ORIGINAL ARTICLE

Conditional mouse mutants highlight mechanisms ofcorticotropin-releasing hormone effects on stress-copingbehaviorA Lu1,6,7, MA Steiner1,7, N Whittle2, AM Vogl1, SM Walser1, M Ableitner1, D Refojo1, M Ekker3,

JL Rubenstein4, GK Stalla1, N Singewald2, F Holsboer1, CT Wotjak1, W Wurst1,5 and JM Deussing1

1Max Planck Institute of Psychiatry, Munich, Germany; 2Department of Pharmacology and Toxicology, University of Innsbruck,Innsbruck, Austria; 3Department of Biology, Center for Advanced Research in Environmental Genomics, University of Ottawa,Ottawa, ON, Canada; 4Nina Ireland Laboratory of Developmental Neurobiology, University of California, San Francisco, CA,USA and 5Institute for Developmental Genetics, Helmholtz Zentrum Munchen, German Research Center for EnvironmentalHealth, Neuherberg, Germany

Hypersecretion of central corticotropin-releasing hormone (CRH) has been implicated in thepathophysiology of affective disorders. Both, basic and clinical studies suggested thatdisrupting CRH signaling through CRH type 1 receptors (CRH-R1) can ameliorate stress-related clinical conditions. To study the effects of CRH-R1 blockade upon CRH-elicitedbehavioral and neurochemical changes we created different mouse lines overexpressing CRHin distinct spatially restricted patterns. CRH overexpression in the entire central nervoussystem, but not when overexpressed in specific forebrain regions, resulted in stress-inducedhypersecretion of stress hormones and increased active stress-coping behavior reflected byreduced immobility in the forced swim test and tail suspension test. These changes wererelated to acute effects of overexpressed CRH as they were normalized by CRH-R1 antagonisttreatment and recapitulated the effect of stress-induced activation of the endogenous CRHsystem. Moreover, we identified enhanced noradrenergic activity as potential molecularmechanism underlying increased active stress-coping behavior observed in these animals.Thus, these transgenic mouse lines may serve as animal models for stress-elicited pathologiesand treatments that target the central CRH system.Molecular Psychiatry (2008) 13, 1028–1042; doi:10.1038/mp.2008.51; published online 13 May 2008

Keywords: corticotropin-releasing hormone; depression; forced swim test; HPA axis; ROSA26;DMP696

Introduction

Corticotropin-releasing hormone (CRH)—also desig-nated as corticotropin-releasing factor (CRF)—isimportant in coordinating the neuroendocrine,autonomic, behavioral and immunological responsesto various stressful stimuli.1 Besides its function asthe major physiological regulator of the hypothala-mic–pituitary–adrenocortical (HPA) system, CRHis capable of modulating a wide range of behaviors,including anxiety-related behavior, arousal, sensoryinformation processing, learning and memory as well

as locomotor activity.2–4 Most behavioral effects ofCRH are attributed to extrahypothalamic neuronalcircuits including neocortical, limbic and brainstemstructures where CRH functions as a neuromodulator.Dysregulation of the CRH system and accompanyingchronically elevated levels of CRH are implicated inhuman stress-related and affective disorders, includ-ing anxiety disorders and major depression.5–7 In thisline, elevated levels of CRH in the cerebrospinalfluid,5 increased numbers of CRH and CRH/argininevasopressin-expressing neurons and elevated CRHmRNA levels in the paraventricular nucleus of thehypothalamus (PVN),8 as well as decreased CRHbinding sites in the frontal cortex have been demon-strated in depressed patients. In addition, animalstudies involving central application of CRH revealedphenotypic alterations reminiscent of symptomsobserved in affected subjects.4 Moreover, CRH recep-tor antagonists are capable of attenuating the beha-vioral consequences of stress, underscoring the role ofendogenous CRH in mediating many stress-induced

Received 26 August 2007; revised 18 February 2008; accepted 2April 2008; published online 13 May 2008

Correspondence: Dr JM Deussing, Max Planck Institute ofPsychiatry, Kraepelinstrasse 2-10, Munich 80804, Germany.E-mail: deussing@mpipsykl.mpg.de6Current address: Clinical Endocrinology Branch, National

Institute of Diabetes and Digestive and Kidney Diseases, National

Institutes of Health, Bethesda, MD 20892, USA.7These authors contributed equally to this work.

Molecular Psychiatry (2008) 13, 1028–1042& 2008 Nature Publishing Group All rights reserved 1359-4184/08 $30.00

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behaviors. Finally, clinical trials have demonstratedthe efficacy of a selective CRH type 1 receptor (CRH-R1)antagonist in treating depressed patients.9

Unlike classical antidepressants that modulate themonoaminergic system, CRH receptor antagonistshave not produced comparable results in antidepres-sant screening paradigms in rodents, such as theforced swim test (FST) or the tail suspension test.10 Inthese tests animals were studied under basal condi-tions. However, according to the hypothesis byHokfelt and Lundberg,11 neuropeptides, unlike mono-amines, are only secreted at substantial amountsunder pathological conditions or severe stress.Accordingly, anxiolytic-like activities of CRH-R1antagonists such as DMP696 and R121919 are bestobserved in animals that have increased levels ofCRH, and are hyperresponsive or more susceptible tostress.12 These findings are in agreement with ablunted stress hormone response in healthy humanspretreated with a CRH-R1 antagonist.13 To study theeffects of central CRH hyperactivity in an animalmodel, CRH transgenic mouse lines were establishedexpressing CRH either under the control of thebroadly active metallothionein (CRH-Tg) or thecentral nervous system (CNS)-restricted Thy-1.2(CRH-OE2122) promoter.14,15 In both cases, unrestrictedCRH overexpression resulted in elevated adrenocorti-cotropin (ACTH) and corticosterone levels accompa-nied by symptoms of Cushing-like syndrome,complicating the interpretation of stress-related be-havioral results. To circumvent these problems wedeveloped a mouse model that permits the over-expression of CRH without producing marked neu-roendocrine disturbances under basal conditions.Combining the knock-in of a single copy of themurine Crh cDNA into the ROSA26 (R26) locus16

with the Cre/loxP system has enabled us to over-express CRH in a spatio-temporally regulated fashionat different dosages. We activated CRH expression inthe entire brain and in specific types of neuronswithin the forebrain using Nes-cre,17 Camk2a-cre18

and Dlx-cre.19 We demonstrated that this approachis an effective way to study the behavioral andneuroendocrine effects of spatially confined CRHoverexpression in mice.

