected brain areas, such as the septum and hippocampus and at lower levels in the amygdala, nucleus accumbens, midbrain and frontal cortex (Kostich et al., 1998). Because neither CRF1 nor CRF2 have yet been conclusively identified in some important stress-sensitive brain structures such as the locus coeruleus and the central

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nucleus of the amygdala it is possible that a novel CRF receptor may actually be cloned (Hauger et al., 2003). Another important regulator of HPA system function is the CRF-binding protein (CRF-BP), a 37 kD secreted glycoprotein that is found in the brain, pituitary areas, placenta and related tissues and adrenals (Seasholtz et al., 2001). It binds CRF and other CRF-related peptides with considerably high affinity and constrains the biological activity of CRF and Ucn 1 (Kemp et al., 1998). Although evidence so far is equivocal, neither Ucn 2 nor Ucn 3 bind to human CRF-BP, whereas CRF and Ucn 1 do (Lewis et al., 2001a). Mouse Ucn 2, but not mouse Ucn 3, however, binds with high affinity to the rat CRF-BP and such affinity had previously not been observed for the human version of this protein (Jahn et al., 2004). Binding of CRF/Ucn 1 by CRF-BP results in CRF/Ucn 1 inactivation and decreased ACTH release in vitro (Cortright et al., 1995). Besides pituitary corticotropes, the CRF-BP is predominantly expressed in the cerebral cortex, subcortical limbic structures, the raphe´ nuclei, and several brainstem nuclei (Potter et al., 1992). CRF-BP is present in both neurons and astrocytes, and its expression overlaps with CRF and CRF receptor expression in several areas. These areas of co-expression suggest that the CRF-BP exerts a modulatory effect on CRF-CRF receptor-interaction, which is corroborated by 40–60% of CRF in the brain being bound to the CRF-BP (Behan et al., 1995).

7. The role of CRF receptors in anxiety Numerous investigations in animals have described anxiogenic-like effects after central CRF administration (Dunn and Berridge, 1990). These effects are likely to be mediated through the CRF1 receptor, as CRF1 antagonistic approaches have anxiolytic-like properties in most, but not all anxiety paradigms (Griebel et al., 1998; Keck et al., 2001; Liebsch et al., 1995). The effectiveness of CRF1 blockade to reduce anxiety is likely to depend on the animal’s stress level as it has been shown that CRF1 antagonists acted anxiolytic-like after stress exposure, but not under basal conditions in ‘normal’ rats (e.g. Griebel et al., 1998). However, in selectively bred rats with innate hyperanxiety and a hyperactive HPA system, administration of a CRF1 antagonist exerted anxiolytic properties under basal conditions (Keck et al., 2001). On the other hand, studies investigating the effects of CRF-BP inhibitors, which will increase CRF activity specifically in cortical areas including the hippocampus, failed to find significant increases in anxiety-related behavior (Behan et al., 1995; Heinrichs and Joppa, 2001), suggesting that CRF mediates anxiety at a subcortical level, e.g. within the lateral septum, the central amygdala and periaqueductal gray. Beyond CRF1, recent pharmacological data point towards a complex involvement of the CRF2 in anxiety and stress-related behaviors and there is a controversy in

the current literature about it’s precise functional role. Central administration of Ucn 1, an endogenous ligand for CRF2, has been shown to induce a variety of effects, including behavioral consequences such as increased anxiety (Moreau et al., 1997; Slawecki et al., 1999). However, as Ucn 1 can bind and activate both CRF receptor subtypes, i.c.v. administered Ucn 1 might activate receptors non-selectively in areas where endogenous Ucn 1 may not exist. Interestingly, activation of the CRF2 can result in either anxiolysis or anxiogenesis depending on when the animal is tested and, possibly, where the receptor is localized (Reul and Holsboer, 2002; Takahashi, 2001). Acute antagonism of CRF2(a) in the rat lateral septum, which abundantly expresses CRF2(a) but not CRF1, produced a behaviorally, anatomically and pharmacologically specific reduction in stress-induced defensive behavior as measured by shock-induced freezing (Bakshi et al., 2002). In the same study, the highly selective CRF1 antagonist NBI 27914 reduced freezing after infusion into the central amygdala but failed to affect freezing when infused into the lateral septum (Bakshi et al., 2002). Similarly, an antisense oligonucleotide directed against the CRF2 mRNA reduced expression of CRF2 in the rat lateral septum by 60–80% and resulted in a reduction in shock-induced freezing duration (Ho et al., 2001). CRF2 activation in the lateral septum, in turn, increased anxiety-like behavior after 30 min, which could be prevented by pretreatment with the CRF2-selective antagonist antisauvagine-30 (Radulovic et al., 1999a). In contrast, i.c.v. administration of the selective CRF2 agonist Ucn 2 had no short-term effects but after four hours resulted in reduced anxiety-related behavior (Valdez et al., 2002). Thus, CRF2 in the brain is capable of reducing anxiety in a delayed fashion. It has to be considered, however, that this delay might be also due to the pharmacokinetics of Ucn 2 with the protein taking a long time to reach target sites that are far from the lateral ventricles in which it is injected. The anxiogenic and anxiolytic properties of CRF2 are certainly not paradoxical, because they operate in different time domains after stress. The role of CRF2 in anxiety is likely complicated, and the site of action appears to be critical. In a recent study it could be shown that CRF2 in the dorsal raphe´ nucleus, where serotonergic neurons innervating the hippocampus emerge, mediates the behavioral consequences, i.e. learned helplessness and increased anxiety, of uncontrollable stress (Hammack et al., 2003). Rats injected i.c.v. with murine Ucn 3 showed locomotor suppressive effects and decreases in anxiolytic-like behaviors following a more acute time course compared to Ucn 2 (Valdez et al., 2003). The underlying mechanisms for this difference may be the affinity of these neuropeptides for the CRF receptor subtypes. Although both are highly selective for the CRF2, Ucn 2 shows some ability to activate the CRF1 whereas murine Ucn 3 fails to activate the CRF1 at high doses (Lewis et al., 2001b). The functional effects of Ucn 3 at the CRF2, therefore, may occur unopposed by the concomitant activation of CRF1 (Lewis et al., 2001b).

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Combining molecular genetics with behavioral pharmacology, however, studies with antisense probes that selectively reduce CRF receptor subtype levels and transgenic mouse models have indicated that CRF1 might be the primary target of interest at which selective compounds should be directed to treat pathological anxiety (Keck and Holsboer, 2001; Liebsch et al., 1995, 1998; Skutella et al., 1998; Timpl et al., 1998). It has to be kept in mind, however, that, in addition to CRF1 hyperfunction, CRF2 hypofunction might play an important role in both causality and treatment of mood and anxiety disorders and that an impaired CRF2-mediated ‘anxiolysis’ might result in an extended state of anxiety and arousal. A pathological imbalance between CRF1- and CRF2-mediated ‘anxiogenic’ versus ‘anxiolytic’ effects may further result from differences in the normally rapidly occurring receptor desensitization, internalization and downregulation during continued agonist presence as has been hypothesized to be the case in mood and anxiety disorders (Holsboer, 2003b; Keck and Holsboer, 2001; Nemeroff, 1996). There is also evidence, however, that CRF1 and CRF2 play similar or parallel roles in the regulation of stress-related behavior, and that this regulation may occur through different brain regions. It has also been demonstrated that subchronic exposure to benzodiazepines decreases CRF1 levels but increases CRF2 levels in rat brain, again indicating that CRF1 and CRF2 may act in an opposing manner (Skelton et al., 2000). It may, therefore, be that the CRF receptor subtypes are differentially affected by long-term drug treatment but that their roles in mediating acute stress-induced behavioral effects are rather similar. This may ultimately lead to the hypothetical conclusion that depending on whether or not a stress-related pathology is considered either chronic or acute nonpeptide CRF2(a) agonists or antagonists, respectively, may prove clinically useful.

8. Genetically engineered mice to elucidate CRF neurocircuitry-related psychopathology During the past decade, genetically-engineered laboratory animals started to play an increasingly important role as research tools in psychiatry. After they have proven to be indispensable tools for the advancement of most medical disciplines, they may ultimately help to increase our knowledge about the neurobiology of psychiatric disorders. Ideally, an animal model should homologously mimic the human condition of interest with respect to its etiology, symptomatology and treatment (Geyer and Markou, 1995). In the context of complex psychiatric disorders, meeting such requirements is infeasible and clearly out of reach. This is particularly true for affective disorders such as depression, where the presence of some of the cardinal features (e.g. feelings of worthlessness and guilt and suicidal ideation) is defined by a subjective verbal report, something that will never be modeled in an animal.

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The presence of other features of depression can be defined operationally (e.g. loss of appetite and weight, sleep disturbances, reproductive behavior, and psychomotor changes). However, a model that is limited to a decrease in appetite and psychomotor activity, for example, would be a very superficial reflection of the clinical condition of depression (Sillaber and Holsboer, 2004). Pathologies having homology with those in humans can be induced in animals more readily if the etiology of the disease is known. Unfortunately, this is not the case with psychiatric diseases. Instead of developing a perfectly homologous model, more pragmatic approaches are now being pursued, in which animal models are developed for distinct purposes: (1) as behavioral tests to screen for potential antidepressant effects of new pharmaceutical drugs and (2) as tools to investigate specific pathogenetic aspects of cardinal symptoms of depression. In this context it is important to note that the technical term ‘animal model’ is used for both animals that display certain behavioral alterations, and for test assays conceptualized to assess these changes in behavior such as, e.g. tests for anxiety or stress coping-behavior like the elevated plus-maze or the forced swim test, respectively (Ohl, 2003). The traditional routes to animal models of depression have recently been expanded by the possibility of studying mice that show behavioral changes, which are not primarily experience-related, but rather are secondary to the insertion of a transgene or to a targeted disruption of a single gene. Importantly, genetic changes always manifest in an environment but by use of this approach the gene and its product under investigation are being brought under experimental control. Complementary to the ‘gene-driven’ analysis of gene function, where the function of a distinct gene is manipulated according to a certain hypothesis (Mu¨ller and Keck, 2002), hypotheses-free ‘phenotypedriven’ approaches such as ethyl-nitrosurea (ENU)-mutagenesis can be performed (Ohl and Keck, 2003). Whatever the route to an animal model, important key requirements for such a model are reliability, predictive, face and construct validity (Geyer and Markou, 1995). The more criteria an animal model satisfies, the greater its utility and relevance to the human condition. Reliability refers to the consistency and stability of the observed variable. Ideally, an animal model for a human clinical condition should display certain symptoms characteristic for a disease (face validity), it should respond with reduced symptoms when treated with a clinically efficacious drug (predictive validity), and, finally, the neurobiological mechanisms underlying symptomatology as well as the psychological causes should be identical (construct validity). Clearly, meeting such requirements is difficult if not impossible for an animal model of depression. A pragmatic approach is to identify those behaviors that are frequently disturbed in the clinical condition and that can be studied confidently in animals (e.g. Willner et al., 1992). Such behaviors include anxiety-related behavior and stress coping strategies.

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9. The use of rodents to investigate depression Although early attempts at model development focused on reproducing in animals a psychiatric syndrome in its entirety, it is now very unlikely that we will ever be able to diagnose a rodent according to the algorithms given by ICD-10 or DSMIV. So far, classic diagnostic algorithms have not been helpful for neurobiological approaches to causality of depression and other affective syndromes (Holsboer, 2004; Hyman, 2000). For studies of causality such common categorical diagnostic classification systems are of very limited value as the subsamples they create are inhomogeneous according to pathogenesis, which in almost all psychiatric disorders depends on multiple genes interacting to confer risk and interrelating with nongenetic environmental factors (Holsboer, 2003a). Each diagnostic entity is rather to be seen as a segment on a continuum without a clear-cut separation by natural boundaries. Moreover, the defining symptoms of psychiatric disorders and even the diagnostic categories themselves are being revised and redefined continuously. The validity of such psychiatric syndromes for research purposes, therefore, is questionable when studies of causality are concerned. Instead, it has been increasingly recognized that beyond the categorical classifications such as ‘major depression’ or ‘generalized anxiety disorder’ today’s task is to define functional phenotypes or endophenotypes, which may be available, for example, by neuroendocrine testing, neuroimaging or neuropsychology (Gottesman and Gould, 2003; Holsboer, 2001a). It is of note that we do not need to have a diagnostic entity such as major depression to construct an endophenotype that is valuable for basic research. On the contrary, in order to learn more about the underlying neurobiological mechanisms, it is even more suitable to investigate alterations in specific behaviors regardless of whether or not they are changed in the same direction (e.g. decreased vs. increased anxiety-related behavior) as in the psychiatric disease of interest. In light of the most recently recognized striking similarities between men and rodents in their genetic endowment, the use of animals that model certain aspects of depression and fulfill the requirement of inherited psychopathology and responsiveness to antidepressant treatment is legitimate. The mouse is an adequate model organism to investigate the neurobiological and genetic basis of psychiatric disorders as approximately 99% of its genes have homologues in humans (Seong et al., 2002; Tecott, 2003). Moreover, mice and men share common neuroanatomical key features such as hippocampus, amygdala and cortex.

