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Neuroscience 183 (2011) 47–63

TOPOGRAPHICAL DISTRIBUTION OF CORTICOTROPIN-RELEASINGFACTOR TYPE 2 RECEPTOR-LIKE IMMUNOREACTIVITY IN THE RATDORSAL RAPHE NUCLEUS: CO-LOCALIZATION WITH TRYPTOPHAN
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J. L. LUKKES,a* D. R. STAUB,b1 A. DIETRICH,c

W. TRUITT,c,d A. NEUFELD-COHEN,e A. CHEN,e

P. L. JOHNSON,b,c A. SHEKHARc AND C. A. LOWRYa,b

aDepartment of Integrative Physiology and Center for Neuroscience,niversity of Colorado Boulder, Boulder, CO 80309, USA

bHenry Wellcome Laboratories for Integrative Neuroscience and En-ocrinology, University of Bristol, Bristol, BS1 3NY, UK

cDepartment of Psychiatry and Pharmacology and Toxicology, Indiananiversity School of Medicine, Indianapolis, IN 46223, USA

dDepartment of Anatomy and Cell Biology, Indiana University Schoolf Medicine, Indianapolis, IN 46223, USA

eDepartment of Neurobiology, Weizmann Institute of Science, Re-ovot, Israel, 76100

Abstract—Corticotropin-releasing factor (CRF) and CRF-re-lated neuropeptides are involved in the regulation of stress-related physiology and behavior. Members of the CRF familyof neuropeptides bind to two known receptors, the CRF type1 (CRF1) receptor, and the CRF type 2 (CRF2) receptor. Al-hough the distribution of CRF2 receptor mRNA expression

has been extensively studied, the distribution of CRF2 recep-tor protein has not been characterized. An area of the brainknown to contain high levels of CRF2 receptor mRNA expres-sion and CRF2 receptor binding is the dorsal raphe nucleusDR). In the present study we investigated in detail the distri-ution of CRF2 receptor immunoreactivity throughout the

rostrocaudal extent of the DR. CRF2 receptor-immunoreac-tive perikarya were observed throughout the DR, with thehighest number and density in the mid-rostrocaudal DR. Dualimmunofluorescence revealed that CRF2 receptor immunore-ctivity was frequently co-localized with tryptophan hydrox-lase, a marker of serotonergic neurons. This study providesvidence that CRF2 receptor protein is expressed in the DR,

1 Present address: Department of Anatomy and Cell Biology, Templeniversity School of Medicine, Philadelphia, PA 19140, USA.

Corresponding author. Tel: �1-303-492-8154; fax: �1-303-492-0811.-mail address: [email protected] (J. L. Lukkes).bbreviations: aq, cerebral aqueduct; Arc, arcuate nucleus; ASV-30,ntisauvagine-30; bv, blood vessel; CP, caudate putamen; CRF, cor-icotropin-releasing factor; CRF1 receptor, CRF type 1 receptor; CRF2

receptor, CRF type 2 receptor; dDRC, dorsal raphe nucleus, caudalpart, dorsal part; dlPAG, dorsolateral periaqueductal gray; DR, dorsalraphe nucleus; DRD, dorsal raphe nucleus, dorsal part; DRI, dorsal raphenucleus, interfascicular part; DRV, dorsal raphe nucleus, ventral part;DRVL, dorsal raphe nucleus, ventrolateral part; DTgP, dorsal tegmentalnucleus, pericentral part; EGFP, enhanced green fluorescent protein; En,entorhinal cortex; ir, immunoreactive; KO, knockout; LDTg, laterodorsaltegmental nucleus; LS, lateral septum; LV, lateral ventricle; mlf, mediallongitudinal fasciculus; MnR, median raphe nucleus; mUcn 2, urocortin 2(mouse); M1, motor cortex; Pa4, paratrochlear nucleus; PBS, phosphatebuffered saline; PV, paraventricular nucleus of the thalamus; RT, roomtemperature; TrpOH, tryptophan hydroxylase; Ucn1, 2, 3, urocortin 1, 2, 3;

svDRC, dorsal raphe nucleus, caudal part, ventral part; VMH, ventromedialhypothalamus; WT, wild type; 4V, fourth ventricle.

0306-4522/11 $ - see front matter © 2011 IBRO. Published by Elsevier Ltd. All righdoi:10.1016/j.neuroscience.2011.03.047

47

nd that CRF2 receptors are expressed in topographicallyrganized subpopulations of cells in the DR, including sero-onergic neurons. Furthermore, these data are consistentith the hypothesis that CRF2 receptors play an important

role in the regulation of stress-related physiology and behav-ior through actions on serotonergic and non-serotonergicneurons within the DR. © 2011 IBRO. Published by ElsevierLtd. All rights reserved.

Key words: CRF2 receptor, immunofluorescence, immuno-istochemistry, raphe, serotonin, tryptophan hydroxylase.