Materials and methods

Targeting vectorThe targeting vector was based on pROSA26-1bearing 5.5-kb homology to the murine ROSA26 locus(R26) and a diphtheria toxin (DTA) expressioncassette.20 It was constructed by introducing thefollowing components into the unique XbaI site ofpROSA26-1: adenovirus splice acceptor (SA), loxP,PGK-Neo—including PGK polyadenylation sequence(pA) and two copies of the SV40 pA (PGK-Neo-3�pA), loxP, IRES-LacZ and bovine growth hormone(bGH) pA (from 50 to 30). The SA and bGH pA weresubcloned from pSAbgeo.21 The IRES-LacZ wasisolated and modified from ETLpA-/LTNL.22 The

loxP-flanked PGK-Neo-3�pA cassette was amplifiedby PCR from genomic tail DNA of R26 reporter mice.20

The DTA cassette was inverted and concomitantly anSwaI site was introduced for linearization. Themurine Crh cDNA was inserted in a unique PacI sitebetween the second loxP site and the IRES-LacZcassette. External probes used for identification ofhomologous recombination events were amplified byPCR from genomic DNA and cloned using the TOPOTA cloning kit (Invitrogen, Karlsruhe, Germany).50-Probe: forward 50-GCG-AGA-CTC-GAG-TTA-GGC-30 and reverse 50-GCG-GCC-GCC-GCC-CGC-CTG-CG-30

(150 bp); 30-probe: forward 50-GTT-GAG-CCA-CTG-AGA-ATG-G-30 and reverse 50-GAA-ACT-ACA-ACC-ATT-GTT-CAT-30 (662 bp).

Generation of conditional CRH overexpressing miceThe linearized targeting vector was electroporatedinto TBV2 embryonic stem (ES) cells (129S2). MutantES cell clones were identified by Southern blotanalysis of genomic ES cell DNA digested with EcoRVor ApaI using the external 50- or 30-probe respectively.Mutant ES cells were used to generate chimeric miceby blastocyst injection. Germ-line transmission of themodified R26 allele (R26flopCrh floxed stop) wasconfirmed in offspring from male chimeras bred towild-type C57BL/6J mice. For the conditional, CNS-restricted overexpression of CRH (CRH-COE-Nes),obtained R26þ /flopCrh mice were crossed to transgenicnestin (Nes)-cre mice.17 Resulting heterozygousR26þ /flopCrh and R26þ /flopCrh Nes-cre F1 animals wereintercrossed to obtain in the F2 generation animals ofthe desired genotypes: R26þ /þ (CRH-COEwt-Nes), R26flopCrh/flopCrh (CRH-COEcon-Nes), R26þ /flopCrh

Nes-cre (CRH-COEhet-Nes) and R26flopCrh/flopCrh Nes-cre(CRH-COEhom-Nes). For the forebrain-restricted over-expression of CRH in principal neurons (CRH-COE-Cam), R26þ /flopCrh mice were crossed to transgenicCamk2a-cre mice.18 As above, R26þ /þ (CRH-COEwt-Cam), R26flopCrh/flopCrh (CRH-COEcon-Cam), R26þ /flopCrh

Camk2a-cre (CRH-COEhet-Cam) and R26flopCrh/flopCrh

Camk2a-cre (CRH-COEhom-Cam) animals wereobtained in the F2 generation. CRH-COE-Dlx miceoverexpressing CRH in GABAergic neurons of theforebrain were accordingly generated using Dlx5/6(Dlx)-cre mice.19

Genotyping was performed by PCR usingprimers: ROSA-1, 50-AAA-GTC-GCT-CTG-AGT-TGT-TAT-30; ROSA-2, 50-GCG-AAG-AGT-TTG-TCC-TCA-ACC-30 and ROSA-4, 50-GGA-GCG-GGA-GAA-ATG-GAT-ATG-30. Standard PCR conditions resulted in a398-bp wild-type and a 320-bp mutant PCR product.The presence of Nes-, Camk2a- and Dlx-cre wasevaluated using primers CRE-F, 50-GAT-CGC-TGC-CAG-GAT-ATA-CG-30 and CRE-R 50-AAT-CGC-CAT-CTT-CCA-GCA-G-30 resulting in a PCR product of 574bp.Genotypes were confirmed by Southern blot analysisof EcoRV-digested tail DNA using the 50-probe and aCre-recombinase-specific probe.23 The efficiency ofNes-cre-mediated excision of the transcriptionalterminator sequence was demonstrated by Southern

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blot analysis of EcoRV-digested genomic DNAprepared from cortex, hippocampus, thalamus, cere-bellum, tail and liver using the external 50-probe.Mice used for this study were kept on a mixed 129S2/Sv�C57BL/6J background.

X-Gal stainingAnimals (2- to 3-month old; n = 4–5 per genotype)were killed by an overdose of isoflurane and transcar-dially perfused with 4% paraformaldehyde, 2 mM

MgSO4 and 5 mM EGTA. Subsequent X-Gal stainingwas performed on free floating 50- or 100-mm thickvibratome sections or on intact organs as previouslydescribed.22

In situ hybridizationMice (10-week old) were killed in the morning(10:00 am) by an overdose of isoflurane. For quantifi-cation of immediate early genes (IEGs) c-fos and zif268animals were either killed under basal conditions orsubjected to 10 min of forced swimming 30 min beforekilling. Brains were carefully removed and immedi-ately shock-frozen on dry ice. Frozen brains were cuton a cryostat in 20-mm thick sections. For quantitativein situ hybridization cryostat sections of CRH-COEcon

and CRH-COEhom brains were mounted side by sideon SuperFrost Plus slides (Menzel GmbH, Braunsch-weig, Germany). This procedure allowed for parallelin situ hybridization of sections under identicalconditions assuring meaningful quantification andcomparison of hybridization signals. All sectionswere processed for in situ hybridization according toa modified version of the procedure described byDagerlind et al.24 The following riboprobes were used:CRH, nucleotides 1306–1661 of GenBank accessionno. AY128673; endogenous CRH (30-UTR), nucleo-tides 1816–2107 of GenBank accession no. AY128673;CRH-R1, nucleotides 1570–2273 of GenBank acces-sion no. NM_007762; c-fos, nucleotides 608–978 ofGenBank accession no. NM_010234; zif268, nucleo-tides 245–786 of GenBank accession no. NM_007913and LacZ, nucleotides 192–569 of GenBank accessionno. U46489. Specific riboprobes were generated byPCR applying T7 and T3 or SP6 primers usingplasmids containing above-mentioned cDNAs astemplates. Antisense and sense cRNA probes weretranscribed from 200 ng of respective PCR productand directly used as a template for the synthesis ofradiolabeled transcripts by in-vitro transcription with35S-UTP (Amersham Biosciences, Piscataway, NJ,USA) using T7 and T3 RNA polymerase (Roche,Penzberg, Germany), respectively. After 20 min ofDNase I (Roche) treatment, the probes were purifiedby the RNeasy Clean up protocol (Qiagen, Hilden,Germany) and measured in a scintillation counter. Forhybridization, sections were pretreated and prehy-bridized as previously described.24 Subsequently,they were hybridized overnight with a probe concen-tration of 7� 1 06 c.p.m. ml�1 at 57 1C and washed at64 1C in 0.1� saline sodium citrate (SSC) and 0.1 mM

dithiothreitol. The hybridized slides were dipped in

autoradiographic emulsion (type NTB2; EastmanKodak, Rochester, NY, USA), developed after 3–6weeks and counterstained with cresyl violet.

For quantification, autoradiographs were digitizedand relative levels of mRNA were determined bycomputer-assisted optical densitometry (ImageJ;http://rsb.info.nih.gov/ij/). For in situ hybridizationsroutinely three different exposure times were appliedto assure that the signals to be quantified were in thelinear range.