10. Genetically engineered mice-the ‘genotype-driven’ approach An important criterion for developing animal models to study psychopathology involves establishing the validity of

the model as a true representation of the process being studied (Geyer and Markou, 2002). Despite the availability of a long list of approximately 4500 mouse mutants created using genetic technologies (www.informatics.jax.org; Mouse Genome Informatics coordinated by the Jackson Laboratory) there remains an appreciable lack of behavioral phenotypes in mouse models enabling us to learn more about the etiology of human psychiatric disorders. In human depression, the most common symptoms associated with depressive symptoms are anxiety and cognitive disturbances. Thus, recognizing that depression is composed of many different symptoms, which vary from individual to individual, and further recognizing that depression is a multigenetic disease, it makes sense to study the effects of single genes on specific behaviors only if the animal generated is not viewed as offering anything but modeling of changes in behaviors that are frequently altered in depression. In search of mouse mutants modeling certain aspects of psychiatric disorders we have to face the problem that the clear minority of such disorders are caused by single gene defects. Psychiatric disorders are almost always nonmendelian, heterogeneous, implicate multiple genetic loci and are influenced significantly by environmental factors. Most of the common psychiatric disorders are characterized by a combination of symptoms, which can be separated into so called endophenotypes. The term endophenotype refers to a set of behavioral and/or physiological characteristics that accompany a basic process that is altered in relation to the illness that is being studied (Freedman et al., 1999; Gottesman and Gould, 2003). At the behavioral level, for example, depression is associated with anxiety. Although it is unrealistic to screen for mouse models that display the whole complexity of behavioral alterations characterizing a psychiatric disorder, specific behavioral traits resembling symptoms or endophenotypes of these disorders, can readily be modeled in mice (Holsboer, 1997; Mu¨ller and Keck, 2002; Piazza and Le Moal, 1998; Seong et al., 2002). It is, therefore, important to note that this more narrowly defined endophenotype approach does not necessarily have to capture specific symptoms that are a part of the clinical diagnosis, but rather may focus on a core and basic process or function that is abnormal in the clinical population under study and that is thought to be related to the manifestation of the illness.

11. Is it reasonable to test for anxiety when investigating for depression? Pathological anxiety is a frequent concomitant of major depression. Anxiety disorders display a substantial lifetime and episode comorbidity between each other and between other psychiatric conditions, particularly mood disorders (Hettema et al., 2001). Although anxiety disorders and depression have been classified as separate types of disorders for decades, there is a longstanding debate about

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whether anxiety and depression constitute different aspects of the same disorder (‘continuum hypothesis’) or distinct, yet overlapping, conditions (Kendler, 2001; Nemeroff, 2002; Persons et al., 2003). The absence of a clear therapeutic demarcation is also of considerable interest: A variety of classic antidepressants such as, e.g. serotonin reuptake inhibitors and tricyclics, have been successfully used for many years to treat anxiety disorders. Animal models conceptualized to elucidate mechanisms underlying depression, therefore, often show altered anxiety-related behavior. With respect to CRF neuronal circuits, the possibility exists, however, that CRF might be mostly related to the anxiety symptomatology seen in many depressed patients. This could explain the wide variability observed in depressed patients in terms of HPA system function, with some patients having anxiety symptoms more than others (Holsboer, 1999a,b, 2000b, 2003a). Anxiety-related behavior is one of the most important, highly conserved behaviors among all kind of species, which can be reliably measured in specific experimental paradigms in rodents (Millan, 2003; Ohl, 2003). In interaction with cognitive parameters anxiety regulates many aspects of behavior in humans and animals. The assessment of anxiety-related behavior in animal models is based on the assumption that anxiety in animals is comparable to anxiety in humans. As a matter of fact, however, it cannot be proven that rodents, the prime species in basic research, experience anxiety in the same way as human beings. In any case, it is relatively clear that distinct behavioral and physiological patterns in rodents indicate anxiety, i.e. behavioral and peripheral changes presumed to accompany high sympathetic nervous activity and activation of the HPA system. From this, an analogy, if not a homology, between anxiety in humans and rodents may be assumed. When testing for anxiety, it should be taken into account that anxiety is not a unitary phenomenon as it includes innate (trait) anxiety, which is considered to be an enduring feature of an individual, and situation-evoked or experiencerelated (state) anxiety. Since tests for anxiety in rodents are always restricted to the evaluation of situation-evoked behavior, it might be difficult to investigate trait anxiety in animals. However, both phenomena are not separable from each other, as individuals with a high trait-anxiety often will show an increased tendency to also display high state anxiety. Thus, the term ‘anxiety’ will be used without an a priori assumption of trait or state anxiety. It is also important to keep in mind that modeling anxiety in animals is critically dependent on the test systems used. Various test paradigms, often termed ‘animal models of anxiety’, have been developed to assess behavioral parameters indicating anxiety. Among the most frequently used paradigms the test apparatus represents unconditioned avoidance tests, where test-naive animals are exposed to a novel environment. This novel environment (e.g. an elevated plus- maze, a dark/light box, an open field) consists of an

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‘aversive’ and a ‘non-aversive’ compartment, i.e. open versus closed arms or brightly lit versus dark compartments. Index of the level of anxiety is the approach behavior of the animal towards the aversive compartment. Overall, those tests have been shown to be very useful in studying changes of unconditioned anxiety-related behavior, either induced by psychoactive compounds or by psychological or genetic manipulations (for review e.g. (Belzung and Griebel, 2001; Hascoet et al., 2001; Hogg, 1996; Menard and Treit, 2001). These and also other test paradigms, not explicitly mentioned here, have been extremely useful as initial screens for drugs potentially affecting aspects of anxiety. They are also valuable tools in determining the implication of genetic factors in the whole complexity of behavior, and specifically the profile of anxiety-related behavior. As standard behavioral test assays for anxiety were validated by classical anxiolytic drugs such as benzodiazepines they might be, however, of limited use to discover novel treatment strategies (Ohl, 2003).

12. Gene targeting methods: short overview 12.1. ‘Conventional’ transgenic mice ‘Gene targeting’ refers to homologous recombination between a specifically designed targeting construct and the chromosomal target of interest. Homologous recombination in embryonic stem cells (ES cells) is now a routine technique that is used to modify the mouse genome at a chosen locus. Targeting constructs are most commonly used to disrupt a target by inserting a heterologous sequence and/or making a small deletion. More specialized design and use of the targeting constructs can allow a whole range of pre-defined chromosomal alterations to be made, ranging from single base-pair changes to megabase-pair deletions, truncations or translocations. Conventional gene disruption (conventional ‘knock-out’) utilizes homologous recombination of a target DNA and the chromosomal gene of totipotent mouse embryonic stem (ES) cells (Capecchi, 1989). Genetically altered ES-cells are reintroduced into a developing blastocyst and contribute to the developing embryo. When the germ cells of the resulting chimerical mouse are ES-cell derived (germ-line mutation), breeding can be used to generate mice that are heterozygous and homozygous for the desired mutation. Homozygous mutants do not express the gene of interest in any cell of the body. Transgenic mice are generated by injection of DNA into a zygote pronucleus of a fertilized egg and the foreign DNA integrates into the genome, leading to an animal that overexpresses a certain gene product (Jaenisch, 1988). Transgenic animals produced by this method are generally gain-of-function mutants since the transgene is designed either to express a novel gene product or to misexpress a normal gene. The transgene can be brought under the control of a promoter, which allows more specific

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expression in those regions where the promotor is normally expressed. Alternatively, it is possible to clone a DNA fragment in an opposite direction and hence to produce transgenic animals expressing antisense RNA, which will result in disrupted posttranslational processes and therefore reduced gene function (Munir et al., 1990). A major disadvantage of this method is the unpredicted localization where the transgene is inserted into the mouse genome. Thus, all lines generated need to be analyzed separately, as the expression activity of the transgene is determined by the neighboring sequences. 12.2. Conditional gene-silencing methods (‘conditional knock-out’) Mouse geneticists have searched for a long time for experimental ‘switches’ that allow genes to be activated or silenced in specific tissues or at selected time points, or both. A variety of methods can be envisaged for achieving such conditional gene inactivation (Morozov et al., 2003). Provided they are specific, inhibitory molecules (e.g. antibodies or antisense oligonucleotides) that are introduced directly into cells can be used for assessing essential gene function. Stable gene-silencing methods, in contrast, make use of regulatory systems that allow the target gene to be controlled by an inducer molecule, either added to the culture medium or to the mouse (‘inducible knock-out’). The tetracycline-controlled transcriptional transactivator (tTA) system, for example makes use of time-sensitive regulation of gene expression, allowing to induce or inhibit gene expression after tetracycline treatment only (Gossen et al., 1995). Exposure to tetracycline in the drinking water at any time during ontogeny deactivates the transgene under study in a matter of days (Mayford et al., 1997). Equally important for the studies of gene function in the mouse is the use of tissuespecific regulatory systems that allow gene silencing to be restricted to specific tissues and, in some cases, to specific times during development. More recently, strategies exploiting site-specific DNA recombination have been incorporated into transgenic and gene-targeting procedures to allow in vivo manipulation of DNA in embryonic stem cells or living animals. In the most widely adopted approach, this is achieved by use of the Cre/lox system (Ku¨hn et al., 1995, for review: Sauer, 1998)). The gene encoding the site-specific bateriophage recombinase Cre is introduced as a regulated transgene, while gene targeting is used to ‘flox’ the target gene, that is, to flank a key region of the target gene with loxP sites, the 34 bp recognition sequence for Cre. The target gene remains expressed until the cre gene is induced; Cre then catalyses site-specific recombination between the loxP sites, deleting part of the target gene and thereby silencing it. Placement of recombination sites into the genome and subsequent targeted expression of recombinase have allowed the development of genetic switches that can either ablate or

turn on any desired gene in transgenic or gene-modified mice. This is achieved by breeding responder mice, carrying the floxed target gene (e.g. CRF1), with regulator mice that bear the regulated Cre transgene under the control of a tissue-specific promotor (e.g. calcium calmodulindependent kinase-Cre) (Fig. 2). Tissue-specific deletion then leads to offspring in which the absence of CRF1 is limited to the forebrain and limbic system (Mu¨ller et al., 2003). A particularly powerful feature of the conditional gene inactivation strategy using Cre is that the same loxPtagged mouse can be used for gene ablation independently in a large number of different tissues, or at different developmental times, by simply mating it with a corresponding Cre transgenic that displays the desired tissue or temporal specificity of expression. Thus, the same genetically modified animal can be used to answer a variety of different questions relating to the expression and function of the target gene.

13. Highlights from mutant mice 13.1. CRF overexpression Transgenic mice overexpressing CRF (genetic background: C57/B6!SJL) exhibit prominent endocrine abnormalities involving the HPA system, such as high plasma levels of ACTH and corticosterone, and display physical changes similar to the stigmata seen in patients with Cushing’s syndrome, such as excess fat accumulation, muscle atrophy, thin skin, and hair loss (Stenzel-Poore et al., 1992). Behavioral analysis revealed decreased exploratory behavior and increased anxiety-related behavior when transgenic mice were tested on a 16-hole board task, in a dark/light box or on the elevated plus-maze (Heinrichs et al., 1997; Van Gaalen et al., 2002). In the latter paradigm, increased anxiety-related behavior in transgenic animals could be reversed by administration of the non-selective CRF antagonist alpha-helical CRF (Stenzel-Poore et al., 1994). Interestingly, adrenalectomy did not attenuate the anxiogenic effect of CRF overproduction, although it normalized plasma corticosterone levels in these animals (Heinrichs et al., 1997), suggesting that the behavioral effects of CRF overexpression are centrally- and CRF receptor-mediated rather than being an effect of enhanced GR activation due to increased corticosterone levels. CRF overexpressing transgenic mice have been reported to be impaired in learning forced-choice alternation and water maze place navigation tasks. Interestingly, however, the place navigation deficit seen in transgenic mice was attenuated by administration of the potent anxiolytic benzodiazepine chlordiazepoxide. This in turn suggests that the navigation deficit seen in transgenic animals was possibly confounded by heightened anxiety or overarousal (Heinrichs et al., 1996).

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Fig. 2. Generation of conditional knockout mice using the Cre/loxP system (A). The gene encoding the site-specific bacteriophage recombinase Cre is introduced as a regulated transgene, while gene targeting is used to ‘flox’ the target gene, that is, to flank a key region of the target gene with loxP sites, the 34 bp recognition sequence for Cre. The target gene remains expressed until the cre gene is induced; Cre then catalyses site-specific recombination between the loxP sites, deleting part of the target gene and thereby silencing it. Placement of recombination sites into the genome and subsequent targeted expression of recombinase have allowed the development of genetic switches that can either ablate or turn on every desired gene in transgenic or gene-modified mice. This is achieved by breeding responder mice, carrying the floxed target gene (e.g. Crf1), with regulator mice that bear the regulated Cre transgene under the control of a tissuespecific promotor (e.g. calcium calmodulin-dependent kinase IIa, CaMKIIa). Tissue-specific deletion then leads to offspring in which the absence of Crf1 is limited to the forebrain and limbic system. B.-E. Spatial pattern of Cre recombinase activity in the Crf1loxP=loxP CaMKIIaCre mouse line and verification of the region-specific CRF1 knockout. (B)–(D) Cre immunohistochemistry in the CaMKIIaCre transgenic line reveals strong expression of Cre recombinase in hippocampal pyramidal neurons (insert: CA1 pyramidal cells) and the granule neurons of the dentate gyrus. (C) Strong Cre-like immunoreactivity was also observed in the cortex (I–VI indicate the cortical layers) and the amygdaloid nuclei (D) BLAZ basolateral nucleus of the amygdala; cAZcentral amygdaloid nucleus, PirZpiriform cortex). This pattern of CaMKIIa-driven Cre expression matches very well the murine expression of functional Crf1 receptors in neuronal circuitries which play a major role in anxiety-related behavior. (E) and (F): Verification of conditional inactivation of CRF1 by in situ hybridization. Crf1loxP=loxP animals show the normal widespread expression pattern of CRF1 in the central nervous system (E), whereas in Crf1loxP=loxP CaMKIIaCre conditional mutants (F), CRF1 expression is selectively inactivated in the anterior forebrain, including the hippocampal formation

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Transgenic mice selectively overexpressing rat CRF in the brain (mainly PVN, central nucleus of the amygdala and BNST) under the control of the Thy-1 promotor on a C57BL/6J background (CRF-OE2122) showed increased body temperature and heart rate during the inactive light phase together with a decreased heart rate variability during the active dark phase herewith displaying chronic stress-like autonomic and physiological alterations (Dirks et al., 2002a, b). Moreover, basal plasma corticosterone, but not ACTH, concentrations were found to be increased. HPA system responsiveness to acute stress showed no differences between mutant and wildtype mice (Groenink et al., 2002). Interestingly, despite elevated plasma corticosterone concentrations, adrenal gland hypertrophy and reduced corticosterone suppression by the synthetic corticosteroid dexamethasone, CRF-OE2122 mice have no Cushing-like phenotype. Ucn 1 expression in the Edinger-Westphal nucleus, the most dominant site of Ucn 1-immunoreactivity, was reported to be down-regulated in these animals (Kozicz et al., 2004). At the behavioral level, reduced acoustic startle activity and habituation, increased motor activity during startle testing and impairments of prepulse inhibition were reported (Dirks et al., 2002). These behavioral alterations resemble sensorimotor gating deficits commonly associated with schizophrenia. Accordingly, these startle gating deficits could be improved by treatment with various antipsychotics (Dirks et al., 2003). The answer to the intriguing question of whether or not these mice display alterations in anxiety-related behavior or stress coping, unfortunately, has not been reported yet. Taken together, considering the similarities in HPA system dysregulation observed in CRF-OE2122 mice and patients suffering from major depression, these mutants may be suitable as animal model for the HPA system changes associated with major depression. The fact that sensorimotor gating deficits are present in these animals might render them useful to model certain aspects of psychotic depression, a disease state which is associated with a particularly dysregulated HPA system (Lindley et al., 1999).