Corticotropin-releasing factor (CRF) is a 41-amino acidneuropeptide that has been linked to the regulation ofstress-related physiology and behavior, including regula-tion of the hypothalamic-pituitary-adrenal (HPA) axis (Valeet al., 1981, 1983). The CRF family of neuropeptides alsoincludes urocortin (Ucn) 1, Ucn 2, and Ucn 3; the role ofthese CRF-related neuropeptides in the HPA axis andstress-related physiological and behavioral responses isless clear, although evidence suggests that they also playan important role in modulating these responses (Reul andHolsboer, 2002). CRF and its family of neuropeptides areknown to bind to two distinct G protein-coupled receptors,the CRF type 1 (CRF1) receptor, and the CRF type 2(CRF2) receptor (Perrin et al., 1993; Lovenberg et al.,1995b), for which they have different binding affinities.CRF preferentially binds to CRF1 receptors, Ucn 1 binds

ith high affinity to both receptors, and Ucn 2 and Ucn 3referentially bind to CRF2 receptors (Lewis et al., 2001;

Reyes et al., 2001). Whereas CRF1 receptors have aidespread distribution in the CNS, including subcorticalnd cortical regions, CRF2 receptor distribution is mainly

imited to subcortical structures (Chalmers et al., 1995;adulovic et al., 1998; Sanchez et al., 1999; Van Pett etl., 2000; Chen et al., 2000; Korosi et al., 2006).

Multiple isoforms of the CRF2 receptor are known to bexpressed including the alpha, beta, and gamma (foundnly in humans) isoforms, a soluble isoform, and a trun-ated isoform (currently described only in rats). These

soforms have distinct expression patterns and bindingffinities for CRF and its family of peptides (Lovenberg etl., 1995a; Kostich et al., 1998; Miyata et al., 1999, 2001;hen et al., 2005b; Evans and Seasholtz, 2009).

A subcortical area of the rat CNS that contains a highensity of cells expressing CRF2 receptor mRNA expres-ion is the dorsal raphe nucleus (DR). The DR is a brain-
tem region that, together with the median raphe nucleus
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J. L. Lukkes et al. / Neuroscience 183 (2011) 47–6348

(MnR), contains the majority of serotonergic neurons withascending projections to forebrain structures (Steinbuschet al., 1978; Steinbusch, 1981). In situ hybridization studieshave shown that the DR is one of a few brain regions thatcontain more CRF2 receptor mRNA expression than CRF1

receptor mRNA expression in rats (Van Pett et al., 2000;Day et al., 2004; Korosi et al., 2006). Co-localization stud-ies in rat brain have shown that CRF2 receptor mRNAexpression within the DR is localized predominantly inserotonergic neurons, although, in caudal parts of the DR,CRF2 receptor mRNA expression is commonly found inon-serotonergic neurons, including GABA-synthesizingABAergic neurons (Day et al., 2004). Consistent with

hese findings, and consistent with expression of functionaleceptor protein in the DR, dense CRF2 receptor binding

has been described in the DR of several vole species (Limet al., 2005).

Although the distribution of CRF2 receptor mRNA ex-pression has been extensively studied, the distribution ofCRF2 receptor protein has not been characterized. Thiscould be due to (1) difficulties generating CRF2 receptor-specific antibodies, (2) antibodies used in many previousstudies, such as the CRF1/2 receptor antibody sc-1757Santa Cruz Biotechnology, Santa Cruz, CA, USA), recog-ize both CRF1 and CRF2 receptors (Campbell et al.,

2003; Chen et al., 2000; Hinkle et al., 2003, also see Fig.4), and (3) some CRF2 receptor-specific antibodies used inprevious studies (Waselus et al., 2009; Wang et al., 2007)are no longer commercially available. Recent studies usingimmunoelectron microscopy have demonstrated thatCRF2 receptor immunoreactivity is predominantly intracel-lular in DR neurons under basal unstressed conditions,and shifts toward a greater expression at the plasma mem-brane following stress exposure (Waselus et al., 2009).The shift in intracellular versus plasma membrane local-ization of CRF2 receptors is associated with a shift fromnhibitory to excitatory neuronal firing rate responses toRF administration (Waselus et al., 2009).

The hypothesis that functional CRF2 receptors are ex-pressed in the DR is supported by studies using immedi-ate-early gene expression (e.g. nuclear c-Fos inductionrepresenting increased cellular responses) and electro-physiology to investigate the effects of CRF2 receptor li-gands on responses of serotonergic neurons in theDR. I.c.v. injections of the CRF2 receptor ligand mouse

cn 2 (mUcn 2) (Staub et al., 2005) or microinjections ofUcn 2 directly into the DR (Amat et al., 2004) increase

-Fos expression in DR serotonergic neurons and increaseerotonin release in DR projection sites, while pretreat-ent with the CRF2 receptor antagonist [DPhe11,

His12]sauvagine(11-40) (antisauvagine-30; ASV-30) blocksthese effects (Amat et al., 2004; Staub et al., 2006). Like-wise, electrophysiological studies in anesthetized ratshave shown that injections of mUcn 2 directly into the DRcan increase the firing rates of serotonergic neurons andthese effects can be prevented by pretreatment withASV-30 (Pernar et al., 2004). Together, these studies areconsistent with the finding that activation of CRF2 recep-
tors in the DR increases extracellular serotonin concentra- t
tions within the basolateral amygdaloid nucleus (Amat etal., 2004) and nucleus accumbens (Lukkes et al., 2008),forebrain targets of serotonergic neurons arising from theDR (Abrams et al., 2005; Van Bockstaele et al., 1993).

Understanding the distribution of CRF2 receptorswithin the DR is important because there is increasingevidence that the DR may include anatomically and func-tionally distinct subpopulations of serotonergic neurons(Imai et al., 1986; Abrams et al., 2004; Lowry et al., 2005,2008; Lowry and Hale, 2010). Indeed, serotonergic neu-rons can be divided into several different types, some ofwhich are restricted to specific regions of the DR, based ontheir physiological properties and behavioral correlates(Rasmussen et al., 1984; Fornal et al., 1996; Jacobs andFornal, 1999; Sakai and Crochet, 2001; Kocsis et al.,2006).