CRH radioimmunoassayMice (2- to 3-month old) were killed by cervicaldislocation at 10:00 am. Brains were carefullyremoved and used in total or further dissected forselective preparation of cortex, hippocampus andthalamus. The CRH-specific radioimmunoassay (RIA)on tissue homogenates was performed after priorextraction as previously described.25

Endocrine analysesTwo weeks before the experiments, 3- to 5-month-oldanimals were separated and singly housed with a12:12 h light:dark schedule (lights off at 07:00 pm).All experiments and data analyses were performedseparately for male and female animals. To determinethe basal hormone plasma levels, mice were leftundisturbed throughout the night before the experi-ment. Blood sampling was performed in the earlymorning (07:30–09:30 am) and afternoon (04:30–05:30pm) by collecting trunk blood from animals rapidlydecapitated under light isoflurane anesthesia or byincision of the tail, with the time from first handlingof the animal to completion of bleeding not exceeding45 s. For evaluation of the endocrine response tostress, we collected blood samples immediately afterand 30 min after 10-min restraint stress, for whichanimals were placed in a 50-ml conical tube with thebottom removed. Stress experiments were performedin the morning (07:30–10:00 am). Plasma corticoster-one and ACTH concentrations were measured induplicate by commercially available RIA kits (ICNBiomedicals, Irvine, CA, USA).

Subjects for behavioral testingMice were singly housed 2 weeks before experimentsunder standard laboratory conditions (22±1 1C,55±5% humidity) with food and water ad libitumunder a 12:12 h inverted light:dark schedule (lightsoff at 09:00 am). Age of tested animals ranged between3 and 6 months. Animal experiments were conductedin accordance with the Guide for the Care and Use ofLaboratory Animals of the Government of Bavaria.Experiments were performed during the dark, activephase of the animals between 01:00 pm and 06:00 pmhours under red light conditions, unless otherwisestated. Animals’ behavior in the tail suspension andFST was analyzed online by trained observers whowere blind to treatment and genotype. If not statedotherwise, male mice were used for the experiments.Experiments to assess the behavioral consequences of

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stress-regulated activation of endogenous CRH wereperformed on previously described CRH-R1 knockoutmice,26 which were kept on a mixed 129/Ola�CD1background.

Open field

Animals were tested under red light in an open field(26� 26� 38 cm high) made of white floor and clearplastic walls, and equipped with infrared photocellsensors. Testing lasted for 30 min. Distance travelledand rearings were measured using the Tru ScanSoftware version 1.1 A (Coulbourn Instruments,Allentown, PA, USA) (sampling frequency, 4 Hz).

Forced swim test

Each mouse was placed in a 5-l glass beaker (height,23.5 cm; diameter, 16.5 cm) containing 15 cm of waterat 25±1 1C or 32±1 1C for 6 min. The water waschanged between subjects. During each trial, floating(immobility) and struggling time was scored bypressing preset keys on a computer keyboard, usinga customized freeware software (EVENTLOG). Theresulting l-channel ethogram was further processedby customized software (Winrat version 2.31; HeinzBarthelmes, MPI Munich, Germany). A mouse wasjudged floating when it stopped any movementsexcept those that were necessary to keep its headabove water. Vigorous swimming movements invol-ving all four limbs of the mouse with the front pawsbreaking the surface of the water, usually at the wallsof the beaker, were regarded as struggling. The testwas carried out on 2 consecutive days.

For the analysis of the influence of restraint stressexperience on FST behavior, animals were subjectedto 1 h of restraint stress in a 50-ml conical tube 4 hbefore exposure to forced swimming according to apreviously established protocol for stress-inducedactivation of CRH-R1.27

Tail suspension test

Animals were suspended by the end of their tail withadhesive tape to a steel bar that was 35 cm above thefloor. Each session lasted 6 min and was videotaped.The duration of immobility was scored by a trainedobserver who was blind to the animals’ genotype,using EVENTLOG software. Mice were consideredimmobile only when they hung passively withoutmoving the limbs or the head.

Pharmacology

All drugs were freshly prepared in a volume of10 ml kg�1. DMP696 (Bristol-Myers Squibb, Munich,Germany) was suspended in a 0.9% saline solutioncontaining 5% dimethyl sulfoxide, 5% polyethyleneglycol 400 and 10 ml of Tween 80 per 1.5 ml (allchemicals from Sigma-Aldrich, Steinheim, Germany),and injected i.p. 1 h before the FST on days 1 and 2.For the analysis of DMP696 influences on stress-

induced ACTH levels, mice were decapitated imme-diately after the FST on day 2.

Para-chlorophenylalanine methyl ester (PCPA) anda-methyl-para-tyrosine methyl ester (AMPT) (alldrugs from Sigma-Aldrich, Steinheim, Germany) weresuspended in a 0.9% saline solution containing 1%dimethyl sulfoxide and administered i.p. PCPA(250 mg per kg) was administered twice daily (every12 h) for 3 days with the last dose given 18 h beforethe FST.28 AMPT (200 mg per kg) was administered asa single dose 4 h before the FST.28

Neurochemical analysisMice treated with AMPT and PCPA were killeddirectly after the FST. Brains were freshly dissectedon ice and hippocampi removed, weighed and storedat �80 1C until analysis. Hippocampal tissue sampleswere diluted 20-fold w/v with HCl (0.1 M) in an icebath and homogenized by sonication (40 s, 75% dutycycle, 3.5 micro tip limit; Branson sonifier 250, SonicPower Company, Danbury, CT, USA). Homogenizedtissue was then ultracentrifuged (35 000 g, 20 min,4 1C; Beckman L-60 Ultracentrifuge, Munich,Germany), and the resulting aqueous layer filtered(0.22 mm� 13 mm, polyvinylidene fluoride, MillexFilters; Millipore, Bedford, TX, USA). Filtrate wasaliquoted in 20 ml samples into separate Eppendorftubes and stored at �80 1C until further analysis.

Determination of serotonin (5-hydroxytryptamine),5-hydroxyindoleacetic acid, 3,4-dihydrophenylaceticacid and 4-hydroxy-3-methoxyphenylacetic acid wasperformed by reverse-phase high-performance liquidchromatography with electrochemical detection aspreviously described.29 Hippocampal filtrates (20 mlstock solutions) were diluted and 50ml were auto-matically injected by a CMA 200 refrigerated auto-sampler (CMA Microdialysis AB, Stockholm,Sweden). The mobile phase consisted of 93% phos-phate buffer (0.1 M NaH2PO4, 1 mM sodium octane-sulphonic acid, 10 mM NaCl and 0.5 mM Na2-EDTA)and 7% acetonitrile, and the pH was adjusted to 4.0with o-phosphoric acid (all chemicals were fromMerck, Darmstadt, Germany). For noradrenaline (NA)and dopamine (DA) tissue quantification, hippocam-pal filtrates (20 ml stock solutions) were diluted andconcentration was determined by a radioenzymaticassay as previously described. This assay involvesCOMT-catalyzed O-methylation using [3H]S-adeno-sylmethionine as methyl donor and separation of theresulting [3H]normetanephrine by thin-layer chromato-graphy (TLC).