14. CRF knockout To further evaluate the role of CRF in both neuroendocrine and behavioral functions, a mammalian model of CRF deficiency has been generated by targeted mutation in embryonic stem cells (Muglia et al., 1995). CRF deficient mice reveal a fetal glucocorticoid requirement for lung maturation. Postnatally, they display marked glucocorticoid deficiency and an impaired endocrine response to stress (Jacobson et al., 2000; Muglia et al., 1995). 3 and the amygdaloid nuclei (sagittal and coronal sections). CRF1 expression in Crf1loxP=loxP CaMKIIaCre conditional mutants is not affected in the cerebellum where CaMKIIa is absent.

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Surprisingly, no gross behavioral abnormalities have been reported with these animals. Anxiety-related behavior is comparable between mutants and wild-type animals, regardless as to whether it is measured under basal conditions or after stress exposure (Dunn and Swiergiel, 1999; Weninger et al., 1999), despite the fact that glucocorticoid levels are greatly reduced, what might have been also expected to influence anxiety-related and cognitive behavior (Korte, 2001a). Interestingly, both the non-selective CRF antagonist alpha-helical CRF and the CRF1 antagonist CP-154,526 showed comparable anxiolytic activities in mutant and wild-type mice when tested in fear conditioning (Weninger et al., 1999). This in turn suggests that blockade of CRF1 is anxiolytic, but that another CRF-related peptide, a yet unidentified CRF receptor ligand or AVP could have compensated for CRF deficiency in these animals. Conversely to CRF overexpressing mice (see above), Ucn 1 mRNA was found to be up-regulated in the EdingerWestphal nucleus of CRF-deficient mice (Weninger et al., 2000). These findings support the notion that CRF and Ucn 1 neuronal systems represent two interrelated entities that inversely- regulate adaptation to chronically altered concentrations of CRF.

15. Urocortin 1 (Ucn 1) knockout: conflicting behavioral data Mice carrying a null mutation of the Ucn 1 gene were independently generated in 2002 by two groups (Vetter et al., 2002; Wang et al., 2002). As with mice deficient for the CRF2 (see below) behavioral data are not unequivocal: by use of various testing paradigms Wang et al. described an unchanged anxiety-related behavior (Wang et al., 2002) whereas Vetter and colleagues showed heightened anxietylike behaviors in Ucn 1-deficient animals (Vetter et al., 2002). HPA system regulation was characterized in response to restraint stress and was found to be normal in either line (Vetter et al., 2002; Wang et al., 2002). These findings support the view that endogenous Ucn 1 is not involved in regulation of the HPA system in response to acute stress, or has a minor or redundant role in such responses. A discrepancy observed between Ucn 1-null mice and studies where Ucn 1 was administered centrally concerns the role of Ucn 1 in anxiety. Central administration of Ucn 1 in rats can elicit anxiety-like behavior (Moreau et al., 1997; Slawecki et al., 1999), and it has been hypothesized that this could be due to activation of CRF1. The discrepancy between the pharmacological studies and genetically engineered mice may be explained by considering that centrally administered Ucn 1 might non-selectively activate several receptor systems in the brain, resulting in the described anxiety-like behavior. Another explanation could be the fact that Ucn 1 knockouts have a reduction in levels of CRF2 mRNA in the lateral septum (Vetter et al.,

2002). Anatomical studies show that abundant Ucn 1-expressing fibers originating from Ucn 1-immunoreactive fibers in the Edinger-Westphal nucleus terminate in the lateral septum (Bittencourt et al., 1999). It has been demonstrated that a significant increase of Ucn 1 mRNA in the Edinger-Westphal nucleus and of CRF2 mRNA in the lateral septum occurs in rats treated chronically with benzodiazepine anxiolytics (Skelton et al., 2000). Therefore, these data derived from Ucn 1-null mutants and from pharmacological studies suggest that Ucn 1-immunoreactive neurons in the Edinger-Westphal nucleus may modulate anxiety in opposition to the actions of CRF itself, and possibly through CRF2 in the lateral septum.

16. CRF-binding protein 16.1. CRF-binding protein overexpressing mice Two different transgenic mouse mutants overexpressing CRF-BP have been independently generated (Burrows et al., 1998; Lovejoy et al., 1998). The mouse line by Lovejoy and colleagues shows increased CRF-BP level in brain and plasma. In contrast to wild-type mice, these animals also express CRF-BP ectopically in peripheral tissues including liver, kidney, heart, lung, adrenals and spleen (Lovejoy et al., 1998). Basal plasma ACTH and corticosterone levels in these mutants are indistinguishable from those of wild-type littermates, but a significantly lower ACTH secretion was seen in male but not female transgenic mice following HPA system challenge with lipopolysaccharide (LPS). In contrast, no difference in corticosterone levels could be detected following LPS administration (Lovejoy et al., 1998). It should be noted, however, that the expression profile for CRF-BP in these transgenic mice is very different from the physiological expression pattern in wildtype mice, which could easily lead to myriad unforeseen alterations. Unfortunately, behavioral data on this transgenic mouse line have not yet been reported. A second CRF-BP transgenic mouse line with overexpression of CRF-BP has been published by Burrows and colleagues (Burrows et al., 1998). These animals express CRF-BP under the control of the pituitary glycoprotein hormone a-subunit (a-GSU) promoter, which is thought to limit transgene expression to the developing anterior pituitary, although occasional expression was also detected in additional brain regions such as the lateral septum. Given that excess CRF-BP will bind more CRF at the level of the anterior pituitary, these transgenic animals should suffer from attenuation of CRF receptor activation on pituitary corticotropes, what might be expected to lead to decreased activity of the HPA system. However, these transgenic animals have normal plasma ACTH and corticosterone levels under basal conditions and following restraint stress. Hypothalamic CRF and AVP expression are increased in the PVN, most likely reflecting potential

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compensatory mechanisms to maintain HPA system activity (Burrows et al., 1998). Behavioral analyses revealed that the mice overexpressing CRF-BP exhibit increased locomotor activity in a novel environment (Burrows et al., 1998). In addition, a tendency towards decreased anxiety-related behavior was observed on the elevated plus maze, which would be in line with limited availability of free CRF due to enhanced binding by CRF-BP in these animals. 16.2. CRF-binding protein knockout To investigate directly the CRF-BP function, a mouse model of CRF-BP deficiency has been created by gene targeting (Karolyi et al., 1999). Under basal conditions as well as following stress exposure, HPA axis function is normal in these animals. However, increased anxiety-like behavior was observed in an open field, on the elevated plus maze and the dark-light test, consistent with the possibility that lack of CRF-BP would increase free CRF and Ucn 1, but this was not directly demonstrated. Moreover, hypothalamic CRF and AVP levels have not yet been examined in these mice. The data suggest, however, that the increased anxiety-like behavior in CRF overexpressing transgenic mice (Heinrichs et al., 1997; Stenzel-Poore et al., 1994) is likely due to central effects, as CRF-BP knockout mice showed altered anxiety-related behavior despite unaltered HPA axis activity.

17. Genetic targeting of CRF receptors 17.1. Conventional inactivation of CRF1 decreases anxietyrelated behavior Two mouse lines deficient for the CRF1 have been independently generated (Smith et al., 1998; Timpl et al., 1998; for a detailed review see Keck et al., 2004). Homozygous CRF1 mutants display a severe impairment of stress-induced HPA system activation and marked glucocorticoid deficiency. Despite the lack of functional CRF1 on pituitary corticotropes, basal plasma ACTH concentrations in homozygous CRF1 mutants are similar to those found in wildtype controls (Timpl et al., 1998), suggesting that basal ACTH secretion can be stimulated via signaling pathways other than CRF/CRF1. Since the discovery of CRF by Vale et al. (Vale et al., 1981a), it was rapidly established that vasopressin potently synergizes with CRF to stimulate pituitary ACTH release: when CRF and vasopressin are given together, hormone output is well above the added effects of the two peptides alone, both in rodents and in humans (Gillies et al., 1982; von Bardeleben et al., 1985). Indeed, the hypothalamic vasopressinergic system was shown to be significantly activated to maintain pituitary ACTH secretion in homozygous CRF1 mutants (Mu¨ller et al., 2000a): Basal plasma vasopressin

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concentrations were significantly elevated in homozygous CRF1 mutants. Vasopressin mRNA expression was increased in the paraventricular nucleus of CRF1 mutants, together with a marked increase in vasopressin-like immunoreactivity in the median eminence. Administration of a vasopressin V1-receptor antagonist significantly decreased basal plasma ACTH levels in mutant mice. Following continuous treatment with corticosterone, plasma vasopressin levels in homozygous CRF1-knockout mice were indistinguishable from those of wild-type littermates, thus providing evidence that glucocorticoid deficiency is the major driving force behind compensatory activation of the vasopressinergic system in CRF1 mutants. At the behavioral level, these mutants exhibit increased exploratory activity and significantly reduced anxietyrelated behavior under both basal conditions and following alcohol withdrawal (Timpl et al., 1998). It has also been reported that CRF1 knockout mice display altered spatial performance: using a Y-maze spontaneous non-matching to position paradigm based on novelty exploration, it was shown that wild-type mice, but not mutants, spent more time exploring the previously not explored arm of the maze (Contarino et al., 1999). This might indicate a spatial recognition memory deficit related to altered CRF neurotransmission. 17.2. Conditional inactivation of CRF1 in the limbic system reduces anxiety-related behavior with normal basal HPA activity Studies on conventional knockout mice deficient for CRF1 firmly established the requirement of pituitary CRF1 for endocrine responses to stress (Smith et al., 1998; Timpl et al., 1998): in CRF1 null mutants, both basal and stressinduced HPA system activity are markedly impaired (Mu¨ller et al., 2000b; Preil et al., 2001; Timpl et al., 1998). Moreover, conventional knock-out of CRF1 results in reduced anxiety-related behavior (Smith et al., 1998; Timpl et al., 1998). The behavioral analyses of CRF1 null mutants, however, are hampered by the fact that CRF1 knockout mice display severe glucocorticoid deficiency (Smith et al., 1998; Timpl et al., 1998). As glucocorticoids play important roles in modulating fear and anxiety-related behavior (Korte, 2001b; Korte et al., 1996), the anxiolytic effect observed in conventional CRF1 knockout mice may, therefore, result from either CRF1 deficiency itself or be influenced by a marked reduction in circulating glucocorticoid hormone levels in these animals. The lack of circulating glucocorticoids may also result in developmental deficiencies what could easily influence behavioral characteristics. To address this question and to dissect CRF/CRF1 central nervous system pathways modulating behavior from those regulating neuroendocrine function, a conditional CRF1 knockout using the Cre/loxP system (Lewandoski, 2001) driving Cre recombinase expression by a Calcium Calmodulin-kinase IIa (CaMKIIa) promotor