Several studies suggest that interactions between CRFor CRF-related ligands and serotonergic systems may playan important role in the regulation of anxiety-related be-haviors in rats (Maier and Watkins, 2005; Lowry et al.,2005) and anxiety and affective disorders in humans (Ar-borelius et al., 1999; Austin et al., 2003). These data,together with (1) the potential for mismatch between CRF2

receptor mRNA and protein expression in neurons withinthe DR, (2) the potential for expression of multiple isoformsof the CRF2 receptor, and (3) the evidence for anatomicaland functional heterogeneity within the DR, led us to char-acterize the detailed topographical organization of CRF2

receptor protein expression within this brainstem regionand the extent of its co-localization with cytoplasmic tryp-tophan hydroxylase (TrpOH), a marker of serotonergicneurons. These studies used an antibody directed againstthe N-terminus of the CRF2 receptor, including an epitopethat is highly conserved among CRF2� and CRF2� recep-or isoforms, and therefore is likely to detect all knownRF2 receptor isoforms in rat brain, including truncated

soforms of the receptor.

EXPERIMENTAL PROCEDURES

DR immunohistochemistry

Animals. Adult male Sprague Dawley rats (n�8; 250–300g; Harlan Laboratories, Indianapolis, IN, USA) were used. The ratswere group housed, two per cage, in wire cages (17�35�45 cm3;

lternative Design, Siloam Springs, AR, USA) and were main-ained with free access to food and water under a 12 h light/darkycle with lights on at 0700 h in a room with standard temperature21 °C) and humidity (22%). Rats were acclimated for at least 1eek before experimental manipulation. All procedures were ap-roved by the University of Colorado at Boulder Institutional Ani-al Care and Use Committee. In addition, all studies were con-

istent with the NIH Guide for the Care and Use of Laboratorynimals (N.I.H. Publication No. 85–23). All possible efforts wereade to minimize the number of animals used and their suffering.

Mice lacking functional CRF1 receptors (n�3) and wild typelittermates (n�3) (Smith et al., 1998) were maintained with freeaccess to food and water under a 12 h light/dark cycle. All exper-imental protocols were approved by The Weizmann Institute ofScience Institutional Animal Care and Use Committee. Mice lack-ing functional CRF2 receptors were not used as methods used todevelop the existing CRF mutants did not delete the 5’region of
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region that is targeted by the anti-CRF2 receptor antibody used inhis study.

Tissue processing. Rats or mice were anesthetized with 0.5l sodium pentobarbital (200 mg/ml; Vortech Pharmaceuticals,earborn, MI, USA). Animals were transcardially perfused with50 ml of 0.1 M phosphate buffered saline (PBS; pH 7.4) at roomemperature (RT), followed by 200 ml of cold (4 °C) 4% parafor-aldehyde (prepared using 40 g paraformaldehyde, 404 ml 0.2 Ma2HPO4, 96 ml 0.2 M NaH2PO4, and 500 ml dH2O, brought to pH

7.4 with sodium hydroxide pellets). Brains were removed from thecranium, postfixed for 1 h in the same fixative at 4 °C, thenimmersed in 0.1 M PBS with 25% sucrose and stored for 2 daysat 4 °C. Following cryopreservation with sucrose, brains were thenblocked in a coronal plane using a rat (RBM-4000C, ASI Instru-ments, Warren, MI, USA) or mouse (RBM-2000C, ASI Instru-ments) brain matrix; brains were cut in the coronal plane at thecaudal border of the mammillary bodies (approximately �5.30 mmbregma in rat brains or �2.80 mm bregma in mouse brains) andrapidly frozen in liquid isopentane chilled on dry ice. The brainswere stored at �80 °C until sectioning. Brain sections (30 �m)

ere made using a cryostat and stored as six alternate sets ofections at �20 °C in cryoprotectant (prepared using 270 mlthylene glycol, 160 ml glycerol, 202 ml 0.2 M Na2HPO4 · 7H2O,8 ml 0.2 M NaH2PO4 · H2O, and 320 ml dH2O) until immuno-

staining was performed.

Immunohistochemistry. For immunostaining, every sixthsection of the midbrain containing the DR (from approximately�5.8 to �10.3 mm bregma in rat brains; approximately �3.5 to�5.7 mm bregma in mouse brains) was removed from the cryo-protectant and washed in 0.01 M PBS for 15 min. The tissue wasthen placed in 0.01 M PBS containing 1% H2O2 for 15 min andthen sections were washed twice in 0.01 M PBS for 15 min each.Sections were then preincubated in blocking buffer (0.01 M PBS,pH 7.4, containing 1% normal rabbit serum, Cat. No. 011-000-120,Jackson ImmunoResearch, West Grove, PA, USA) at RT for 2 hto reduce non-specific staining. Sections were then incubated ingoat anti-CRF2 receptor-selective polyclonal antibody (1:300; Cat.No. sc-1826; Santa Cruz Biotechnology, Santa Cruz, CA, USA) in0.01 M PBS containing 0.04% normal rabbit serum and 0.2%Triton X-100 for 20 h at 4 °C with gentle agitation (Lukkes et al.,2009). Sections were washed in 0.01 M PBS containing 0.04%normal rabbit serum two times for 15 min, and incubated for 2 h atRT in biotin-conjugated donkey anti-goat secondary antibody (1:200; Cat. No. 705-066-147, Jackson ImmunoResearch) in 0.01 MPBS containing 0.04% normal rabbit serum and 0.03% TritonX-100 to visualize CRF2 receptor-like immunoreactivity. After in-ubation with secondary antibody, sections were washed in 0.01

