Molecular Brain Research 89 (2001) 29–40 www.elsevier.com / locate / bres Research report Attenuation of fear conditioning by antisense inhibition of brain corticotropin releasing factor-2 receptor * Siew Peng Ho , Lorey K. Takahashi, Valentin Livanov, Karen Spencer, Treena Lesher, Carla Maciag, Mark A. Smith, Kenneth W. Rohrbach, Paul R. Hartig, Stephen P. Arneric CNS Diseases Research, DuPont Pharmaceuticals, Experimental Station E400, Wilmington, DE 19880-0400, USA Accepted 30 January 2001 Abstract Corticotropin releasing factor (CRF) is an important regulator of the endocrine, behavioral, autonomic and immune responses to stress. Two high affinity CRF receptors have been identified, which are distributed in distinct anatomical regions. CRF receptors have been 1 relatively well characterized and antagonists to this receptor effectively block stress-induced behaviors in rodents. The function of CRF 2 receptors, which are highly expressed in limbic brain regions, is less well understood. Therefore, an antisense oligonucleotide approach was used to study the role of CRF receptors in the lateral septum in rats. An antisense oligonucleotide directed against the CRF receptor 2 2 mRNA reduced expression of CRF receptors by 60–80%. In shock-induced freezing tests, animals administered the antisense 2 oligonucleotide exhibited a significant reduction in freezing duration. However, pain sensitivity and locomotor activity were unaltered. A four-base mismatch of the antisense sequence had no significant effects on CRF receptor density and on freezing behavior. These data 2 support the involvement of CRF receptors in fear conditioning. CRF receptor antagonists also reduce freezing in this test. Additional 2 1 studies to determine the effects of simultaneous inhibition of both receptor subtypes show that rats receiving both CRF receptor antisense 2 oligonucleotide and CRF receptor antagonist froze significantly less than animals treated with either agent alone. These results provide 1 additional evidence for the role of CRF receptors in mediating the stress-induced actions of endogenous CRF.  2001 Published by 2 Elsevier Science B.V. Theme: Neurotransmitters, modulators, transporters and receptors Topic: Peptide receptor structure and function Keywords: CRF receptor; Corticotropin releasing factor; Fear conditioning; Stress; Antisense oligonucleotide; Shock-induced freezing 2 1. Introduction dysregulation of CRF systems may underlie a variety of stress-related disorders including anxiety and depression Corticotropin releasing factor (CRF), a 41-amino-acid [2,3,28]. peptide hormone, plays a prominent role in regulating the The physiological actions of CRF are mediated through synthesis and secretion of adrenocorticotropic hormone activation of at least two high affinity receptors, CRF and 1 (ACTH) from the anterior pituitary [58]. In addition, the CRF , which are members of the seven-transmembrane 2 neuropeptide regulates autonomic, immune and behavioral family of receptors [15,54]. Evidence from transgenic responses to stress [15]. Central administration of CRF in knockouts [11,53,57], antisense oligonucleotide studies animals produces general autonomic and behavioral activa- [23,37,52] and CRF receptor antagonists [21,39,40,59] 1 tion [16,56], and transgenic mice over-expressing CRF provide evidence for the involvement of CRF receptors in 1 exhibit increased anxiogenic-like behavior [55]. In vivo mediating the anxiogenic effects of CRF. data as well as evidence from clinical studies suggest that The CRF receptor was identified more recently 2 [32,34,38,44] and exists as three splice variants, a, b and g, in humans. CRF and CRF receptor subtypes are 70% 1 2 *Corresponding author. Tel.: 11-302-695-1646; fax: 11-302-695- homologous in their amino acid sequences but appear to be 4162. E-mail address: siew.p.ho@dupontpharma.com (S.P. Ho). pharmacologically [6,15] and anatomically distinct [9,47]. 0169-328X / 01 / $ – see front matter  2001 Published by Elsevier Science B.V. PII: S0169-328X(01)00050-X