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(Minichiello et al., 1999) was generated recently (Mu¨ller et al., 2003). The CaMKIIa gene is expressed with tissuespecificity predominantly in the mouse anterior forebrain during postnatal development with high expression levels in hippocampal neurons (pyramidal and granule cell layer), cortical layers and the amygdala (Sola` et al., 1999). Selective disruption of CRF/CRF1 signaling pathways in behaviorally relevant limbic neuronal circuitries significantly reduces anxiety-related behavior (Mu¨ller et al., 2003). The robust anxiety-reduced phenotype of Crf1loxP=loxP CaMKIIaCre conditional mutants could be confirmed in two different behavioral paradigms based on the natural avoidance behavior of mice, the dark-light box paradigm and the elevated plus-maze test. In contrast to CRF1 null mutants, basal plasma ACTH and corticosterone levels are similar to wildtype levels in Crf1loxP=loxP CaMKIIaCre conditional mutants. The behavioral phenotype of conditional CRF1 mutants, therefore, is not likely to be influenced by central nervous system effects of circulating stress hormones. Plasma ACTH and corticosterone levels are virtually identical between wild-type mice and Crf1loxP=loxP CaMKIIa Cre conditional mutants under basal conditions and immediately following 2, 5 or 10 min of acute immobilization stress. However, hormone levels remain significantly elevated in Crf1loxP=loxP CaMKIIaCre conditional mutants 30 and 90 min. following a 5 min period of restraint stress. These data provide the first evidence that limbic CRF1 is required for central control of HPA system feedback and hormonal adaptation to stress. Taken together, the data from Crf1loxP=loxP CaMKIIaCre conditional mutants underline the importance of limbic CRF1 in modulating anxiety-related behavior. Furthermore, the findings underline the clinical assumption that central CRF/CRF1 neuropeptidergic circuits, other than those driving the peripherally accessible HPA system, may well be overactive and could be therapeutic targets (review: (Holsboer, 2003b)). 17.3. CRF2 knockout: increased anxiety-related behavior? Interestingly, significant differences in aspects of both the endocrine and behavioral phenotype were described between the three independently created knockout mouse lines deficient for the CRF2 (Bale et al., 2000; Coste et al., 2000; Kishimoto et al., 2000). This phenomenon points towards the fact that most likely the genetic background of genetically engineered mice plays a crucial role, especially when dealing with subtle behavioral alterations (Crusio, 2004; Lathe, 1996). Moreover, it is possible that environmental interactions in different laboratories and housing facilities are involved (Crabbe et al., 1999) and differences in compensatory gene expression are also likely to play a role (Bale et al., 2002). Not surprisingly, therefore, behavioral and endocrine analysis of CRF2 knockout mice has provided a less clear

picture as compared to CRF1 mutants and, consequently, the physiological role of CRF2 in mediating anxiety-like behavior has been the subject of controversy. Indeed, the behavioral performance reveals significant differences between the three independently created CRF2 deficient mouse lines: whereas Coste et al. (Coste et al., 2000) found no differences in anxiety-related behavior (genetic background: 129/SvJ!C57/B6J), Bale and co-workers (Bale et al., 2000) (genetic background: 129J!C57/B6) and Kishimoto et al. (Kishimoto et al., 2000) (genetic background: 129/Sv!C57/B6J) detected a significant increase in anxiety-like behavior in their CRF2 mutants. Interestingly, the latter behavioral phenotype could be observed only in male, but not in female CRF2 deficient mice. Recently, the CRF2 knockouts developed by Bale and coworkers were tested in the forced swim test where they displayed increased immobility as an indicator of depression-like behavior. When treated with the CRF1 antagonist antalarmin the time spent immobile was found to be decreased while swimming and climbing, i.e. active stress coping behaviors, increased (Bale and Vale, 2003). Although there were no controls to indicate whether antalarmin reduces depression-like behavior in CRF2 wildtype mice, the effectiveness of CRF1 antagonism might be explained by the previous finding that CRF2 deficient mice show increased CRF levels in the central nucleus of the amygdala and increased Ucn 1 levels in the Edinger Westphal nucleus (Bale et al., 2000). The specific interaction, however, between CRF2 deletion effects and CRF1 on depression-like behavior remains unproven. Locomotor behavior in response to novelty was unaltered in animals lacking CRF2, both under basal conditions and following intracerebroventricular (icv) Ucn 1 administration, suggesting that it is primarily the CRF1 through which CRF modulates motor function (Contarino et al., 2000; Coste et al., 2000; Kishimoto et al., 2000). Homozygous CRF2 mutants have elevated basal blood pressure, demonstrating that CRF2 participates in cardiovascular homeostasis (Coste et al., 2000). Basal food intake was unaltered in CRF2 knockout mice (Bale et al., 2000; Coste et al., 2000; Kishimoto et al., 2000), while food intake following food deprivation was reduced (Bale et al., 2000) and mutants recovered faster from the anorectic effects of icv Ucn 1 administration (Coste et al., 2000). These findings are in line with the purported role of CRF2(a) in modulating feeding behavior (Spina et al., 1996). With respect to HPA system regulation, in two of the three CRF2 deficient mouse lines ACTH and corticosterone release in response to stress was found to be augmented (Bale et al., 2000; Coste et al., 2000). Therefore, the picture emerges that CRF1 and CRF2 act in an antagonistic manner: CRF1 activates and CRF2 attenuates the release of ACTH and corticosterone. The plasma ACTH levels, however, decreased within 10 min of stress onset, earlier than in wild-type animals, a phenomenon which may reflect higher negative feedback inhibition. On the contrary, ten minutes

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after stress onset, the corticosterone levels still continued to rise in the mutants herewith suggesting that the adrenal cortex of the mutant mice may be hypersensitive to ACTH (Bale et al., 2000; Coste et al., 2000). Kishimoto and coworkers, in contrast, failed to detect any significant phenotype in basal and stress-induced HPA system regulation in their CRF2 knockout line. This is, however, most likely due to the fact that their endocrine analysis was limited in that they examined one time point after the exposure to stress only (Kishimoto et al., 2000). 17.4. Life without CRF receptors: CRF1/CRF2 double knockout mice Mice deficient for both CRF1 and CRF2 were generated to investigate the HPA system regulation in the absence of functionally active CRF receptors under basal conditions and in response to different ethologically relevant stressors (Bale et al., 2002; Preil et al., 2001; for a detailed review see Keck et al., 2004). Taken together, data derived from detailed neuroendocrine and behavioral characterizations of these animals point towards the fact that mammals might not necessarily need a functional CRF/CRF receptor system to live, rendering this a suitable system for drug targeting.

18. First steps towards therapy: CRF1 antagonists for the treatment of affective disorders The findings summarized above support the hypothesis that efficient strategies to characterize the deleterious effects of CRF hypersecretion in psychiatric diseases should include antagonism of CRF effects through CRF1 blockade. This may ultimately open a new lead in the treatment of stress-related disorders such as depression, anxiety, and sleep disorders. Among others (for a selection see Table 1; review: (Heinrichs and Koob, 2004; Saunders and Williams, 2003; Takahashi, 2001)), one compound recently examined is the high-affinity non-peptide CRF1 antagonist NBI 30775 (also termed R121919) developed by Neurocrine Biosciences (La Jolla, USA). This compound binds with high affinity to cloned human and rat CRF1 and inhibits CRF1-mediated signal transduction in transfected cells whilst binding to other receptors known to be present in the central nervous system is extremely low (Gutman et al., 2003; Keck et al., 2001, 2003). In preclinical studies with two selectively bred rat lines, in high anxiety-trait (HAB) animals NBI 30775/R121919 reduced anxiety-related behavior in a dose-dependent manner, whereas it had virtually no behavioral effect in low anxiety-trait (LAB) rats. At the highest dose tested, HAB rats reached the performance level of LAB animals (Keck et al., 2001, 2003). In contrast, the stress-induced activity of the HPA system, as determined by simultaneously measured plasma ACTH levels, was similarly blunted by NBI 30775/R121919 in both HAB and LAB

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animals. This finding further underlines the fact that the behavioral effects are mediated via cerebral CRF1 independently of the peripheral secretion of ACTH. The lack of effect of NBI 30775/R121919 in LAB rats is consistent with the hypothesis that neuropeptides are preferentially released at significant quantities under potentially pathogenic conditions (Ho¨kfelt et al., 2000). Neuropeptide receptors, therefore, are only targets in the presence of pathophysiological mechanisms (review: Ho¨kfelt et al., 2003). The fact that CRF decreases slow wave sleep activity in humans and rats (Holsboer et al., 1988), prompted to investigate the sleep EEG effects of NBI 30775/R121919 in HAB and LAB rats after stress exposure. It was found that HAB and LAB rats differed in their sleep-wake behavior and that the sleep EEG was more markedly affected by stress exposure in HAB rats. Following administration of NBI 30775/R121919, these differences disappeared. Particularly, the suppressive effect of stress-elicited CRF effects on slow wave sleep was avoided when NBI 30775/ R121919 was administered (Lancel et al., 2002). Based on the promising preclinical findings an openlabel trial (phase IIa) in patients suffering from major depression was conducted. Treatment with NBI 30775/R121919 led to a 50% reduction in anxiety and depression scores comparable to that obtained with the selective serotonin reuptake inhibitor paroxetine (Keck and Holsboer, 2001; Zobel et al., 2000). As part of this exploratory study, a thorough endocrine evaluation and detailed clinical laboratory analysis was assessed several times during 30 days of treatment with two different dose escalation regimens of NBI 30775/R121919 (Ku¨nzel et al., 2003). As the anterior pituitary is richly endowed with CRF1, antidepressant and anxiolytic effects were achieved at dosages that did not hamper the ACTH and cortisol response to CRF stimulation or blunted basal serum ACTH and cortisol concentrations (Ku¨nzel et al., 2003). Most importantly, no effects of NBI 30775/R121919 on clinical laboratory parameters including liver enzymes were monitored in the low dose dose panel (Ku¨nzel et al., 2003). In the high dose panel, 3 of 10 patients showed a moderate increase in liver transaminase levels (Ku¨nzel et al., 2003), a phenomenon well known to occur quite often also when patients are treated with conventional antidepressants. In addition, a random subgroup of patients underwent several sleep-EEG recordings. Slow wave sleep time increased significantly compared to baseline after 1 week and after 4 weeks (Held et al., 2004). The number of awakenings and REM density decreased during the same time period. In combination with the animal data outlined above (Lancel et al., 2002), these findings indicate that NBI 30775/R121919 has a normalizing influence on sleep EEG changes as they occur among patients with depression. While the clinical development program for NBI 30775/ R121919 has been discontinued due to liver enzyme elevations in probands which were monitored in a separate dose-escalation safety trial, all major pharmaceutical

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Table 1 Selection of studies with selective CRF1 (A) and CRF2 (B) antagonists in relation to emotional behavior and antidepressant-like effects in both animals and humans (oral application except NBI 27914) Compound

Species

(A) Selective CRF1 antagonists R121919/ Rat NBI 30775

Test methods/study design

Results

References

Elevated plus-maze (basal conditions)

Reduced anxiety in high-anxiety animals only R121919 avoided the suppressive effects of stress-elicited CRF action on slow-wave sleep Dose-dependent attenuation of stressinduced, CRF-induced and spontaneous anxiety-related behavior Reduced anxiety-related behavior

(Keck et al., 2001)

Sleep analysis

Swim-stress induced anxiety in the elevated plus-maze

NBI 27914

Rat

Defensive withdrawal, defensive burying Defensive withdrawal paradigm Open-label clinical trial for safety and tolerability; effects on depression- and anxietyrating scales Open-label clinical trial for safety and tolerability; effects on sleep-EEG Shock-induced freezing

CP-154,526

Mouse

Mouse defense test battery

Rat

Elevated plus-maze (basal conditions) Vogel conflict test

Human

Social interaction in unfamiliar environment Light-dark box (basal conditions)

Elevated plus-maze (basal conditions) Learned helplessness CRF-induced anxiety in the elevated plus-maze Fear-potentiated startle Antalarmin

Mouse Rat

Chronic mild stress model, followed by test in the light-dark box CRF-induced anxiety in the elevated plus-maze

Defensive withdrawal

CRA1000 CRA1001

Primate (rhesus monkey) Mouse

Rat

SSR-125543A

Mouse

Rat

Social stress

Swim-stress induced anxiety in the light-dark box Light-dark box (basal conditions) Emotional stress CRF-induced anxiety in the elevated plus-maze Social defeat-induced anxiety in the elevated plus-maze Light-dark box (basal conditions) Chronic mild stress model, followed by elevated plus-maze and light-dark test Mouse defense test battery Forced swim test Elevated plus-maze (basal conditions)

(Lancel et al., 2002)

(Heinrichs et al., 2002)

(Gutman et al., 2003)

Significant improvements in Hamilton-Depression and Anxiety-scales

(Zobel et al., 2000)

Significant increase in slow-wave sleep Intra-central amydala application: reduction in shock-induced freezing Intra-lateral septum application: no effects Reduced flight, defensive biting and contextual defense No significant change Dose-dependent increase in punished responses Increase in active social interaction Reduced anxiety-related behavior (increase in time spent in lit compartment) No significant changes Dose-dependent reversion of escape deficit Blockade of anxiogenic effect of CRF Attenuation of enhancement of startle amplitude Increased in time spent in the lit compartment (pZ0.16) Blockade of anxiogenic effect of CRF; no effect in vehicle-treated animals Anxiolytic-like effect on spontaneous defensive withdrawal behavior Reduction in anxiety-related behavior

(Held et al., 2004)

Reversion of swim-stress induced reduction of time spent in the light area No effect under nonstress conditions Reversal of behavioral changes induced by acute emotional stress Reversal of behavioral changes Increase in time spent in open arms No significant effect Reversion of stress-induced effects on anxiety measures Decrease in avoidance distance and aviodance frequency Significant decrease in immobility time No significant effect

(Bakshi et al., 2002)

(Griebel et al., 1998) (Millan et al., 2001)

(Griebel et al., 1998)

(Griebel et al., 1998) (Mansbach et al., 1997) (Okuyama et al., 1999) (Chen et al., 1997) (Ducottet et al., 2003) (Zorrilla et al., 2002)

(Habib et al., 2000)

(Okuyama et al., 1999)

(Okuyama et al., 1999) (Hotta et al., 1999) (Okuyama et al., 1999) (Griebel et al., 2002)

(Griebel et al., 2002)

(continued on next page)

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Table 1 (continued) Compound