PBS containing 0.04% normal rabbit serum two times for 15in, and then placed in an avidin-biotin-peroxidase complex

ABC) reagent (Cat. No. PK-6106; Vector Laboratories, Burlin-ame, CA, USA) at 1:200 in 0.01 M PBS for 90 min. The tissueas then rinsed in 0.01 M PBS two times for 15 min each. The

issue was then placed in a peroxidase-based substrate reactionVector SG chromogen kit; Cat. No. SK-4700; Vector Laborato-ies) for 6 min. The reaction was stopped by rinsing the tissue in.01 M PBS for 15 min. After this the tissue was briefly rinsed in.1 M sodium phosphate buffer (PB) and mounted onto cleanlass microscope slides. Once mounted the sections were dehy-rated, cleared with xylene, and mounted with cover slips.

Immunocytochemistry. Human embryonic kidney (HEK-93) cells that stably express the mouse CRF2� receptor andnhanced green fluorescent protein (EGFP) and wild type (WT)EK-293 cells were used to determine the specificity of the anti-RF2 receptor antibody (Chen et al., 2005a). Cells were main-

tained in Dulbecco’s Modified Eagle Medium (DMEM; Cat. No.
10938025; Invitrogen, Paisley, UK) solution supplemented with A
10% fetal calf serum. For immunocytochemistry cells were grownon glass cover slips in a 90 cm2 petri dish. After reaching thedesired confluency the cover slips were placed in 0.05 M PBS,and then placed in 4% paraformaldehyde in 0.05 M PBS for 30min. The cover slips were then rinsed in 0.05 M PBS and thenplaced in 0.05 M PBS containing 0.3% Triton X-100 (PBST) for 5min. Cover slips were then placed in 1% bovine serum albumin(BSA) in 0.05 M PBS for 30 min. Some cover slips were thenplaced in either goat anti-CRF2 receptor antibody (1:100; Cat. No.sc-1826; Santa Cruz Biotechnology) in 1% BSA, goat anti-CRF1/2

receptor antibody (1:100; Cat. No. sc-1757; Santa Cruz Biotech-nology) in 1% BSA, or in 1% BSA overnight at 4 °C. Cover slipswere then placed in 0.05 M PBS and then incubated in either arabbit anti-goat antibody conjugated to Alexa Fluor 555 (1:500;Cat. No. A21431; Invitrogen, Paisley, UK) in 1% BSA, or 1% BSAfor 2 h. The cover slips were then placed in 0.05 M PBS andmounted onto slides using a fluorescent mounting medium (Cat.No. S3023; DAKO, Ely, UK).

Western blot, microdissected brain regions

Adult male Sprague Dawley rats (n�2; 250–300 g; Harlan Labo-ratories) were maintained as described above for immunohisto-chemical procedures and used for western blots. Western blotswere performed to determine the relative molecular weight of theprotein(s) recognized by the CRF2 receptor antibody used, and todetermine the relative amounts of CRF2 receptor protein in differ-ent brain regions. Rats were decapitated, brains were then re-moved, frozen and stored at �80 °C, and then sectioned frozen(300 �m) using a cryostat (Leica CM 1900; North Central Instru-

ents, Plymouth, MN, USA) at �12 °C. The motor cortex (M1),ntorhinal cortex (En), lateral septum (LS), ventromedial hypothal-mus (VMH), median raphe nucleus (MnR), and DR were dis-ected at �10 °C using the Palkovits punch technique (Palkovitsnd Brownstein, 1988) and a 22 gauge cannula (Cat. No. 18036-2; 0.41 mm internal diameter; Fine Science Tools, Foster City,A, USA). Tissue was then homogenized in 60 �l of HEPES

buffer (Cat. No. H3375; Sigma-Aldrich, St. Louis, MO, USA) con-taining 2.4 �l protease inhibitor stock “complete” (1:25 dilution;Roche Diagnostics Ltd, Indianapolis, IN, USA). Total protein con-centrations were determined within 5 �l sample duplicates using aPierce protein assay (Cat. No. 1856210; Thermoscientific, Rock-ford, IL, USA) and a microplate reader (MultiSkan EX 355; ThermoElectron Corporation, Waltham, MA, USA). Samples (30 �g totalrotein) were processed for western blotting, and CRF2 receptor

and actin levels were detected using methods described previ-ously (Lukkes et al., 2009). Briefly, protein was mixed in 1�sodium dodecyl sulfate (SDS)/�-mercaptoethanol loading buffer,ortexed and boiled for 3 min prior to separation by 8% SDS-olyacrylamide gel electrophoresis. Following electrophoresisMini-PROTEAN 3 cell, Bio-Rad Laboratories, Hercules, CA,SA), proteins were transferred to a polyvinylidene fluoride