Statistical analysisData were analyzed for multiple comparisons usingone-, two- or three-way analyses of variance (ANO-VAs) for repeated measures where appropriate,followed by post-hoc Newman–Keuls multiple com-parison test. For two-group comparisons unpairedStudent’s t-test was used. Differences were consid-ered statistically significant when P < 0.05. Data arepresented as mean±s.e.m.

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Results

Generation of mutant mice conditionallyoverexpressing CRH in the CNS (CRH-COE-Nes)

We used homologous recombination in ES cells totarget the ubiquitously expressed ROSA26 (R26) locuswith a single copy of the murine Crh cDNA precededby a loxP-flanked (floxed) transcriptional ‘Stop’sequence (Figures 1a–c). As previously reported,20

mice homozygous for the modified R26 allele(R26flopCrh/flopCrh floxed Stop), which is Cre-recombi-nase-sensitive, were indistinguishable from wild-typelittermates, behaviorally (data not shown), as well aswith respect to endogenous CRH mRNA (Figures2e and f) and protein levels (Figure 3a). Homozygous

R26flopCrh/flopCrh mice were crossed to transgenic Nes-cremice17 allowing for a CNS-restricted overexpressionof CRH in double transgenic animals (CRH-COE-Nes).In this mouse line Cre expression is controlled by thenestin promoter and neural enhancer, which drive creexpression in neuronal and glial precursors as earlyas embryonic day 10.5.30 In the F2 generation weobtained R26þ /þ , R26flopCrh/flopCrhR26þ /flopCrh Nes-creand R26flopCrh/flopCrh Nes-cre mice (Figure 1d), whichwe will refer to as CRH-COEwt-Nes, CRH-COEcon-Nes,CRH-COEhet-Nes and CRH-COEhom-Nes, respectively.On the genomic level, Cre-mediated deletion of thetranscriptional terminator sequence was observedonly in the CNS, but not in peripheral organs ofCRH-COEhom-Nes mice (Figure 1e).

Figure 1 Generation of mice overexpressing corticotropin-releasing hormone (CRH) restricted to the central nervous system(CNS). (a) Strategy for conditional, Cre-mediated expression of CRH from the R26 locus. Partial restriction maps of wild-typeR26 locus, targeting vector, recombined R26flopCrh allele and activated R26Crh allele (WT, wild-type fragment; MT, mutantfragment following homologous recombination; DEL, deletion fragment resulting from Cre-mediated excision of the ‘Stop’cassette; A, ApaI; E, EcoRV; S, SwaI; S, splice acceptor; loxP sites are indicated as black arrowheads). (b) Southern blotanalysis of wild-type and targeted embryonic stem (ES) cell clones. The R26 50-probe was hybridized to EcoRV-digestedgenomic ES cell DNA. The targeted allele was indicated by the presence of an additional mutant 4.1-kb fragment. (c) The R2630-probe was hybridized to ApaI-digested DNA from the same ES cell clones confirming homologous recombination bydetection of an additional mutant fragment at 11.4 kb. (d) Southern blot analysis of EcoRV-digested tail DNA of CRH-COE-Nesmice simultaneously hybridized with the 50-probe and a Cre-recombinase-specific probe. The hybridizing fragments obtainedcorrespond to the indicated genotypes. (e) Southern blot analysis of EcoRV-digested genomic DNA from various tissues of aCRH-COEhom-Nes animal hybridized with the 50-probe, showing the extent of Cre-mediated deletion of the transcriptionalterminator sequence as indicated by the presence of an additional 5.2-kb fragment.

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Figure 2 Verification of the central nervous system (CNS)-restricted overexpression of corticotropin-releasing hormone(CRH) in CRH-COE-Nes mice. Intact organs of (a) control, (b) heterozygous and (c) homozygous CRH-COE-Nes mice werestained overnight with X-Gal. (left: brain with spinal cord; right, from top: liver, lung, heart, spleen, testis with epididymis;inlay: adrenal gland (left) and pituitary (right). Note the background staining of the epididymis. Intense blue staining in thebrain and spinal cord of CRH-COEhet- and CRH-COEhom-Nes mice reflects the brain-specific overexpression of the IRES-LacZreporter gene. No staining was observed in peripheral organs of CRH-COE-Nes mice. Determination of IRES-LacZ reportergene expression by X-Gal staining on coronal sections of (d) CRH-COEcon-, (g) CRH-COEhet- and (j) CRH-COEhom-Nes mice.Crh overexpression was demonstrated by in situ hybridization using a specific radiolabeled riboprobe detecting bothendogenous and exogenous CRH expression. Representative dark-field photomicrographs of coronal and sagittal brainsections of CRH-COE-Nes mice are depicted. (e, f) CRH-COEcon-Nes mice display the characteristic, heterogeneous Crhexpression throughout the entire CNS with strong expression in the paraventricular nucleus (PVN) of the hypothalamus,central nucleus of the amygdala (CeA), bed nucleus of stria terminalis (BST), olfactory bulb (OB) and nuclei of the brain stem.In addition, CRH-expressing neurons are found scattered within the cortex (CX) and hippocampus (HIP). In (h, i) CRH-COEhet- and (k, l) CRH-COEhom-Nes mice exogenous CRH is expressed throughout the brain corresponding to the pattern ofIRES-LacZ reporter gene expression. The level of exogenous Crh mRNA expression is gene dosage-dependent asdemonstrated by the stronger in situ hybridization signals detected in CRH-COEhom- versus CRH-COEhet-Nes animals(ac, anterior commissure).

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Verification of the CNS-restricted overexpression ofCRH in CRH-COE-Nes mice

To assess the spatial distribution of exogenous CRHexpression in detail, we made use of the introducedIRES-LacZ reporter gene, which is co-activated uponCre-mediated excision of the transcriptional termina-tor sequence (Figure 1a). X-Gal staining of intactorgans revealed the absence of any specific staining inCRH-COEcon-Nes mice (Figure 2a). CRH-COEhet-Nesand CRH-COEhom-Nes mice exhibited an intensestaining in the brain and spinal cord whereasperipheral organs were devoid of LacZ-dependentstaining. Increased staining intensities in the intactbrain and spinal cord as well as on coronal brainsections of CRH-COEhom-Nes compared to CRH-COEhet-Nes animals reflected the assumed gene dosageeffect (Figures 2b, c, g and j). The conditionaloverexpression of CRH was verified by in situhybridization using a CRH-specific riboprobe. InCRH-COEcon-Nes mice, endogenous CRH expressionwas detected heterogeneously throughout the entireCNS as previously described31 (Figures 2e and f). InCRH-COEhet- and CRH-COEhom-Nes mice, the patternof CRH induction paralleled the activation of theIRES-LacZ reporter gene as demonstrated by X-Galstaining. Expression of exogenous CRH was detected

at varying levels throughout the brain and attributedto the CNS-wide expression of Nes-cre and to theubiquitous activity of the R26 locus. CRH mRNA wasdetected at highest levels in the olfactory bulb, cortexand hippocampus, again in a gene dosage-dependentmanner (Figures 2h, i, k and l). Moreover, weconfirmed the increased CRH peptide content in theentire brain and various brain areas of CRH-COEhet-Nes and CRH-COEhom-Nes mice using a CRH-specificRIA (Figures 3a–e). No difference in CRH peptidecontent was observed between CRH-COEwt- and CRH-COEcon-Nes mice (Figure 3a).