Species

Test methods/study design

Results

References

DMP695

Rat

Elevated plus-maze (basal conditions) Vogel conflict test

(Millan et al., 2001)

DMP696

Rat

No significant change Dose-dependent increase in punished responses Increase in active social interaction Increase in time spent in the open arms Increase in social interaction Reduced latency to exit the dark chamber Reduced latency to exit the dark chamber Reduced latency to exit the dark chamber Reduction in stereotypical behavior

Social interaction in unfamiliar environment Maternal separation paradigm, followed by elevated plus-maze and social interaction test in adulthood Rat situational anxiety test Defensive withdrawal test Defensive withdrawal test

DMP904

Primate (rhesus monkey) Rat

(B) Selective CRF2 antagonist Anti-sauvagine 30 Mouse

Human intruder test

(Maciag et al., 2002)

(He et al., 2000) (McElroy et al., 2002) (Li et al., 2003) (He et al., 2000)

elevated-plus maze, defensive withdrawal

Increase in time spent in the open arms Reduction in exit latency

(Lelas et al., 2004)

Marble burying task (basal conditions)

Significant reduction in marble burying Increased percentage of time spent in the center Increased percentage of time spent in the open arms No significant effect Intraseptal administration of antisauvagine 30 completely antagonized anxiety induced by intraseptal injection of CRF Increased number of entries and time spent in the open arms Increase in time spent in the large illuminated field Decreased percentage of time spent in the open arms and decreased number of entries into the open arms

(Pelleymounter et al., 2002)

Open field (basal conditions) Elevated plus-maze (basal conditions) CRF-induced anxiety in the elevated plus-maze CRF-induced anxiety in the elevated plus-maze

Elevated plus-maze (basal conditions) Defensive-withdrawal (basal conditions) Elevated plus-maze (basal conditions)

(Brauns et al., 2001) (Radulovic et al., 1999b)

(Takahashi et al., 2001) (Takahashi et al., 2001) (Kishimoto et al., 2000)

Non-selective CRF receptor antagonists (e.g. alpha-helical CRF and astressin) were not included.

industries are now catching up on CRF1 antagonists as a new treatment modality that emerged from closely interrelated clinical and preclinical research.

19. Clinical outlook and summary Targeted gene mutation has become an established tool for increasing our knowledge about neuropeptide functions in the central nervous system. Genetically engineered mice overexpressing a neuropeptide or deficient in a neuropeptide or it’s receptor subtype are helpful in elucidating specific biological actions of a neuropeptidergic circuit and in complementing current knowledge. As currently available treatments for depression, which is one of the most pervasive and costly brain diseases (Michaud et al., 2001), are not fully satisfactory in many patients there is a need for novel treatment strategies based on novel neurobiological concepts. The integrated information especially from

the newer conditional mutant mice together with the increasing knowledge on the role of single nucleotide polymorphisms in psychiatric disorders prophesies an enormous progress (Holsboer, 2001a,b). Most antidepressants in current clinical use have well-documented effects on the disposition of biogenic amines that are readily demonstrable both in vitro and in vivo. Converging lines of evidence such as the delayed onset of action common to all antidepressants, however, have led us beyond the usual suspects, i.e. the monoaminergic synapses, and for strategies to improve antidepressant therapy-although there is evidence that CRF modulates monoamine release. Based on the hypotheses that have, for example, emerged from neurobiological research in genetically engineered mouse models the strategy of CRF1 antagonism has been developed and pursued. So far, beyond preclinical research, one selective non-peptide high-affinity CRF1 antagonist, NBI 30775/R121919, has proven safety and efficacy in an openlabel phase IIa trial in depressed patients. Large-scale,

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placebo-controlled, double-blind clinical trials with CRF1 antagonists are eagerly awaited to enable us to judge whether or not this interesting concept will keep its promise.

References Antoni, F.A., 1993. Vasopressinergic control of pituitary adrenocorticotropin secretion comes of age. Front. Neuroendocrinol. 14, 76–122. Asaba, K., Makino, S., Hashimoto, K., 1998. Effect of urocortin on ACTH secretion from rat anterior pituitary in vitro and in vivo: comparison with corticotropin-releasing hormone. Brain Res. 806, 95–103. Bakshi, V.P., Smith-Roe, S., Newman, S.M., Grigoriadis, D., Kalin, N.H., 2002. Reduction of stress-induced behavior by antagonism of corticotropin-releasing hormone 2 receptors in lateral septum or corticotropin-releasing hormone 1 receptors in amygdala. J. Neurosci. 22, 2926–2935. Bale, T.L., Vale, W.W., 2003. Increased depression-like behaviors in corticotropin-releasing factor receptor-2-deficient mice: sexually dichotomous responses. J. Neurosci. 23, 5295–5301. Bale, T.L., Contarino, A., Smith, G.W., Chan, R., Gold, L.H., Sawchenko, P.E., et al., 2000. Mice deficient for corticotropin-releasing hormone receptor-2 display anxiety-like behaviour and are hypersensitive to stress. Nat. Genet. 24, 410–414. Bale, T.L., Picetti, R., Contarino, A., Koob, G.F., Vale, W., Lee, K.F., 2002. Mice deficient for both corticotropin-releasing factor receptor 1 (CRFR1) and CRFR 2 have an impaired stress response and display sexually dichotomous anxiety- like behavior. J. Neurosci. 22, 193–199. Barden, N., Reul, J.M., Holsboer, F., 1995. Do antidepressants stabilize mood through actions on the hypothalamic-pituitary-adrenocortical system?. [Review] [53 refs]. Trends Neurosci. 18, 6–11. Behan, D.P., De Souza, E.B., Lowry, P.J., Potter, E., Sawchenko, P., Vale, W.W., 1995. Corticotropin releasing factor (CRF) binding protein: a novel regulator of CRF and related peptides. Front. Neuroendocrinol. 16, 362–382. Belzung, C., Griebel, G., 2001. Measuring normal and pathological anxiety-like bevaviour in mice: a review. Behav. Brain Res. 125, 141–149. Bissette, G., Klimek, V., Pan, J., Stockmeier, C., Ordway, G., 2003. Elevated concentrations of cortocotropin-releasing factoF in the locus coeruleus of depressed subjects. Neuropsychopharmacology 28, 1328–1335. Bittencourt, J.C., Vaughan, J., Arias, C., Rissman, R.A., Vale, W.W., Sawchenko, P.E., 1999. Urocortin expression in rat brain: evidence against a pervasive relationship of urocortin-containing projections with targets bearing type 2 CRF receptors. J. Comp. Neurol. 415, 285–312. Brauns, O., Liepold, T., Radulovic, J., Spiess, J., 2001. Pharmacological and chemical properties of astressin, antisauvagine-30 and alphahelCRF: significance for behavioral experiments. Neuropharmacology 41, 507–516. Burrows, H.L., Nakajima, M., Lesh, J.S., Goosens, K.A., Samuelson, L.C., Inui, A., et al., 1998. Excess corticotropin releasing hormone-binding protein in the hypothalamic-pituitary-adrenal axis in transgenic mice. J. Clin. Invest. 101, 1439–1447. Capecchi, M.R., 1989. The new mouse genetics: altering the genome by gene targeting. Trends Genet. 5, 70–76. Chalmers, D.T., Lovenberg, T.W., De Souza, E.B., 1995. Localization of novel corticotropic-releasing factor receptor 2 (CRF2) mRNA expression to specific subcortical nuclei in rat brain: comparison with CRF1 receptor mRNA expression. J. Neurosci. 15, 6340–6350. Chalmers, D.T., Lovenberg, T.W., Grigoriadis, D.E., Behan, D.P., De Souza, E.B., 1996. Corticotropin-releasing factor receptors: from molecular biology to drug design. Trends Pharmacol. Sci. 17, 166–172. Chen, Y.L., Mansbach, R.S., Winter, S.M., Brooks, E., Collins, J., Corman, M.L., et al., 1997. Synthesis and oral efficacy of

a 4-(butylethylamino)pyrrolo[2,3-d]pyrimidine: a centrally active corticotropin-releasing factor1 receptor antagonist. J. Med. Chem. 40, 1749–1754. Contarino, A., Heinrichs, S.C., Gold, L.H., 1999. Understanding corticotropin releasing factor neurobiology: contributions from mutant mice. Neuropeptides 38, 1–12. Contarino, A., Dellu, F., Koob, G.F., Smith, G.W., Lee, K., Vale, W.W., et al., 2000. Dissociation of locomotor activation and suppression of food intake induced by CRF in CRFR1-deficient mice. Endocrinology 141, 2698–2702. Cortright, D.N., Nicoletti, A., Seasholtz, A.F., 1995. Molecular and biochemical characterization of the mouse brain corticotropin-releasing factor (CRF)-binding protein. Mol. Cell Endocrinol. 111, 147–157. Coste, S.C., Kesterson, R.A., Heldwein, K.A., Stevens, S.L., Heard, A.D., Hollis, J.H., et al., 2000. Abnormal adaptations to stress and impaired cardiovascular function in mice lacking CRH receptor-2. Nat. Genet. 24, 403–409. Crabbe, J.C., Wahlsten, D., Dudek, B.C., 1999. Genetics of mouse behavior: interactions with laboratory environment. Science 284, 1670–1672. Crusio, W.E., 2004. Flanking gene and genetic background problems in genetically manipulated mice. Biol. Psychiatry 56, 381–385. Dautzenberg, F.M., Hauger, R.L., 2002. The corticotropin-releasing factor peptide family and their receptors: yet more partners discovered. Trends Pharmacol. Sci. 23, 71–77. Dautzenberg, F.M., Kilpatrick, G.J., Hauger, R.L., Moreau, J.L., 2001. Molecular biology of the corticotropin-releasing hormone receptors-in the mood. Peptides 22, 753–760. Dayas, C.V., Day, T.A., 2001. Opposing roles for medial and central amygdala in the initiation of noradrenergic cell responses to a psychological stressor. Eur. J. Neurosci. 15, 1712–1718. De Bellis, M.D., Gold, P.W., Geracioti, T.D.J., Listwak, S.J., Kling, M.A., 1993. Association of Fluoxetin treatment with reductions in CSF concentrations of corticotropin-releasing hormone and arginine vasopressin in patients with major depression. Am. J. Psychiatry 150, 656–657. Dirks, A., Groenink, L., Bouwknecht, J.A., Hijzen, T.H., Van der Gugten, J., Ronken, E., et al., 2002a. Overexpression of corticotropinreleasing hormone in transgenic mice and chronic stress-like autonomic and physiological alterations. Eur. J. Neurosci. 16, 1751–1760. Dirks, A., Groenink, L., Schipholt, M.I., van der Gugten, J., Hijzen, T.H., Geyer, M.A., et al., 2002b. Reduced startle reactivity and plasticity in transgenic mice overexpressing corticotropin-releasing hormone. Biol. Psychiatry 51, 583–590. Dirks, A., Groenink, L., Westphal, K.G.C., Olivier, J.D.A., Verdouw, P.M., Van der Gugten, J., et al., 2003. Reversal of startle gating deficits in transgenic mice overexpressing corticotropin-releasing factor by antipsychotic drugs. Neuropsychopharmacology 28, 1790–1798. Donaldson, C.J., Sutton, S.W., Perrin, M.H., Corrigan, A.Z., Lewis, K.A., Rivier, J.E., et al., 1996. Cloning and characterization of human urocortin. Endocrinology 137, 2167–2170. Ducottet, C., Griebel, G., Belzung, C., 2003. Effects of the selective nonpeptide corticotropin-releasing factor receptor 1 antagonist antalarmin in the chronic mild stress model of depression in mice. Prog. Neuropsychopharmacol. Biol. Psychiatry 27, 625–631. Dunn, A.J., Berridge, C.W., 1990. Physiological and behavioral responses to corticotropin-releasing factor administration: is CRF a mediator of anxiety or stress responses?. Brain Res. Rev. 15, 71–100. Dunn, A.J., Swiergiel, A.H., 1999. Behavioral responses to stress are intact in corticotropin-releasing factor-deficient mice. Brain Res. 845, 14–20. Erkut, Z., Pool, C., Swaab, D.F., 1998. Glucocorticoids suppress corticotropin-releasing hormone and vasopressin expression in human hypothalamic neurons. J. Clin. Endocrinol. Metab. 83, 2066–2073. Freedman, R., Adler, L., Leonard, S., 1999. Alternative phenotypes for the complex genetics of schizophrenia. Biol. Psychiatry 45, 551–558.