PVDF) membrane (Immuno-Blot; 0.2 �m, Bio-Rad Laboratories).he membranes were blocked with 5% non-fat dry milk in Tris–uffered saline containing 0.1% Tween-20 (TBS-T) for 20 min atT and incubated with goat anti-CRF2 receptor polyclonal anti-ody (1:100; Cat. No. sc-1826; Santa Cruz Biotechnology) in 5%on-fat dry milk in TBS-T overnight at 4 °C. The membranes wereinsed three times for 10 min each time at RT in TBS-T. After theinsing procedure, the membranes were incubated for 2 h at RT inffinity-purified rabbit anti-goat IgG (whole molecule)-peroxidaseonjugated secondary antibody (1:30,000; Cat. No. A5420; Sig-a-Aldrich) in 5% non-fat dry milk in TBS-T. Control for protein

oading was achieved by using mouse anti-actin primary antibody1:2000; Cat. No. MAB1501R; Chemicon International, Billerica,A, USA) and goat anti-mouse IgG (Fab specific)-peroxidase

onjugated secondary antibody (1:5000; Cat. No. A3682, Sigma-
ldrich) in 5% non-fat dry milk in TBS-T. Proteins were detected

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using a ChemiDoc-IT imaging system with cooled CCD camera(UVP Bioimaging Systems, Upland, CA, USA).

Antibodies

The goat anti-CRF2 receptor antibody used (N-20, Cat. No. sc-1826, Santa Cruz Biotechnology) is an affinity-purified polyclonalantibody raised against a peptide corresponding to amino acidsequence 47–66 mapping at the N-terminus of the CRF2� recep-tor of mouse origin (RTTIGNFSGPYTYCNTTLDQ) (Fig. 1). Thereis no similarity with the amino acid sequence of the CRF1 receptoronly four of 20 amino acids are conserved; Fig. 1). The sequencesed to generate the antibody is unique based on a search of theasic local alignment search tool (BLAST) database; this does notreclude the possibility that there are peptides that have not beenharacterized that have similar sequences.

The goat anti-CRF1/2 receptor antibody used (C-20, Cat.No. sc-1757, Santa Cruz Biotechnology) is a polyclonal anti-body raised against a peptide corresponding to amino acidsequence 396 – 415 mapping at the C-terminus of the CRF1

receptor of human origin (SIPTSPTRVSFHSIKQSTAV). Thereis a three-amino acid difference between the rat CRF1 and ratCRF2 receptor at the region that corresponds to this sequence.

estern blot analysis indicates that this antibody recognizes aand corresponding to the predicted molecular weight of theRF1 receptor in mice 77– 80 kDa (Radulovic et al., 1998; Chent al., 2000).

Analysis of the distribution of CRF2 receptorimmunoreactivity

For analysis, the DR was divided into rostral (�6.92 to �7.64mm bregma), mid-rostrocaudal (�7.73 to �8.45 mm bregma),and caudal (�8.54 to �9.26 mm bregma) regions (Abrams etal., 2004). Analysis of CRF receptor immunostaining in the DR

Fig. 1. Amino acid sequences of the N-termini, up to the first predicteRF2 receptor, as well as a soluble form of the mouse and rat CRF2� r

Black line indicates the amino acid sequence corresponding to the 20he antigen for development of the polyclonal CRF2� receptor antibodfollowing sources: mCRF2� (Perrin et al., 1995) (NCBI Protein databEDL88098), mCRF2� (Chen et al., 2005a) (NCBI Protein database acc009), (NCBI Protein database accession no.: AAU94301), rCRF2�-tratabase accession no.: NP_073205), rat CRF2�sol (Evans and Seaccession no.: NP_112261). Abbreviations: mCRF2�, mouse CRF2�

receptor; rCRF2�, rat CRF2� receptor; rCRF2�, rat CRF2� receptor; rCat CRF1 receptor. Black shading indicates conserved amino acids; lere obtained using: www.ebi.ac.uk/Tools/clustalw2/index.html.

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was performed at 12 anatomical levels at 180 �m intervals s

rostral DR; mid-rostrocaudal DR; and caudal DR; one section/evel/rat). At each level the DR was further divided into differentegions. These regions were the dorsal part of the DR (DRD),he ventral part of the DR (DRV), the ventrolateral part of theR and ventrolateral periaqueductal gray (DRVL/VLPAG), theaudal part of the DR (DRC; subdivided into dorsal (dDRC) andentral (vDRC) parts at �9.08 mm bregma), and the interfas-icular part of the DR (DRI). The rostrocaudal levels and sub-ivisions were defined based on a standard rat brain stereo-axic atlas (Paxinos and Watson, 1998).

CRF2 receptor/TrpOH double immunofluorescence

Adult male Sprague Dawley rats (n�6; 250–300 g; Harlan Labo-ratories) were maintained as described above for immunohisto-chemical procedures and used for CRF2 receptor/TrpOH doubleimmunofluorescence.

Immunofluorescence was used to identify CRF2 receptor-xpressing and TrpOH-expressing cells because this method pro-ides the cellular resolution required to determine co-localizationf CRF2 and TrpOH immunoreactivity. Perfusion and postfixationethods were as described above for CRF2 receptor immunohis-

ochemistry.For immunofluorescence, every sixth section of the mid-

rain containing the DR (from approximately �7.64 to �8.54m bregma) was removed from the cryoprotectant and washed

n 0.05 M PBS twice, for 15 min each time. Sections were thenncubated in 0.05 M PBS containing 0.3% Triton X-100 (PBST)nd 0.01% sodium azide for 60 min at RT and then placed inouse anti-TrpOH monoclonal antibody (1:2000; Cat. No.-0678, Sigma-Aldrich) in PBST containing 0.01% sodiumzide overnight at RT. Following incubation with primary anti-ody, sections were rinsed two times for 15 min each time in.05 M PBS and then placed in Cy5-conjugated donkey anti-ouse IgG secondary antibody (1:200; Cat. No. 115-175-205,eak emission 670 nm, red fluorescence; Jackson ImmunoRe-