Expression of endogenous CRH and CRH-R1 is alteredin the brain of CRH-COE-Nes miceTo explore alterations in the expression of endogen-ous CRH and CRH-R1 in response to exogenous CRHexpressed from the R26 locus, we used in situhybridization. We observed a strong decrease ofendogenous CRH mRNA levels in the PVN (Supple-mentary Figure 1c), in all areas of the hippocampusproper (CA1-CA3) and in the dentate gyrus (Supple-mentary Figure 1a) of CRH-COEhom-Nes mice com-pared to control littermates. However, in the CeA(Supplementary Figure 1b) endogenous CRH mRNAlevels were significantly increased. CRH-COEhom-Nes

Figure 3 Corticotropin-releasing hormone (CRH) overexpression from the R26 locus results in a gene dosage-dependentincrease of CRH protein content in the (a) entire brain, (b) cortex, (c) hippocampus, (d) thalamus and (e) cerebellum of maleCRH-COEhet-Nes (het) and CRH-COEhom-Nes (hom) mice in comparison to CRH-COEcon-Nes (con) mice (n = 5–12). The CRHcontent in the brain of CRH-COEcon-Nes mice is indistinguishable from wild-type CRH-COEwt-Nes (wt) mice. CRH content isgiven as pg mg�1 tissue wet weight. *P < 0.05, **P < 0.01, ***P < 0.001.

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mice exhibited increased CRH-R1 mRNA levels in thehippocampus and dentate gyrus (SupplementaryFigure 1d). Surprisingly, CRH-R1 mRNA levels inthe hippocampus were only increased in the CA2 andCA3, however not in CA1 (Supplementary Figure 1d).In addition, CRH-R1 expression was significantlyincreased in the basolateral amygdala of homozygousCRH overexpressing mice (Supplementary Figure 1e).

HPA axis of male but not female CRH-COE-Nes mice ishypersensitive to stressBasal plasma ACTH and corticosterone levels did notdiffer significantly between male CRH-COEcon-, CRH-COEhet- and CRH-COEhom-Nes mice over the circadiancycle, neither at the diurnal trough, nor at the diurnalpeak (Supplementary Figures 2a and c). To examinethe response of the HPA axis to stress, animals weresubjected to 10 min of restraint stress in the morningand killed either directly or 30 min after the end of thestressor (Supplementary Figures 2b and d). Restraintstress resulted in significantly elevated ACTH(F2,32 = 7.5; P < 0.01) and corticosterone (F2,32 = 4.10;P < 0.05) levels in male CRH-COEhet- and CRH-COEhom-Nes mice compared to CRH-COEcon-Nes lit-termates. In CRH-COEhom-Nes mice, corticosteronelevels remained significantly elevated 30 min afterstress compared to CRH-COEcon-Nes and CRH-COEhet-Nes mice (F2,27 = 4.10; P < 0.05).

Similar to males, female CRH-COEhom-Nes animalsshowed no difference in basal ACTH and corticoster-one levels compared to CRH-COEcon-Nes mice (Supple-mentary Figures 2e and f). However, in contrast tomales, female CRH-COEhom-Nes mice also did notshow elevated ACTH and corticosterone secretion inresponse to restraint stress in comparison with CRH-COEcon-Nes mice (Supplementary Figures 2e and f).

CNS-restricted overexpression of CRH results indecreased immobility in the FST and the tailsuspension testIn the open field, CRH-COEhom mice showed nodifference in locomotor activity compared to CRH-COEcon littermates (Figure 4a). CRH-COEhom mice didshow increased exploratory behaviors compared toCRH-COEcon littermates as indicated by an elevatednumber of vertical movements (rearings) (t18 = 2.14,P < 0.05; Figure 4a). To examine stress-coping beha-viors, CRH-COE-Nes mice were exposed to the FST. Inmale mice, CRH overexpression resulted in a genedosage-dependent decrease in floating in the FSTboth on day 1 (F2,30 = 20.0; P < 0.001) and on day 2(F2,30 = 15.0; P < 0.001) with both CRH-COEhom-Nesand CRH-COEhet-Nes mice floating significantly lessthan CRH-COEcon-Nes littermates (Figure 4b). Further-more, CRH overexpression resulted in a dose-depen-dent increase in struggling both on day 1 (F2,30 = 3.4;P < 0.05) and on day 2 (F2,30 = 4.3; P < 0.05) with CRH-COEhom-Nes mice struggling generally more thanCRH-COEcon-Nes littermates (data not shown). FemaleCRH-COEhom-Nes mice showed essentially the samephenotype in the FST as males: they floated signifi-

cantly less than their CRH-COEcon-Nes littermatesboth on days 1 and 2 (Supplementary Figure 3a).

Exposure to cold water (25 1C) in the FST leads tofast body cooling in mice. In order to exclude that thephenotype of CRH-COEhet- and CRH-COEhom-Nesmice in the FST did not solely derive from a fasterreduction of body temperature compared to CRH-COEcon-Nes littermates, we repeated the FST at 32 1Cwater temperature. Also at 32 1C, CRH-COEhom-Nesmice floated significantly less than CRH-COEcon-Neslittermates (Supplementary Figure 3b).

To further corroborate the finding that CRH pro-motes a reduction of immobility in the FST, wesubjected male animals to the tail suspension test(TST).32 Again, CRH-COEhom-Nes mice displayedsignificantly decreased immobility compared toCRH-COEcon-Nes and CRH-COEhet-Nes littermates(F2,83 = 8.9; P < 0.001), no difference was observedbetween CRH-COEhet-Nes and CRH-COEcon-Nes ani-mals (Figure 4c).

Effects of CRH overexpression on FST behavior can bereversed by the selective CRH-R1 antagonist DMP696

To ascertain that the behavioral as well as theneuroendocrine consequences of CRH overexpressionrather relate to acute effects of CRH than to long-termchanges in brain physiology caused by the life-longoverexpression of CRH, we treated male CRH-COEhet

mice with the selective CRH-R1 antagonist DMP696.33

Antagonist treatment dose-dependently reversed theFST phenotype by increasing floating time both onday 1 (F2,35 = 4.4; P < 0.05; Figure 4d) and on day 2(F2,35 = 4.4; P < 0.05; Figure 4d). Furthermore, DMP696attenuated the swim stress-induced hyperactivationof the HPA axis (F2,14 = 10.6; P < 0.01; Figure 4e).