M.E. Keck et al. / Neuroscience and Biobehavioral Reviews 29 (2005) 867–889 Geriacoti, T.D., Orth, D.N., Ekhator, N.N., Blumenkopf, B., Loosen, P.T., 1992. Serial cerebrospinal fluid corticotropin-releasing hormone concentrations in healthy and depressed humans. J. Clin. Endocrinol. Met. 74, 1325–1330. Geriacoti, T.D., Loosen, P.T., Orth, D.N., 1997. Low cerebrospinal fluid corticotropin-releasing hormone concentrations in eucortisolemic patients. Biol. Psychiatry 42, 166–174. Gesing, A., Bilang-Bleuel, A., Droste, S.K., Linthorst, A.C.E., Holsboer, F., Reul, J.M.H.M., 2001. Psychological stress increases hippocampal mineralocorticoid receptor levels: Involvement of corticotropin-releasing hormone. J. Neurosci. 21, 4822–4829. Geyer, M.A., Markou, A., 1995. Animal models of psychiatric disorders. In: Bloom, F.E., Kupfer, D.J. (Eds.), Psychopharmacology: The Fourth Generation of Progress. Raven Press, New York, pp. 787–798. Geyer, M.A., Markou, A., 2002. The role of preclinical models in the development of psychotropic drugs. In: Davis, K.L., Charney, D.S., Coyle, J.T., Nemeroff, C.B (Eds.), Neuropsychopharmacology. The Fifth Generation of Progress. Lippincott, Williams and Wilkins, Philadelphia, pp. 445–456. Gillies, G.E., Linton, E.A., Lowry, P.J., 1982. Corticotropin releasing activity of the new corticotropin-releasing factor is potentiated several times by vasopressin. Nature 299, 355–357. Gold, P.W., Chrousos, G.P., 2002. Organization of the stress system and its dysregulation in melancholic and atypical depression: high vs. low CRH/NE states. Mol. Psychiatry 7, 254–275. Gold, P.W., Chrousos, G.P., Kellner, C., Post, R.M., Roy, A., Augerinas, P., et al., 1984. Psychiatric implications of basic and clinical studies with corticotropin-releasing factor. Am. J. Psychiatry 141, 619–627. Gossen, M., Freundlieb, S., Bender, G., Mu¨ller, G., Hillen, W., Bujard, H., 1995. Transcriptional activation by tetracyclines in mammalian cells. Science 268, 1766–1769. Gottesman, Gould, T.D, 2003. The endophenotype concept in psychiatry: etymology and strategic intentions. Am. J. Psychiatry 160, 636–645. Griebel, G., Perrault, G., Sanger, D.J., 1998. Characterization of the behavioral profile of the non-peptide corticotropin-releasing factor receptor antagonist CP-154,526 in anxiety models in rodents. Comparison with diazepam and buspirone. Psychopharmacology 138, 55–66. Griebel, G., Simiand, J., Steinberg, R., Jung, M., Gully, D., Roger, P., et al., 2002. 4-(2-Chloro-4-methoxy-5-methylphenyl)-N-[(1S)-2-cyclopropyl-1-(3-fluoro-4-methylphenyl)ethyl]5-methyl-N-(2-propynyl)-1-3thiazol-2-amine hydrochloride (SSR125543A), a potent and selective corticotrophin-releasing factor(1) receptor antagonist. II. Characterization in rodent models of stress-related disorders. J. Pharmacol. Exp. Ther. 301, 333–345. Groenink, L., Dirks, A., Verdouw, P.M., Schipholt, M.I., Veening, J.G., Van der Gugten, J., et al., 2002. HPA-axis dysregulation in mice overexpressing corticotropin-releasing hormone. Biol. Psychiatry 51, 875–881. Gutman, D.A., Owens, M.J., Skelton, K.H., Thrivikraman, K.V., Nemeroff, C.B., 2003. The CRF1 receptor antagonist R121919 attenuates the behavioral and endocrine responses to stress. J. Pharmacol. Exp. Ther. 304, 874–880. Habib, K.E., Weld, K.E., Rice, K.C., Pushkas, J., Champoux, M., Listwak, S., et al., 2000. Oral administration of a corticotropinreleasing hormone receptor antagonist significantly attenuates behavioral, neuroendocrine, and autonomic responses to stress in primates. PNAS 97, 6079–6084. Hammack, S.E., Schmid, M.J., LoPresti, M.L., Der-Avakian, A., Pellymounter, M.A., Foster, A.C., et al., 2003. Corticotropin-releasing hormone type 2 receptors in the dorsal raphe nucleus mediate the behavioral consequences of uncontrollable stress. J. Neurosci. 23, 1019–1025. Hascoet, M., Bourin, M., Dhonnchadha, B.A., 2001. The mouse light-dark paradigm: a review. Prog. Neuropsychopharmacol. Biol. Psychiatry 25, 141–166.

885

Hauger, R.L., Grigoriadis, D.E., Dallman, M.F., Plotsky, P.M., Vale, W.W., Dautzenberg, F.M., 2003. International Union of Pharmacology. XXXVI. Current status of the nomenclature for receptors for corticotropin-releasing factor and their ligands. Pharmacol. Rev. 55, 21–26. He, L., Gilligan, P.J., Zaczek, R., Fitzgerald, L., McElroy, J., Shen, H.L., et al., 2000. 4-(1,3-Dimethoxyprop-2-ylamino)-2,7-dimethyl-8-(2,4dichlorophenyl)-pyrazolo [1,5- *]-1,3,5- triazine: A potent, orally bioavailable CRF1 receptor antagonist. J. Med. Chem. 43, 449–456. Heinrichs, S.C., Joppa, M., 2001. Dissociation of arousal-like from anxiogenic-like actions of brain corticotropin-releasing factor receptor ligands. Behav. Brain Res. 122, 43–50. Heinrichs, S.C., Koob, G.F., 2004. Corticotropin-releasing factor in brain: a role in activation, arousal, and affect regulation. J. Pharmacol. Exp. Ther. 311, 427–440. Heinrichs, S.C., Stenzel-Poore, M.P., Gold, L.H., Battenberg, E., Bloom, F.E., Koob, G.F., et al., 1996. Learning impairment in transgenic mice with central overexpression of corticotropin-releasing factor. Neuroscience 74, 303–311. Heinrichs, S.C., Min, H., Tamraz, S., Carmouche´, M., Boehme, S.A., Vale, W.W., 1997. Anti-sexual and anxiogenic behavioral consequences of corticotropin-releasing factor overexpression are centrally mediated. Psychoneuroendocrinology 22, 215–224. Heinrichs, S.C., DeSouza, E.B., Schulteis, G., Lapsansky, J., Grigoriadis, D., 2002. Brain penetrance, receptor occupancy and antistress in vivo efficacy of a small molecule corticotropin-releasing factor type I receptor selective antagonist. Neuropsychopharmacology 27, 194–202. Held, K., Ku¨nzel, H.E., Ising, M., Schmid, D.A., Zobel, A.W., Murck, H., et al., 2004. Treatment with the corticotropin-releasing hormone 1 receptor antagonist R121919 improves sleep-EEG in patients with depression. J. Psychiatry Res. 38, 129–136. Hettema, J.M., Prescott, C.A., Kendler, K.S., 2001. A population-based twin study of generalized anxiety disorder in men and women. J. Nerv. Ment. Dis. 189, 413–420. Heuser, I., Yassouridis, A., Holsboer, F., 1994a. The combined dexamethasone/CRH test: a refined laboratory test for psychiatric disorders. J. Psychiatry Res. 28, 341–356. Heuser, I., Yassouridis, A., Holsboer, F., 1994b. The combined dexamethasone/CRH test: a refined laboratory test for psychiatric disorders. J. Psychiatry Res. 28, 341–356. Heuser, I.J.E., Schweiger, U., Gotthardt, U., Schmider, J., Lammers, C.H., Dettling, M., et al., 1996. Pituitary-adrenal system regulation and psychopathology during amitriptyline treatment in elderly depressed patients and in normal control subjects. Am. J. Psychiatry 153, 93–99. Ho, S.P., Takahashi, L.K., Livanov, V., Spencer, K., Lesher, T., Maciag, C., et al., 2001. Attenuation of fear conditioning by antisense inhibition of brain corticotropin releasing factor-2 receptor. Mol. Brain Res. 89, 29–40. Hogg, S., 1996. A review of the validity and variability of the elevated plusmaze as an animal model of anxiety. Pharmacol. Biochem. Behav. 54, 21–30. Ho¨kfelt, T., Broberger, C., Xu, Z.Q.D., Sergeyev, V., Ubink, R., Diez, M., 2000. Neuropeptides-an overview. Neuropharmacology 39, 1337– 1356. Ho¨kfelt, T., Bartfai, T., Bloom, F., 2003. Neuropeptides: opportunities for drug discovery. Lancet Neurol. 2, 463–472. Holsboer, F., 1997. Transgenic mouse models: new tools for psychiatric research. Neuroscientist 3, 328–336. Holsboer, F., 1999a. Animal models of mood disorders. In: Charney, D.S., Nestler, E.J., Bunney, B.S. (Eds.), Neurobiology of Mental Illness. Oxford University Press, New York, pp. 317–332. Holsboer, F., 1999b. The rationale for corticotropin-releasing hormone receptor (CRH-R) antagonists to treat depression and anxiety. J. Psychiatry Res. 33, 181–214.

886

M.E. Keck et al. / Neuroscience and Biobehavioral Reviews 29 (2005) 867–889

Holsboer, F., 1999c. The rationale for the corticotropin-releasing hormone receptor (CRH-R) antagonists to treat depression and anxiety. J. Psychiatry Res. 33, 181–214. Holsboer, F., 2000a. The corticosteroid receptor hypothesis of depression. Neuropsychopharmacology 23, 477–501. Holsboer, F., 2000b. The corticosteroid receptor hypothesis of depression. Neuropsychopharmacology 23, 477–501. Holsboer, F., 2001a. Antidepressant drug discovery in the postgenomic area. World J. Biol. Psychiatry 2, 165–177. Holsboer, F., 2001b. Prospects for antidepressant drug discovery. Biol. Psychol. 57, 47–65. Holsboer, F., 2003a. Corticotropin-releasing hormone modulators and depression. Curr. Opin. Inv. Drug 4, 46–50. Holsboer, F., 2003b. The role of peptides in treatment of psychiatric disorders. J. Neural Transm. 64, 17–34. Holsboer, F., 2004. Diagnostic implications of molecular mechanisms underlying depression and antidepressant action. American Psychiatric Publishing, Inc., Washington. Holsboer, F., von Bardeleben, U., Steiger, A., 1988. Effects of intravenous corticotropin-releasing hormone upon sleep-related growth hormone surge and sleep EEG in man. Neuroendocrinology 48, 32–38. Hotta, M., Shibasaki, T., Arai, K., Demura, H., 1999. Corticotropinreleasing factor receptor type 1 mediates emotional stress-induced inhibition of food intake and behavioral changes in rats. Brain Res. 823, 221–225. Hsu, S., Hsueh, A., 2001. Human stresscopin and stresscopin-related peptide are selective ligands for the type 2 corticotropin-releasing hormone receptor. Nat. Med. 7, 605–611. Hyman, S., 2000. Mental illness: genetically complex disorders of neural circuitry and neural communication. Neuron 28, 321–323. Iino, K., Sasano, H., Oki, Y., Andoh, N., Shin, R.W., Kitamoto, T., et al., 1999. Urocortin expression in the human central nervous system. Clin. Endocrinol. 50, 107–114. Imaki, T., Katsumata, H., Miyata, M., Naruse, M., Imaki, I., inami, S., 2001. Expression of corticotropin-releasing factor, urocortin and corticotropin-releasing factor type 1 receptors in hypothalamichypophyseal systems under osmotic stimulation. J. Neurendocrinol. 13, 328–338. Imaki, T., Katsumata, H., Konishi, S.I., Kasagi, Y., Minami, S., 2003. Corticotropin-releasing factor type-1 receptor mRNA is not induced in mouse hypothalamus by either stress or osmotic stimulation. J. Neurendocrinol. 15, 916–924. Jacobson, L., Muglia, L.J., Weninger, S.C., Pacak, K., Majzoub, J.A., 2000. Corticotropin-releasing hormone deficiency impairs but does not block pituitary-adrenal responses to diverse stressors. Neuroendocrinology 71, 79–87. Jaenisch, R., 1988. Transgenic animals. Science 240, 1468–1472. Jahn, O., Tezval, H., van Werven, J., Eckart, K., Spiess, J., 2004. Threeamino acid motifs of urocortin II and III determine their CRF receptor subtype selectivity. Neuropharmacology 47, 233–242. Kageyama, K., Bradbury, M.J., Zhao, L., Blount, A.L., Vale, W.W., 1999. Urocortin messenger ribonucleic acid: tissue distribution in the rat and regulation in thymus by lipopolysaccharide and glucocorticoids. Endocrinology 140, 5651–5658. Karolyi, I.J., Burrows, H.L., Ramesh, T.M., Nakajima, M., Lesh, J.S., Seong, E., et al., 1999. Altered anxiety and weight gain in corticotropinreleasing hormone-binding protein-deficient mice. Proc. Natl Acad. Sci. USA 96, 11595–11600. Keck, M.E., Holsboer, F., 2001. Hyperactivity of corticotropin-releasing hormone neuronal circuits as a target for therapeutic interventions in affective disorders. Peptides 22, 835–844. Keck, M.E., Hatzinger, M., Wotjak, C., Landgraf, R., Holsboer, F., Neumann, I.D., 2000. Ageing alters intrahypothalamic release patterns of vasopressin and oxytocin in rats. Eur. J. Neurosci. 12, 1487–1494. Keck, M.E., Welt, T., Wigger, A., Renner, U., Engelmann, M., Holsboer, F., et al., 2001. The anxiolytic effect of