mbrane domain, of mouse and rat CRF2� and CRF2� isoforms of thea truncated form of the rat CRF2� receptor, and the rat CRF1 receptor.cid peptide from the mouse CRF2� receptor isoform that was used as6) used in this study. Amino acid sequences were obtained from thession no.: Q60748), rCRF2� (NCBI Protein database accession no.:o.: AAS07021), mCRF2�sol (Chen et al., 2005b; Evans and Seasholtz,(Miyata et al., 1999), rCRF2� (Lovenberg et al., 1995b) (NCBI Protein009), rCRF1 receptor (Perrin et al., 1993) (NCBI Protein databasemCRF2�, mouse CRF2� receptor; mCRF2�sol, mouse soluble CRF2�

at CRF2�sol receptor; rCRF2�tr, rat truncated CRF2� receptor; rCRF1,shading indicates conservative amino acid substitutions. Alignments

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washed in 0.05 M PBS two times for 15 min each time at RTand then incubated in goat anti-CRF2 receptor polyclonal anti-

ody (1:300; Cat. No. sc-1826, Santa Cruz Biotechnology) in.01 M PBS containing 0.04% normal rabbit serum (Cat. No.11-000-120, Jackson ImmunoResearch) and 0.2% Triton-100 for 20 h at 4 °C with gentle agitation (Lukkes et al., 2009).ections were then washed in 0.01 M PBS containing 0.04%ormal rabbit serum two times for 15 min each time, and

ncubated for 2 h at RT in FITC-conjugated donkey anti-goatgG secondary antibody (1:200; Cat. No. 705-095-147, peakmission 520 nm, green fluorescence; Jackson ImmunoRe-earch) in 0.01 M PBS containing 0.04% normal rabbit serum toisualize CRF2 receptor immunofluorescence. After incubation

n secondary antibody, sections were washed in 0.01 M PBSontaining 0.04% normal rabbit serum two times for 15 minach time and mounted onto glass slides, then cover-slippedith 4’,6-diamidino-2-phenylindole DAPI Vectashield (Cat. No.-1200, Vector Laboratories), which fluorescently stains celluclei.

Quantification of CRF2 receptor/TrpOH doubleimmunofluorescence

For each subject (n�6; Sprague Dawley rats; Harlan Labora-tories), 100� and 200� magnification photomicrographs weregenerated for regions of interest using a Nikon 90i microscopeand a Roper Scientific CoolSNAP ES digital camera linked to acomputer with NIS Elements imaging software (A.G. HeinzeInc., Lake Forest, CA, USA). The 100� photomicrographs wereused to identify and document the rostrocaudal level of thesample; 200� photomicrographs were used for quantificationwith each subdivision. For each photomicrograph, the region ofinterest we placed in the center of the field of view using the20� objective lens and then photographs were taken usingFITC, Cy5, and DAPI filters. Analysis of fluorescence in the DRand pontine raphe nucleus (PnR) was performed at six ana-tomical levels (�7.46, �7.64, �8.00, �8.18, �8.54, and �9.16

Fig. 2. Photomicrographs of tryptophan hydroxylase (TrpOH) immuno(DR) at six different rostrocaudal levels (A–F). White boxes indicate reentire fields of view of the boxes indicating the regions sampled at 20periaqueductal gray (DRVL/VLPAG; B–D), and pontine raphe nucleusdorsal part; DRV, dorsal raphe nulceus, ventral part; DRVL/VLPAG, ddorsal raphe nucleus, interfascicular part; DRC, dorsal raphe nucleus, c
�m. For interpretation of the references to color in this figure legend, the read
m bregma; one section/level/rat; Fig. 2), within the DRD, DRV,RVL/VLPAG, DRI, DRC and PnR. The anatomical levels and sub-ivisions were defined based on a rat brain atlas (Abrams et al.,004; Paxinos and Watson, 1998). Cell counts collected from pho-omicrographs were probably not biased by differences in cell num-er or density, as photomicrographs were obtained at a plane ofocus in the middle of the tissue sections, which were 30 �m thick.Separate layers for CRF2 receptor-immunoreactive (ir) and TrpOH-irhotomicrographs were created using Adobe Photoshope CSAdobe Systems Incorporated, San Jose, CA, USA). The numbers ofRF2 receptor-ir and TrpOH-ir neurons were quantified by placing

dots over each CRF2 receptor-ir and TrpOH-ir profile in additional cellounting layers. The cell counting layers were superimposed and theumbers of single-labeled CRF2 receptor-ir cells, single-labeledrpOH-ir neurons, and double-labeled neurons were counted. Dou-le-labeled (CRF2 receptor-ir/TrpOH-ir) neurons were confirmed withhe slides themselves using 400� magnification.