Conditional CRH overexpression mimics the behavioralconsequences of stress-induced activation of theendogenous CRH system

To assess whether overexpression of exogenous CRHmimics the behavioral consequences of stress-regu-lated activation of endogenous CRH, we analyzed FSTbehavior of CRH-R1 knockout (CRH-R1�/�) mice26 andwild-type littermates (CRH-R1þ /þ ) without or withprior 1-h restraint stress. In accordance with ourprevious findings,34,35 ablation of CRH-R1 failed toaffect floating behavior in naive animals, thus arguingagainst a general involvement of endogenous CRH inFST behavior. However, prior restraint stress resultedin a decrease in floating in CRH-R1þ /þ mice com-pared to nonstressed CRH-R1þ /þ controls (t27 = 3.7,P < 0.01) that was similar to the behavior of CRH-COEhet-Nes and CRH-COEhom-Nes mice (Figure 4f).The floating response was also significantly smallerthan that of restraint stressed CRH-R1�/� mice(t29 = 3.1, P < 0.01), which remained unaffected bythe stressor (t27 = 1.2, P < 0.243). These results implythat the CRH system must be activated by priorstressor exposure before endogenous CRH may mod-ulate behavior in the FST via CRH-R1.

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Forebrain-restricted overexpression of CRH inprincipal neurons or GABAergic interneurons does notaffect stress-coping behavior in the FST

In order to specify the brain regions and neurochem-ical substrates involved in the immobility-reducingeffect of exogenous CRH, we exploited the propertiesof the conditional R26flopCrh allele. Breeding CRH-

COEcon mice to Camk2a-cre18 and Dlx-cre19 micerestricted the CRH overexpression to principal neu-rons or GABAergic interneurons respectively, of theanterior forebrain including limbic brain structures.The Dlx5/6 promoter drives cre expression as early asembryonic day 10.5 (JL Rubenstein and M Ekker,unpublished data), whereas the Camk2a promoter

Figure 4 Corticotropin-releasing hormone (CRH) overexpression leads to an increase in active stress-coping behavior inantidepressant screening paradigms. (a) During a 30 min exposure to an open field, male CRH-COEhom-Nes (hom) miceshowed no difference in total distance moved compared to CRH-COEcon-Nes (con) littermates (left panel). However, CRH-COEhom mice showed increased vertical movements (rearings; right panel, n = 8–12 animals per genotype). (b) In the forcedswim test (FST; 25 1C) CRH overexpression resulted in a dose-dependent decrease in floating both on day 1 (d1) and on day 2(d2) with both male CRH-COEhet- (het) and CRH-COEhom-Nes (hom) mice floating significantly less than their male CRH-COEcon-Nes (con) littermates (n = 9–15 animals per genotype). (c) In the tail suspension test male CRH-COEhom-Nes miceshowed significantly decreased immobility compared to male CRH-COEcon- and CRH-COEhet-Nes littermates. Data werecollapsed from two independent experiments, which revealed essentially the same results, resulting in n = 27–30 mice pergenotype and protocol. (d) DMP696 (applied at 10 and 50 mg per kg, i.p. 1 h before testing on days 1 and 2) dose-dependentlyincreased floating behavior of CRH-COEhet-Nes mice compared to vehicle-treated controls (0 mg per kg) both on day 1 (d1)and on day 2 (d2) (n = 12–13 animals per group). (e) DMP696 was able to attenuate the swim stress-induced increase ofadrenocorticotropin (ACTH) on day 2 in CRH-COEhet mice at doses of 10 and 50 mg per kg (n = 5–6 animals per group).(f) Previous exposure to restraint stress resulted in decreased floating behavior in the forced swim test in CRH type 1 receptor(CRH-R1) wild-type (CRH-R1þ /þ ) mice compared to unstressed controls and stressed CRH-R1 knockout (CRH-R1�/�) animalson day 1. Restraint stress failed to significantly affect floating behavior of CRH-R1�/� animals (n = 13–16 animals per group).*P < 0.05, **P < 0.01, ***P < 0.001.

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drives cre expression from around postnatal day 15.18

Analogous to CRH-COE-Nes mice we obtaineddesired control (CRH-COEcon-Cam/-Dlx), heterozygous(CRH-COEhet-Cam/-Dlx) and homozygous (CRH-COEhom-Cam/-Dlx) CRH-COE-Cam/-Dlx mice, respectively inthe F2 generation. In situ hybridization confirmedthe forebrain-restricted expression of exogenousCRH in both overexpressing mouse lines (Figures 5a,c and e). The function of the HPA axis of homozygousCRH-COE-Cam/-Dlx mice was not significantlyaltered compared to control mice, neither underbasal nor under stress conditions (SupplementaryFigure 4).

To examine whether forebrain-restricted expressionof CRH is sufficient to recapitulate the immobility-reducing effect of exogenous CRH expressed throughoutthe CNS in CRH-COE-Nes mice, male CRH-COEhom-Cam and CRH-COEhom-Dlx mice were exposed to theFST. Forebrain-restricted CRH overexpression failedto significantly affect floating or struggling behavior(data not shown) in homozygous CRH-COE-Cam(Figure 5b) or CRH-COE-Dlx (Figure 5d) mice com-pared to control littermates, both on days 1 and 2.

Active stress-coping behavior of CRH overexpressingmice depends on catecholaminergic transmission

CRH-COE-Cam as well as CRH-COE-Dlx mice suggestan involvement of more caudal brain nuclei withinthe mid/hind brain including monoaminergic cellpopulations overexpressing CRH, which promote thereduced immobility in the FST and TST. In an attemptto understand the neurochemical mechanisms under-lying the behavior of CRH-COE-Nes mice in the FST,animals were pretreated with either the tryptophanhydroxylase inhibitor PCPA or the tyrosine hydro-xylase inhibitor AMPT before testing in the FST.PCPA pretreatment reduced hippocampal serotoninlevels by 85% in CRH-COEcon-Nes mice and by 71%in CRH-COEhom-Nes mice (data not shown). Catechola-mine levels were not affected. Blockade of trypto-phane hydroxylase failed to significantly affect floatingbehavior of CRH-COEcon-Nes and CRH-COEhom-Nesmice in the FST (Figure 6a; statistics not shown). Inaccordance with our previous findings in naiveanimals (compare Figure 4b), vehicle-treated CRH-COEhom-Nes mice floated less than vehicle-treatedCRH-COEcon-Nes littermates.

Figure 5 Forebrain-restricted overexpression of corticotropin-releasing hormone (CRH) does not affect forced swimmingbehavior. In situ hybridization using a LacZ-specific riboprobe, which detects the CRH-LacZ fusion transcript, confirmedexogenous CRH expression in the forebrain of CRH-COEhom-Cam and CRH-COEhom-Dlx mice. (a) Sagittal section of a CRH-COEhom-Cam mouse brain shows strong overexpression of CRH in the olfactory bulb, all cortical layers, hippocampus andstriatum. (b) In the forced swim test floating behavior on days 1 (d1) and 2 (d2) did not differ significantly between maleCRH-COEcon-Cam (con) and CRH-COEhom-Cam (hom) mice (n = 10–13 animals per genotype). (c) Sagittal section of a CRH-COEhom-Dlx mouse brain shows strong overexpression of CRH in the olfactory bulb, striatum, reticular nucleus, corticallayers and hippocampus. (d) In the forced swim test floating behavior on days 1 (d1) and 2 (d2) did not differ significantlybetween male CRH-COEcon-Dlx (con) and CRH-COEhom-Dlx (hom) mice (n = 11 animals per genotype). (e) Sagittal section of aCRH-COEwt-Cam mouse brain demonstrating background in situ hybridization signals of the LacZ-specific riboprobe.Sections of CRH-COEwt-Dlx mice exhibited identical background hybridization signals (data not shown).