the corticotropin-releasing hormone 1 receptor antagonist R121919 depends on innate emotionality in rats. Eur. J. Neurosci. 13, 373–380. Keck, M.E., Wigger, A., Welt, T., Mu¨ller, M.B., Gesing, A., Reul, J.M.H.M., et al., 2002. Vasopressin mediates the response of the combined dexamethasone/CRH test in hyper-anxious rats: Implications for pathogenesis of affective disorders. Neuropsychopharmacology 26, 94–105. Keck, M.E., Welt, T., Mueller, M.B., Landgraf, R., Holsboer, F., 2003. The high-affinity non-peptide corticotropin-releasing hormone receptor antagonist R121919 attenuates stress- induced alterations in plasma oxytocin, prolactin, and testosterone secretion in rats. Pharmacopsychiatry 36, 27–31. Keck, M.E., Holsboer, F., Mu¨ller, M.B., 2004. Mouse mutants for the study of corticotropin-releasing hormone receptor function: development of novel treatment strategies for mood disorders. Ann. NY Acad. Sci. 1018, 445–457. Kemp, C.F., Woods, R.J., Lowry, P.J., 1998. The corticotrophin-releasing factor-binding protein: an act of several parts. Peptides 19, 1119–1128. Kendler, K.S., 2001. Twin studies of psychiatric illness. Arch. Gen. Psychiatry 58, 1005–1014. Kessler, R.C., McGonagle, K.A., Zhao, S., Nelson, C.B., Hughes, M., Eshleman, S., et al., 1994. Lifetime and 12-month prevalence of DSMIII-R psychiatric disorders in the United States: results from the National Comorbidity Survey. Arch. Gen. Psychiatry 51, 8–19. Kishimoto, T., Radulovic, J., Radulovic, M., Lin, C.R., Schrick, C., Hooshmand, F., et al., 2000. Deletion of corticotropin-releasing hormone receptor-2 reveals an anxiolytic role for corticotropinreleasing hormone receptor-2. Nat. Genet. 24, 415–419. Kling, M.A., Geracioti, T.D., Licinio, J., Michelson, D., Oldfield, E.H., Gold, P.W., 1994. Effects of electroconvulsive therapy on the CRHACTH-cortisol system in melancholic depression: preliminary findings. Psychopharmacol. Bull. 30, 489–494. Konishi, S., Katsumata, H., Kasagi, Y., Minami, S., Imaki, T., 2003. Regulation of corticotropin-releasing factor (CRF) type-1 receptor gene expression by CRF in the hypothalamus. Endocr. J. 50, 21–36. Korte, S.M., 2001a. Corticosteroids in relation to fear, anxiety and psychopathology. Neurosci. Biobehav. Rev. 25, 117–142. Korte, S.M., 2001b. Corticosteroids in relation to fear, anxiety and psychopathology [Review] [292 refs]. Neurosci. Biobehav. Rev. 25, 117–142. Korte, S.M., Korte-Bouws, G.A., Koob, G.F., De Kloet, E.R., Bohus, B., 1996. Mineralocorticoid and glucocorticoid receptor antagonists in animal models of anxiety. Pharmacol. Biochem. Behav. 54, 261–267. Kostich, W.A., Chen, A., Sperle, K., Largent, B.L., 1998. Molecular identification and analysis of a novel human corticotropin-releasing factor (CRF) receptor: the CRF2gamma receptor. Mol. Endocrinol. 12, 1077–1085. Kozicz, T., Yanaihara, H., Arimura, A., 1998. Distribution of urocortin-like immunoreactivity in the central nervous system of the rat. J. Comp. Neurol. 391, 1–10. Kozicz, T., Korosi, A., Korsman, C., Tilbourg-Ouwens, D., Groenink, L., Veening, J.G., et al., 2004. Urocortin expression in the EdingerWestphal nucleus is down-regulated in transgenic mice over-expressing neuronal corticotropin-releasing factor. Neuroscience 123, 589–594. Ku¨hn, R., Schwenk, F., Aguet, M., Rajewsky, K., 1995. Inducible gene targeting in mice. Science 259, 1427–1429. Ku¨nzel, H.E., Zobel, A.W., Nickel, T., Ackl, N., Uhr, M., Sonntag, A., et al., 2003. Treatment of depression with the corticotropin-releasing hormone-1-receptor antagonist R121919: endocrine changes and side effects. J. Psychiatry Res. 37, 525–533. Lancel, M., Muller-Preuss, P., Wigger, A., Landgraf, R., Holsboer, F., 2002. The corticotropin-releasing hormone1 receptor antagonist R121919 attenuates stress-elicited sleep disturbances in rats, particularly in those with high innate anxiety. J. Psychiatry Res. 36, 197–208. Lathe, R., 1996. Mice, gene targeting and behaviour: more than just genetic background. Trends Neurosci. 19, 183–186.

M.E. Keck et al. / Neuroscience and Biobehavioral Reviews 29 (2005) 867–889 Lelas, S., Wong, H., Li, Y-W., Heman, K.L., Ward, K.A., Zeller, K.L., et al., 2004. Anxiolytic-like effects of the corticotropin-releasing factor1 (CRF1) antagonist DMP904 (4-(3-pentylamino)-2,7-dimethyl8-(2-methyl-4-methoxyphenyl)-pyrazolo-(1,l5-a)-pyrimidine) administered acutely or chronically at doses occupying central CRF1 receptors in rats. J. Pharmacol. Exp. Ther. 309, 293–302. Lewandoski, M., 2001. Conditional control of gene expression in the mouse. Nat. Rev. Genet. 2, 743–755. Lewis, K., Li, C., Perrin, M., Blount, A., Kunitake, K., Donaldson, C., et al., 2001a. Identification of urocortin III, an additional member of the corticotropin-releasing factor (CRF) family with high affinity for the CRF2 receptor. Proc. Natl. Acad. Sci. USA 98, 7570–7575. Lewis, K., Li, C., Perrin, M.H., Blount, A., Kunitake, K., Donaldson, C., et al., 2001b. Identification of urocortin III, an additional member of the corticotropin-releasing factor (CRF) family with high affinity for the CRF2 receptor. Proc. Natl Acad. Sci. USA 98, 7570–7575. Li, C., Vaughan, J., Sawchenko, P.E., Vale, W.W., 2002. Urocortin IIIimmunoreactive projections in rat brain: partial overlap with sites of type 2 corticotrophin-releasing factor receptor expression. J. Neurosci. 22, 991–1001. Li, Y.W., Hill, G., Wong, H., Kelly, N., Ward, K., Pierdomenico, M., et al., 2003. Receptor occupancy of nonpeptide corticotropin-releasing factor 1 antagonist DMP696: correlation with drug exposure and anxiolytic efficacy. J. Pharmacol. Exp. Ther. 305, 86–96. Liebsch, G., Landgraf, R., Gerstberger, R., Probst, J.C., Wotjak, C.T., Engelmann, M., et al., 1995. Chronic infusion of a corticotropinreleasing hormone 1 receptor antisense oligodeoxynucleotide into the central nucleus of the amygdala reduced anxiety-releated behavior in socially defeated rats. Regul. Pept. 59, 229–239. Liebsch, G., Linthorst, A.C.E., Neumann, I.D., Reul, J.M.H.M., Holsboer, F., Landgraf, R., 1998. Behavioral, physiological, and neuroendocrine stress responses and differential sensitivity to diazepam in two wistar rat lines selectively bred for high- and low- anxiety- related behavior. Neuropsychopharmacology 19, 381–396. Liebsch, G., Landgraf, R., Engelmann, M., Lorscher, P., Holsboer, F., 1999. Differential behavioural effects of chronic infusion of CRH 1 and CRH 2 receptor antisense oligonucleotides into the rat brain. J. Psychiatric Res. 33, 153–163. Lindley, S.E., Bengoechea, T.G., Schatzberg, A.F., 1999. Glucocorticoid effects on mesotelencephalic dopamine neurotransmission. Neuropsychopharmacology 21, 399–407. Lovejoy, D.A., Aubry, J.M., Turnbull, A., Sutton, S., Potter, E., Yehling, J., et al., 1998. Ectopic expression of the corticotropinreleasing factor-binding protein: minor impact on HPA axis regulation but induction of sexually dimorphic weight gain. J. Neurendocrinol. 10, 483–491. Lovenberg, T.W., Chalmers, D.T., Liu, C., De Souza, E.B., 1995a. Corticotropin-releasing factor 2 alpha and corticotropin-releasing factor 2 beta receptor mRNAs are differentially distributed between the rat central nervous system and peripheral tissues. Endocrinology 136, 4139–4142. Lovenberg, T.W., Liaw, C.W., Grigoriadis, D.E., Clevenger, W., Chalmers, D.T., De Souza, E.B., et al., 1995b. Cloning and characterization of a functionally distinct corticotropin-releasing factor receptor subtype. Proc. Natl Acad. Sci. USA 92, 836–840. Maciag, C.M., Dent, G., Gilligan, P.J., He, L., Dowling, K., Ko, T., et al., 2002. Effects of a non-peptide corticotropin-releasing factor antagonist (DMP 696) on the behaviour and endocrine sequelae of maternal separation. Neuropsychopharmacology 26, 574–582. Mansbach, R.S., Brooks, E.N., Yuhpyng, L.C., 1997. Antidepressant- like effects of CP-154,526, a selective corticotropin-releasing factor 1 antagonist. Eur. J. Pharmacol. 323, 21–26. Mayford, M., Bach, M.E., Huang, Y.-Y., Wang, L., Hawkins, R.D., Kandel, E.R., 1997. Control of memory formation through regulated expression of a CAMKII transgene. Science 274, 1678–1683. McElroy, J.F., Ward, K.A., Zeller, K.L., Jones, K.W., Gilligan, P.J., He, L., et al., 2002. The CRF(1) receptor antagonist DMP696 produces

887

anxiolytic effects and inhibits the stress-induced hypothalamicpituitary-adrenal axis activation without sedation or ataxia in rats. Psychopharmacology 165, 86–92. Menard, J., Treit, D., 2001. The anxiolytic effects of intra- hippocampal midazolam are antagonized by intra-septal L-glutamate. Brain Res. 888, 163–166. Michaud, C.M., Murray, C.J., Bloom, B.R., 2001. Burden of disease: implications for future research. J. Am. Med. Ass. 285, 535–539. Millan, M.J., 2003. The neurobiology and control of anxious states. Prog. Neurobiol. 70, 83–244. Millan, M.J., Brocco, M., Gobert, A., Dorey, G., Casara, P., Dekeyne, A., 2001. Anxiolytic properties of the selective, non-peptidergic corticotropin-releasing factor 1 antagonists, CP 154,526 and DMP 695: A comparison to other classes of anxiolytic agent. Neuropsychopharmacology 25, 585–600. Minichiello, L., Korte, M., Wolfer, D.P., Ku¨hn, R., Unsicker, K., Cestari, V., et al., 1999. Essential role of trkB receptors in hippocampus-mediated learning. Neuron 24, 401–414. Moreau, J., Kilpatrick, G., Jenck, F., 1997. Urocortin, a novel neuropeptide with anxiogenic-like properties. Neuroreport 8, 1697–1701. Morin, S.M., Ling, N., Liu, X.J., Kahl, S.D., Gehlert, D.R., 1999. Differential distribution of urocortin- and corticotropin-releasing factor-like immunoreactivities in the rat brain. Neuroscience 92, 281–291. Morozov, A., Kellendonk, C., Simpson, E., Tronche, F., 2003. Using conditional mutagenesis to study the brain. Biol. Psychiatry 54, 1125–1133. Muglia, L., Jacobson, L., Dikkes, P., Majzoub, J.A., 1995. Corticotropinreleasing hormone deficiency reveals major fetal but not adult glucocorticoid need. Nature 373, 427–432. Munir, M.I., Rossiter, B.J.F., Caskey, C.T., 1990. Antisense RNA production in transgenic mice. Somat Cell Mol. Genet. 16, 383–394. Mu¨ller, M.B., Keck, M.E., 2002. Genetically engineered mice for studies of stress-related clinical conditions. J. Psychiatr. Res. 36, 53–76. Mu¨ller, M.B., Landgraf, R., Preil, J., Sillaber, I., Kresse, A.E., Keck, M.E., et al., 2000a. Selective activation of the hypothalamic vasopressinergic system in mice deficient for the corticotropin-releasing hormone receptor 1 is dependent on glucocorticoids. Endocrinology 141, 4262–4269. Mu¨ller, M.B., Landgraf, R., Sillaber, I., Kresse, A.E., Keck, M.E., Zimmermann, S., et al., 2000b. Selective activation of the hypothalamic vasopressinergic system in mice deficient for the corticotropinreleasing hormone receptor 1 is dependent on glucocorticoids. Endocrinology 141, 4262–4269. Mu¨ller, M.B., Zimmermann, S., Sillaber, I., Hagemeyer, T.P., Deussing, J.M., Timpl, P., et al., 2003. Limbic corticotropin-releasing hormone receptor 1 mediates anxiety-related behaviour and is required for hormonal adaptation to stress. Nat. Neurosci. 6, 1100–1107. Nemeroff, C.B., 1996. The corticotropin-releasing factor (CRF) hypothesis of depression: new finding and new directions. Mol. Psychiatry 1, 336–342. Nemeroff, C.B., 2002. Comorbidity of mood and anxiety disorders: The rule, not the exception?. Am. J. Psychiatry 159, 3–4. Nemeroff, C.B., Owens, M.J., 2002. Treatment of mood disorders. Nat. Neurosci. 5, 1068–1070. Nemeroff, C.B., Bissette, G., Akil, H., Fink, M., 1991. Neuropeptide concentrations in the cerebrospinal fluid of depressed patients treated with electroconvulsive therapy: corticotropin-releasing factor, b-endorphin, and somatostatin. Br. J. Psychiatry 158, 59–63. Ohl, F., 2003. Testing for anxiety. Clin. Neurosci. Res. 3, 233–238. Ohl, F., Keck, M.E., 2003. Behavioural screening in ENU-mutant mice- in search for novel animal models of psychiatric disorders. Eur. J. Pharmacol. 480, 219–228. Okuyama, S., Chaki, S., Kawashima, N., Suzuki, Y., Ogawa, S.I., Nakazato, A., et al., 1999. Receptor binding, behavioral, and