Image capture

Brightfield photomicrographs were taken using a Nikon 90imicroscope and a Nikon DS-Fi1 digital camera linked to acomputer with NIS Elements 3.00 imaging software (A.G. Hei-nze Inc., Lake Forest, CA, USA). Low magnification images ofimmunofluorescence in cell culture studies were generatedusing a Leica DMRB fluorescence microscope, a Leica DC500camera, and image capture software (Leica Microsystems,Heidelberg, Germany). Confocal images of immunofluores-cence in cell culture studies were generated using a Leica TCSSP2 AOBS laser scanning confocal microscope using FITC,Cy5, and DAPI filter cubes and Leica Confocal Software (v.2.00, Leica Microsystems), presented as 8-�m-thick z-stack

rojections. Contrast and brightness of the photographs weredjusted using Adobe Photoshop CS (Adobe Systems Incorpo-ated, San Jose, CA, USA). Photographic plates were prepared inorelDraw for Windows 12.0 (Viglen Ltd., Wembley, UK).

nce illustrating the subdivisions analyzed in the dorsal raphe nucleustographed at higher magnification (200�) and used for analysis. The

nification for the dorsal raphe nucleus, ventrolateral part/ventrolateralare not visible in the figure. Abbreviations: DRD, dorsal raphe nucleus,he nucleus, ventrolateral part/ventrolateral periaqueductal gray; DRI,rt; PnR, pontine raphe nucleus. Scale bar: 50 �m; inset scale bar: 100

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RESULTS

Immunocytochemistry

In order to determine the specificity of the anti-CRF2 re-eptor antibody used in these studies, we conducted im-unocytochemical staining of wild type HEK-293 cells thatre known to lack CRF2 receptor expression (Dautzenberg

Fig. 4. Photomicrographs illustrate immunofluorescence of HEK-293fluorescent protein (EGFP; C, G) and mCRF2� receptor (D, H). The gmCRF2� receptor. Note that only light green autofluorescence is premmunofluorescence using the goat anti-CRF1/2 receptor antibody (sc-nti-CRF receptor antibody (sc-1826) used in this study, replicatin

Fig. 3. Photomicrographs illustrate immunocytochemical staining ofreen fluorescent protein (EGFP) and mCRF2� receptor (C–G). The gr

the mCRF2� receptor. Note that only light green autofluorescencemmunofluorescence using the goat anti-mCRF2� receptor antibodyillustrating immunofluorescence with the goat anti-mCRF2� receptohotomicrographs of immunofluorescence of HEK-293 cells stably exells, (F) mCRF2� receptor immunofluorescence, (G) photomicrograph

in HEK-293 cells. Scale bar: (A–D), 60 �m; (E–G), 20 �m. For interprthe Web version of this article.

2

nterpretation of the references to color in this figure legend, the reader is refe

et al., 2000), and HEK-293 cells stably expressing themouse CRF2� receptor and EGFP. The anti-CRF2 receptorantibody did not immunostain wild type HEK-293 cells (Fig.3B) but did immunostain HEK-293 cells stably expressingthe mouse CRF2� (mCRF2�) receptor (Fig. 3D) and EGFP(Fig. 3C; for additional controls, see Fig. 4). High magnifi-cation confocal images revealed that the CRF2 receptor

cells (A, B, E, F) or HEK-293 cells stably expressing enhanced greenescence (C, G) results from EGFP expressed in conjunction with theEK-293 wild type cells (A, E). (B, D) Photomicrographs illustrating

–H) Photomicrographs illustrating immunofluorescence using the goatfrom the experiment illustrated in Fig. 3. Scale bar: 50 �m. For

wild type cells (A, B) or HEK-293 cells stably expressing enhancedescence (A, C, E, G) results from EGFP expressed in conjunction witht in HEK-293 wild type cells (A). (B, D, F, G) Illustrate results of

6) used in this study. (B, D) Low magnification photomicrographsy (sc-1826) used in this study. (E–G) High magnification confocalEGFP and mCRF2� receptor: (E) EGFP expression in the HEK-293

ng the cellular distribution of EGFP and mCRF2� receptor expressionf the references to color in this figure legend, the reader is referred to

wild typereen fluorsent in H

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immunostaining was restricted to the plasma membraneand cytoplasmic compartments of the cell, while EGFPwas expressed throughout the cell including the nuclearcompartment (Fig. 3E–G).

As opposed to CRF2 receptors, wild type HEK-293cells are known to express CRF1 receptors (Dautzenberget al., 2000). The anti-CRF1/2 receptor antibody immuno-tained both the wild type HEK-293 cells and the HEK-293ells that stably express the mCRF2� receptor and EGFPFig. 4B, D). The intensity of staining in the HEK-293 cellstably expressing the mCRF2� receptor was greater than

n the wild type HEK-293 cells, suggesting that this antibodyetected both endogenous CRF1 and, in cells stably express-

ing the mCRF2� receptor, CRF2 receptors (Fig. 4).

CRF2 receptor immunostaining in CRF1 receptorknockout mice. Very few, faintly stained, CRF2 recep-tor-ir cells were found in regions of the brain known tocontain little to no CRF2 receptor mRNA expression, in-cluding the caudate putamen (CP; Fig. 5A, B) and para-ventricular nucleus of the thalamus (PV; Fig. 4C, D (VanPett et al., 2000)) in either wild type (Fig. 5A, C) or CRF1

receptor knockout mice (CRF1 KO; Fig. 5B, D). In contrast,large numbers of CRF2 receptor-ir cells were found inegions known to contain high amounts of CRF2 receptorRNA expression, such as the arcuate nucleus (Arc; Fig.E, F), ventromedial hypothalamus (VMH; Fig. 5E, F), andorsal raphe nucleus (DR; Fig. 5G, H; (Van Pett et al.,000)), in both wild type (Fig. 5E, G) and CRF1 receptorO mice (Fig. 5F, H), indicating that the immunostainingas not due to cross-reactivity with CRF1 receptors.

estern blot

he anti-CRF2 receptor antibody recognized a band be-ween 37 and 50 kDa (the predicted molecular weight ofhe full-length CRF2� receptor is about 40–45 kDa) inestern blot analysis using homogenized microdissectedrain regions (Fig. 6). All brain regions examined arenown to contain CRF2 receptor mRNA expression (Vanett et al., 2000), and the density of the bands in differentrain regions corresponded with the density of CRF2 re-

ceptor mRNA expression in the brain (Van Pett et al.,2000). For example, band density was highest in the LS,whereas it was lowest in cortical regions such as the Enand M1 (Fig. 6). There were no differences in the actinlevels in different brain regions (Fig. 6). There was no bandat the predicted molecular weight for the CRF1 receptor76–80 kDa; (Radulovic et al., 1998)) in any region exam-ned (Fig. 6).