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AMPT pretreatment reduced hippocampal NAlevels by 35% in CRH-COEcon-Nes mice and by 43%in CRH-COEhom-Nes mice (data not shown). DA levelswere reduced by 38% in CRH-COEcon-Nes mice andby 47% in CRH-COEhom-Nes mice. Serotonin levelswere not affected. Blockade of tyrosine hydroxylasefailed to affect floating behavior of CRH-COEcon-Nesmice, but led to a significant increase of floating inCRH-COEhom-Nes mice (t13 = 2.5, P < 0.05; Figure 6b),suggesting that decreased floating in CRH-COEhom-Nes mice is at least partly mediated by increasedcatecholaminergic neurotransmission in these ani-mals. Vehicle-treated CRH-COEhom-Nes mice floatedless than vehicle-treated CRH-COEcon-Nes littermates,according to our previous findings in naive animals(compare Figure 4b).

CRH overexpression enhances FST-mediatedactivation of the locus coeruleusTo determine to what extent CRH overexpressionactivates catecholaminergic neurons, we analyzed byin situ hybridization the transcript levels of IEGs c-fosand zif268 with focus on the locus coeruleus (LC),nucleus of the solitary tract, ventral tegmental area(VTA), substantia nigra (SN) and dorsal raphe nucleus(DRN). As previously demonstrated in the rat,expression of c-fos and zif268 in these nuclei wasundetectable under basal conditions (data notshown).36 However, 30 min post forced swim stress,a marked increase of c-fos and zif268 expression wasdetected in the LC (Figures 6c and e) but not in theVTA and SN or DRN (data not shown). Quantificationof the signals revealed a stronger increase of c-fos(1.35-fold; t6 = 4.24, P < 0.01) and zif268 (1.55-fold;t6 = 6.15, P < 0.001) transcript levels in maleCRH-COEhom-Nes mice than in CRH-COEcon-Nesmice (Figures 6d and f), suggesting an enhancedstress-dependent activation of the LC due to CRHoverexpression.

To assess whether LC hyperactivation is indepen-dent of enhanced HPA axis reactivity, we alsoanalyzed zif268 expression in female CRH-COEhom-Nes mice that do not show HPA axis hypersensitivity(compare Supplementary Figures 2e and f). Similar tomales, female CRH-COEhom-Nes mice showed a sig-nificantly stronger increase of zif268 expression(1.30-fold; t10 = 3.12, P < 0.05) in the LC compared toCRH-COEcon-Nes mice 30 min post forced swim stress(Figure 6g).

Discussion

To genetically dissect the impact of excess CRH indistinct brain regions and neuronal cell populationson stress-coping behavior, we used a knock-inapproach to generate a mouse model that allowedCRH overexpression at different levels in a spatiallyrestricted manner. Superior to standard transgenesisthis conditional mouse model provides the opportu-nity to generate and compare different CRH over-expressing mouse lines—as demonstrated by breeding

to Nes-, Camk2a- and Dlx-cre mice—avoidingcommon uncertainties of transgene production suchas copy number or site of transgene insertion.Although the pattern of CRH overexpression exclu-sively depends on the spatial and/or temporal proper-ties of the introduced Cre recombinase, thetranscriptional control via R26 guarantees for iden-tical expression levels.

This approach enabled us to specifically investigatethe CNS effects of different dosages of CRH in CRH-COE mice without affecting the peripheral CRHsystem or the circadian HPA axis regulation underbasal conditions. Nevertheless, chronic overexpres-sion of exogenous CRH activates compensatorymechanisms affecting the expression levels of en-dogenous CRH and CRH-R1 in a brain region-specificmanner as demonstrated in CRH-COE-Nes mice.Alterations of endogenous CRH and CRH-R1 levelswill mutually interfere with existing regulatorycircuits and, in concert with effects of exogenousCRH expression, add another layer of complexity. Forinstance, expression of CRH in the PVN of CRH-COEhom-Nes is significantly decreased under basalconditions and thereby probably sensitizes or upre-gulates CRH-R1 in pituitary corticotrophs. As aconsequence, basal ACTH and corticosterone plasmalevels of male CRH-COEhet- and CRH-COEhom-Nesmice are indistinguishable from those of controllittermates, whereas the HPA axis of these animalsis hyperreactive in response to stress. Interestingly,female CRH-COEhom-Nes mice displayed no suchstress-dependent HPA axis hyperreactivity, support-ing previous observations of gender differences inbiological functions of the endogenous CRH system.37

In CRH-COEhom-Nes mice we observed increasedexpression of CRH in the CeA as well as of CRH-R1in the BLA and hippocampus. In contrast, Thy-1-driven overexpression in projection neurons of CRH-OE2122 mice results in a rather uniform downregula-tion of CRH-R1 in several brain nuclei.38 However, itis of notice that chronically increased corticosteronelevels15 might here dominate the effects on CRH-R1expression compared to transgene CRH expression.

Male CRH-COE-Nes mice exhibited a marked genedosage-dependent reduction of immobility in the FSTand TST, which is not due to excessive stresshormone secretion, as female CRH-COE-Nes mice,which displayed normal HPA axis reactivity, showedsimilar behavioral alterations in the FST. Accord-ingly, neither CRH-R1 deletion35 nor adrenalectomyresulted in altered forced swimming behavior.39 Inline with the observations in our chronic model ofCRH excess, also the acute intracerebroventricular(i.c.v.) injection of CRH or cortagine, a potent CRH-R1agonist, as well as site-directed injection of CRH intothe LC, decreases immobility in the FST in rats andmice.40–42 Furthermore, CRH-Tg mice also showedreduced immobility in the FST.43 Interestingly, CRHhas also been demonstrated to elicit antidepressant-like effects in a differential reinforcement of low-rateschedule in rats.44

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Figure 6 Active stress-coping behavior of CRH-COEhom-Nes mice is partly mediated by increased catecholaminergicneurotransmission, which originates from corticotropin-releasing hormone (CRH)-mediated hyper-activation of the locuscoeruleus. (a) Pretreatment of male CRH-COE-Nes mice with the tryptophane hydroxylase inhibitor Para-chlorophenyla-lanine methyl ester (PCPA) did not affect floating behavior of male CRH-COEcon- or CRH-COEhom-Nes mice in the forced swimtest (25 1C). (b) Inhibiting tyrosine hydroxylase by a-methyl-para-tyrosine methyl ester (AMPT) pretreatment led to asignificant increase in floating behavior of CRH-COEhom-Nes mice, whereas floating behavior of CRH-COEcon-Nes remainedunaffected (n = 7–10 animals per group). (c–g) Forced swim stress induced a significantly stronger expression of c-fos andzif268 mRNA in the locus coeruleus of CRH-COEhom-Nes (hom) mice than in CRH-COEcon-Nes (con) mice. Representativedark-field photomicrographs of in situ hybridizations of coronal brain sections depicting (c) c-fos and (e) zif268 mRNAexpression in the locus coeruleus of male CRH-COEcon-Nes (con) and CRH-COEhom-Nes (hom) mice. Quantification ofrespective (d) c-fos and (f) zif268 mRNA expression in the locus coeruleus as determined by in situ hybridization. (g)Quantification of zif268 mRNA expression in the locus coeruleus of female CRH-COE-Nes mice as determined by in situhybridization. *P < 0.05, **P < 0.01, ***P < 0.001.