888

M.E. Keck et al. / Neuroscience and Biobehavioral Reviews 29 (2005) 867–889

electrophysiological profiles of nonpeptide corticotropin-releasing factor subtype 1 receptor antagonists CRA 1000 and CRA 1001. J. Pharmacol. Exp. Ther. 289, 926–935. Pelleymounter, M.A., Joppa, M., Ling, N., Foster, A.C., 2002. Pharmacological evidence supporting a role for central corticotropin-releasing factor(2) receptors in behavioral, but not endocrine, response to environmental stress. J. Pharmacol. Exp. Ther. 302, 145–152. Persons, J.B., Roberts, N.A., Zalecki, C.A., 2003. Anxiety and depression change together during treatment. Behav. Ther. 34, 149–163. Piazza, P.V., Le Moal, M., 1998. The role of stress in drug selfadministration. Trends Pharmacol. Sci. 19, 67–74. Potter, E., Behan, D.P., Linton, E.A., Lowry, P.J., Sawchenko, P.E., Vale, W.W., 1992. The central distribution of a corticotropin-releasing factor (CRF)-binding protein predicts multiple sites and modes of interaction with CRF. Proc. Natl. Acad. Sci. USA 89, 4192–4196. Preil, J., Mu¨ller, M.B., Gesing, A., Reul, J.M.H.M., Sillaber, I., van Gaalen, M., et al., 2001. Regulation of the hypothalamic-pituitaryadrenocortical system in mice deficient for CRH receptors 1 and 2. Endocrinology 142, 4946–4955. Purba, J.S., Hoogendijk, W.J.G., Hofman, M.A., Swaab, D.F., 1996. Increased number of vasopressin- and oxytocin-expressing neurons in the paraventricular nucleus of the hypothalamus in depression. Arch. Gen. Psychiatry 53, 137–143. Raadsheer, F.C., Hoogendijk, W.J.G., Stam, F.C., Tilders, F.J.H., Swaab, D.F., 1994. Increased number of corticotropin-releasing hormone expressing neurons in the hypothalamic paraventricular nucleus of depressed patients. Neuroendocrinology 60, 436–444. Radulovic, J., Ruhmann, A., Liepold, T., Spiess, J., 1999a. Modulation of learning and anxiety by corticotropin-releasing factor (CRF) and stress: differential roles of CRF receptors 1 and 2. J. Neurosci. 19, 5016–5025. Radulovic, J., Ru¨hmann, A., Liepold, T., Spiess, J., 1999b. Modulation of learning and anxiety by corticotropin-releasing factor and stress: differential roles of corticotropin-releasing factor receptors 1 and 2. J. Neurosci. 19, 5016–5025. Reul, J.M.H.M., Holsboer, F., 2002. Corticotropin-releasing factor receptors 1 and 2 in anxiety and depression. Curr. Opin. Pharmacol. 2, 23–33. Reul, J.M.H.M., deKloet, E.R., 1985. Two receptor systems for corticosterone in the rat brain: microdistribution and differential occupation. Endocrinology 117, 2505–2512. Rex, A., Voigt, J.P., Voits, M., Fink, H., 1998. Pharmacological evaluation of a modified open-field test sensitive to anxiolytic drugs. Pharmacol. Biochem. Behav. 59, 677–683. Reyes, T.M., Lewis, K., Perrin, M.H., Kunitake, K.S., Vaughan, J., Arias, C.A., et al., 2001a. Urocortin II: A member of the corticotropinreleasing factor neuropeptide family that is selectively bound by type 2 corticotropin-releasing factor receptors. Proc. Natl Acad. Sci. USA 98, 2843–2848. Reyes, T.M., Lewis, K., Perrin, M.H., Kunitake, K.S., Vaughan, J., Arias, C.A., et al., 2001b. UrocortinII: A member of the corticotropinreleasing factor (CRF) neuropeptide family that is selectively bound by type 2 CRF receptors. Proc. Natl Acad. Sci. USA 98, 2843–2848. Sanchez, M.M., Young, L.J., Plotsky, P.M., Insel, T.R., 1999. Autoradiographic and in situ hybridization localization of corticotropin-releasing factor 1 and 2 receptors in nonhuman primate brain. J. Comp. Neurol. 408, 365–377. Sauer, B., 1998. Inducible gene targeting in mice using the Cre/lox system. Meth. Enzymol. 14, 381–392. Saunders, J., Williams, J., 2003. Antagonists of the corticotropin releasing hormone receptor. Prog. Med. Chem. 41, 195–247. Schulkin, J., Gold, P.W., McEwen, B.S., 1998. Induction of corticotropinreleasing hormone gene expression by glucocorticoids: implication for understanding the states of fear and anxiety and allostatic load. Psychoneuroendocrinology 23, 219–243. Seasholtz, A.F., Burrows, H.L., Karolyi, I.J., Camper, S.A., 2001. Mouse models of altered corticotropin-releasing hormone-binding protein expression. Peptides 22, 743–751.

Seong, E., Seasholtz, A.F., Burmeister, M., 2002. Mouse models for psychiatric disorders. Trends Genet. 18, 643–650. Sillaber, I., Holsboer, F., 2004. Animal models of mood disorders. In: Charney, D.S., Nestler, E.J. (Eds.), Neurobiology of Mental Illness. Oxford University Press, San Francisco. Skelton, K.H., Nemeroff, C.B., Knight, D.L., Owens, M.J., 2000. Chronic administration of the triazolobenzodiazepine alprazolam produces opposite effects on corticotropin-releasing factor and urocortin neuronal systems. J. Neurosci. 20, 1240–1248. Skutella, T., Probst, J.C., Renner, U., Holsboer, F., Behl, C., 1998. Corticotropin-releasing hormone receptor (type 1) antisense targeting reduces anxiety. Neuroscience 85, 795–805. Slawecki, C.J., Somes, C., Rivier, J.E., Ehlers, C.L., 1999. Neurophysiological effects of intracerebroventricular administration of urocortin. Peptides 20, 211–218. Smith, G.W., Aubry, J.-M., Dellu, F., Contarino, A., Bilezikijan, L.M., Gold, L.H., et al., 1998. Corticotropin releasing factor receptor 1deficient mice display decreased anxiety, impaired stress response, and aberrant neuroendocrine development. Neuron 20, 1093–1102. Sola`, C., Tusell, J.M., Serratosa, J., 1999. Comparative study of the distribution of calmodulin kinase II and calcineurin in the mouse brain. J. Neurosci. Res. 57, 651–662. Spina, M., Merlo-Pich, E., Chan, R.K.W., Basso, A.M., Rivier, J., Vale, W., et al., 1996. Appetite- suppressing effects of Urocortin, a corticotropin-releasing factor-related neuropeptide. Science 273, 1561–1564. Steckler, T., Holsboer, M., 1999. Corticotropin-releasing hormone receptor subtypes and emotion. Biol. Psychiatry 46, 1480–1508. Stenzel-Poore, M.P., Cameron, V.A., Vaughan, J., Sawchenko, P.E., Vale, W., 1992. Development of Cushing’s syndrome in corticotropinreleasing factor transgenic mice. Endocrinology 130, 3378–3386. Stenzel-Poore, M.P., Heinrichs, S.C., Rivest, S., Koob, G.F., Vale, W.W., 1994. Overproduction of corticotropin-releasing factor in transgenic mice: a genetic model of anxiogenic behavior. J. Neurosci. 14, 2579–2584. Swanson, L.W., Sawchenko, P.E., Rivier, J., Vale, W.W., 1983. Organization of ovine corticotropin-releasing factor immunoreactive cells and fibers in the rat brain: an immunohistochemical study. Neuroendocrinology 36, 165–186. Takahashi, L.K., 2001. Role of cortocotropin-releasing factor 1 and corticotropin-releasing factor 2 receptors in fear and anxiety. Neurosci. Biobehav. Rev. 25, 627–636. Takahashi, L.K., Ho, S.P., Livanov, V., Graciani, N., Arneric, S.P., 2001. Antagonism of corticotropin-releasing factor (2) receptors produces anxiolytic behavior in animal models of anxiety. Brain Res. 902, 135– 142. Tecott, L.H., 2003. The genes and brains of mice and men. Am. J. Psychiatry 160, 646–656. Timpl, P., Spanagel, R., Sillaber, I., Kresse, A., Reul, J.M.H.M., Stalla, G.K., et al., 1998. Impaired stress response and reduced anxiety in mice lacking a functional corticotropin-releasing hormone receptor 1. Nat. Genet. 19, 162–166. Valdez, G.R., Inoue, K., Koob, G.F., Rivier, J., Vale, W., Zorrilla, E.P., 2002. Human urocortin II: mild locomotor suppressive and delayed anxiolytic-like effects of a novel corticotropin-releasing factor-related peptide. Brain Res. 943, 142–150. Valdez, G.R., Zorrilla, E.P., Rivier, J., Vale, W.W., Koob, G.F., 2003. Locomotor suppressive and anxiolytic-like effects of urocortin 3, a highly selective type 2 corticotropin-releasing factor agonist. Brain Res. 980, 206–212. Vale, W., Spiess, J., Rivier, C., Rivier, J., 1981a. Characterization of a 41residue ovine hypothalamic peptide that stimulates secretion of corticotropin and b-endorphin. Science 213, 1394–1397. Vale, W., Spiess, J., Rivier, C., Rivier, J., 1981b. Characterization of a 41residue ovine hypothalamic peptide that stimulates secretion of corticotropin and beta-endorphin. Science 213, 1394–1397.

M.E. Keck et al. / Neuroscience and Biobehavioral Reviews 29 (2005) 867–889 Van Bockstaele, E.J., Colago, E.E., Valentino, R.J., 1998. Amygdaloid corticotropin-releasing factor targets locus coeruleus dendrites: substrate for the co-ordination of emotional and cognitive limbs of the stress response. J. Neurendocrinol. 10, 743–757. Van Gaalen, M.M., Stenzel-Poore, M.P., Holsboer, F., Steckler, T., 2002. Effects of transgenic overproduction of corticotropin-releasing hormone on anxiety-like behaviour. Eur. J. Neurosci. 15, 2007–2015. Vaughan, J., Donaldson, C., Bittencourt, J., Perrin, M.H., Lewis, K., Sutton, S., et al., 1995. Urocortin, a mammalian neuropeptide related to fish urotensin I and to corticotropin-releasing factor. Nature 378, 287–292. Venihaki, M., Sakihara, S., Subramanian, S., Dikkes, P., Weninger, S.C., Liapakis, G., et al., 2004. Urocortin III, a brain neuropeptide of the corticotropin-releasing hormone family: modulation by stress and attenuation of some anxiety-like behaviours. J. Neurendocrinol. 16, 411–422. Vetter, D.E., Li, C., Zhao, L., Contarino, A., Liberman, M.C., Smith, G.W., et al., 2002. Urocortin-deficient mice show hearing impairment and increased anxiety-like behavior. Nat. Genet. 31, 363–369. von Bardeleben, U., Holsboer, F., Stalla, G.K., Mu¨ller, O.A., 1985. Combined administration of human corticotropin-releasing factor and lysine vasopressin induces cortisol escape from dexamethasone suppression in healthy subjects. Life Sci. 37, 1613–1618. Wang, X., Su, H., Copenhagen, L.D., Vaishnav, S., Pieri, F., Do Shope, C., et al., 2002. Urocortin-deficient mice display normal stress-induced anxiety behavior and autonomic control but an impaired acoustic startle response. Mol. Cell Biol. 22, 6605–6610. Watts, A.G., 1996. The impact of physiological stimuli on the expression of corticotropin-releasing hormone (CRH) and other neuropeptide genes. Front. Neuroendocrinol. 17, 281–326.

889

Weninger, S.C., Dunn, A.J., Muglia, L.J., Dikkes, P., Miczek, K.A., Swiergiel, A.H., et al., 1999. Stress-induced behaviors require the corticotropin-releasing hormone (CRH) receptor, but not CRH. Proc. Natl Acad. Sci. USA 96, 8283–8288. Weninger, S.C., Peters, L.L., Majzoub, J.A., 2000. Urocortin expression in the Edinger-Westphal nucleus is upregulated by stress and corticotropin-releasing hormone deficiency. Endocrinology 2000;, 256–263. Willner, P., Muscat, R., Papp, M., 1992. Chronic mild stress-induced anhedonia: a realistic animal model of depression. Neurosci. Biobehav. Rev. 16, 525–534. Wong, M.L., Al-Shekhlee, A., Bongiorno, P.B., Esposito, A., Khatri, P., Sterneberg, E.M., et al., 1998. Localization of urocortin messenger RNA in rat brain and pituitary. Mol. Psychiatry 1, 307–312. Yamamoto, H., Maeda, T., Fujimura, M., Fujimiya, M., 1998. Urocortinlike immunoreactivity in the substantia nigra, ventral tegmental area and Edinger-Westphal nucleus of rat. Neurosci. Lett. 243, 21–24. Zhao, L., Donaldson, C.J., Smith, G.W., Vale, W.W., 1998. The structures of the mouse and human urocortin genes (Ucn and UCN). Genomics 50, 23–33. Zobel, A.W., Yassouridis, A., Frieboes, R-M., Holsboer, F., 1999. Prediction of medium-term outcome by cortisol response to the combined dexamethasone/CRH test in patients with remitted depression. Am. J. Psychiatry 156, 949–951. Zobel, A.W., Nickel, T., Ku¨nzel, H.E., Ackl, N., Sonntag, A., Ising, M., et al., 2000. Effects of the high affinity corticotropin-releasing hormone receptor 1 antagonist R121919 in major depression: the first 20 patients treated. J. Psychiatric Res. 34, 171–181. Zorrilla, E.P., Valdez, G.R., Nozulak, J., Koob, G.F., Markou, A., 2002. Effects of antalarmin, a corticotropin-releasing factor type 1 receptor antagonist, on anxiety-like behavior and motor activation in the rat. Brain Res. 952, 188–199.