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The Porsolt FST is highly predictive for clinicallyeffective antidepressants targeting the monoaminergicsystem, however, the construct of reduced immobilityis not understood.45 Immobility is thought to reflecteither a failure of persistence in escape-directedbehavior (that is, behavioral despair) or the develop-ment of passive behavior that disengages the animalfrom active forms of coping with stressful stimuli.46

However, others have suggested that immobilitycould also reflect an adaptive mechanism to conserveenergy.47 Besides, the FST certainly also involves astrong component of arousal, alertness and stress-coping behavior. Therefore, decreased immobility inCRH-COE-Nes mice might rather reflect enhancedresponsiveness to a stressful situation resulting inincreased active coping behavior than antidepressant-like behavior. CRH has been demonstrated to influ-ence neuronal connectivity in the developing hippo-campus.48 Hence, CRH overexpression starting fromearly embryogenesis might have caused adaptivechanges of neuronal physiology. However, the at-tenuation of active coping behavior and of HPA axishyperreactivity by pretreatment of CRH-COEhet-Nesmice with the CRH-R1-selective antagonist DMP696suggests that these phenotypes are an acute conse-quence of CRH overexpression.33 Considering theobservation that CRH-R1 antagonists exhibit highestefficacy in rodents that are hyperresponsive to stressand exhibit increased levels of CRH,12,39 CRH-COE-Nes mice may constitute a mouse model withstrong and predictable responsiveness to CRH-R1antagonists.

To assure that the FST phenotype of CRH-COE-Nesmice is not of artificial nature, arising from excessiveectopic expression of CRH in the brain, we elucidatedthe role of the endogenous CRH system in FSTbehavior. Under basal, nonactivated conditions theendogenous CRH system does not influence FSTbehavior as neither the genetic disruption nor thepharmacological blockade of CRH-R1 affected FSTbehavior.10,34,35 However, we could demonstrate thatstress-mediated activation of the endogenous CRHsystem before the FST decreased immobility incontrol mice, but not in CRH-R1 knockout mice.These findings suggest that ectopic overexpression ofCRH in CRH-COE-Nes mice mimics the behavioralconsequences of stress-mediated activation of theendogenous CRH system, and that CRH-R1-depen-dent signaling pathways promote the increase inactive coping behavior.

The forebrain-restricted disruption of CRH-R1 inCRH-R1lox/lox Camk2a-cre mice decreases anxiety-related behavior,23 suggesting the involvement ofCRH/CRH-R1-dependent pathways in structures ofthe anterior forebrain including those of the limbicsystem in emotionality control. In order to investigatewhether these structures are also causally related toaltered FST behavior, we spatially restricted CRHoverexpression using Camk2a-cre mice. Similar toCRH-OE2122 mice,15 CRH overexpression in the fore-brain did not result in the reduced immobility

observed in CRH-COE-Nes (this study) and CRH-Tgmice,14,43 presumably owing to the overexpression inprincipal neurons only. To date the cellular andsubcellular localization of CRH and its receptorsincluding receptor-mediated effects at synapses arenot well understood. In the hippocampus, endogen-ous CRH expression has been assigned to GABAergicinterneurons from where CRH is released in the causeof acute physiological stress.49 CRH release excitespyramidal cells, which express CRH-R1 postsynapti-cally on their dendritic spines. However, using Dlx-cremice to direct CRH overexpression to GABAergicinterneurons, in order to model more closely endo-genous expression sites in the forebrain, failed toaffect FST behavior. The absence of the FST pheno-type in forebrain-specific CRH overexpressing mouselines argues against volume transmission throughoutthe brain as mediating CRH effects, but clearly favorsa mechanism involving specific synaptic release.Furthermore, our results suggest that CRH overex-pression in more caudal brain nuclei of the mid/hindbrain could promote the effects of CRH on stress-coping behavior.

In the brain stem of mice and rats CRH-R1 isexpressed in serotonergic neurons of the median andDR50 and in dopaminergic neurons of the SN andVTA.51 CRH is expressed in noradrenergic neurons ofthe LC,52 and i.c.v. injection of CRH has beendemonstrated to induce a strong Fos immunoreactiv-ity in the LC indicating the activation of CRH-R-dependent signaling pathways.52 Accordingly, CRH isable to potentiate noradrenergic,40,53 dopaminergic54

and serotonergic55 neurotransmission. Here we coulddemonstrate that the pharmacological blockade ofcatecholamine synthesis by AMPT, but not of seroto-nin synthesis by PCPA, could partially reverse thephenotype of CRH-COEhom-Nes mice. Therefore, it islikely that CRH overexpression activates the endo-genous catecholaminergic system similarly as anti-depressants, which would explain increased arousalin an open field and the reduced immobility in theFST and TST.

Quantification of c-fos and zif268 expression inresponse to forced swim stress further identifies thehyperactivation of noradrenergic neurons of the LC inCRH-COE-Nes mice. Intracoerulear microinfusion ofCRH in rats has demonstrated that CRH can serve asan excitatory neurotransmitter in the LC56 resulting inenhanced NA release in LC projection areas.57 CRH–NA interactions not only occur in the LC, where CRHactivates the LC, but also at the projections of theforebrain noradrenergic system, where NA stimulatesCRH release.58 For instance, stress induces NA releasein the PVN and thereby stimulates secretion of CRH.59

This feed-forward mechanism could be a causal factorto the hyperreactivity of the HPA axis upon stress.

In conclusion, we have created a new, highlyflexible transgenic mouse model, which can help indissecting the contribution of CRH-sensitive path-ways involved in the transition from physiological topathological stress responses, which are thought to

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underlie the etiology of affective and anxiety dis-orders.6 This animal model is also suited for validat-ing drug candidates targeting the central CRH system.

Acknowledgments

We thank Claudia Kuhne, Katja Mayer, Tanja Orschmann,Daniela Kohl and Jessica Koepke for excellenttechnical assistance, Johanna Stalla for performingCRH RIAs, Ursula Habersetzer for endocrinologicalmeasurements, Maik Engeholm for vector construc-tion and Peter Weber for his photographic expertise.We thank Ralf Kuhn, Susanne Bourier and SawoulaMichailidou for blastocyst injection and generation ofchimeric mice. We thank P Soriano and P Mombaertsfor gifts of plasmids pROSA26-1 and ETLpA-/LTNL,respectively. D Refojo is supported by the EuropeanMolecular Biology Organization Fellowship Pro-gramme. This work was partially supported by theBundesministerium fur Bildung und Forschung with-in the framework of the NGFN2 (01GS0481) and bythe Fonds der Chemischen Industrie.

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Supplementary Information accompanies the paper on the Molecular Psychiatry website (http://www.nature.com/mp)

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