RF2 receptor immunostaining in the rostral DR

Rostral regions of the rat DR (�6.92 mm to �7.64 mmbregma) contained a low (in the most rostral sections of therostral DR) to moderate (in the most caudal sections of therostral DR; Fig. 7A–C, Table 1) number of CRF2 receptor-irells. Although some CRF2 receptor-ir cells were apparent

in structures adjacent to the DR, such as the periaqueduc-tal gray region (Fig. 7D) both the density and intensity of
staining of cells were lower compared to regions in the DR.
In the rostral DRD, CRF2 receptor-ir cells were dense nearhe cerebral aqueduct (Aq) at the midline, although someere located outside the boundaries of the DRD laterally

Fig. 7A, B). In the most rostral part of the rostral DR, theajority of CRF2 receptor-ir cells were located along theidline within the DR (with some cells scattered outside

he DR, in the adjacent oculomotor complex and periaq-eductal gray; Fig. 7A, B). Large, densely stained CRF2

receptor-ir cells were observed within the paratrochlearnucleus (Pa4; Fig. 7A, C).

CRF2 receptor immunostaining in themid-rostrocaudal DR

Mid-rostrocaudal regions of the DR (�7.73 mm to �8.45mm bregma) contained the greatest density and numbersof CRF2 receptor-ir cells in the DR (Fig. 8, Table 1). Thelargest numbers of CRF2 receptor-ir cells were located inhe DRV, particularly at and around �8.00 mm bregma104.2�3.9 cells). At this level, although there were alsoany CRF2 receptor-ir cells scattered around the midline

of the DRV, there were bilateral clusters of intenselystained CRF2 receptor-ir cells above the medial longitudi-al fasciculus (mlf) on either side of the midline (Fig. 8A,). The majority of CRF2 receptor-ir cells were dorsal to

the mlf, but some were located between the fiber bundlesof the mlf with a few cells in the region lateral to thesuperior cerebellar peduncle (Fig. 8A, C). A dense centralcluster of CRF2 receptor-ir cells in the midline was locatedin the DRD throughout the mid-rostrocaudal DR (Fig. 8A,B), a region referred to as the DRD core region by Abramset al. (2005). There also were some CRF2 receptor-ir cellscattered outside this dense cluster (Fig. 8A, B), within aegion referred to as the DRD shell region by Abrams et al.2005). Scattered CRF2 receptor-ir cells were present in

the DRVL region (Fig. 8A, D).

CRF2 receptor immunostaining in the caudal DR

Caudal regions of the DR (�8.54 mm to �9.26 mmbregma) contained fewer numbers of CRF2 receptor-ircells (Fig. 9A–C, Table 1), relative to the mid-rostrocaudalDR. CRF2 receptor-ir cells within the caudal DR wereobserved in the DRI where a small number of cells, ori-ented in a vertical plane between the mlf, were arranged inbilateral columns (Fig. 9A, C). There were, however, a fewscattered, intensely stained CRF2 receptor-ir cells dorsalto the DRI, in the DRC (Fig. 9A, B). These were located inthe midline ventrally, extending laterally in clusters of cellslocated just below the cerebral aqueduct in the dorsal partof the DRC (Fig. 9A, B). Lateral to the DRC, scatteredCRF2 receptor-ir cells were observed within the lateral andericentral part of the dorsal tegmental nucleus (LDTg,TgP; Fig. 9A, D).

RF2 receptor/TrpOH double immunofluorescence

CRF2 receptor and TrpOH immunofluorescence wereound throughout the rostrocaudal extent of the DR (Figs.0 and 11). CRF2 receptor immunofluorescence was

found outside of the DR, but it was most prominent in the

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Fig. 5. Photomicrographs illustrating the distribution of CRF2 receptor-immunoreactive (ir) cells in specific brain regions of wild type (A, C, E, G) andRF1 receptor knockout (B, D, F, H) mouse brains. (A, B) Very few, faintly immunostained, CRF2 receptor-ir cells were found in the caudate putamen

of either wild type (A) or CRF1 receptor knockout mice (B). (C, D) Very few, faintly immunostained, CRF2 receptor-ir cells were found in theparaventricular nucleus of the thalamus of either wild type (C) or CRF1 receptor knockout mice (D). (E, F) Numerous and densely packed CRF2

receptor-ir cells were found throughout the arcuate nucleus (Arc) and ventromedial hypothalamus (VMH) of both wild type (E) and CRF1 receptornockout (F) mice. (G, H) CRF2 receptor-ir cells were found throughout the dorsal raphe nucleus (DR) of both wild type (G) and CRF1 receptor

knockout (H) mice. Abbreviations: Arc, arcuate nucleus; DR, dorsal raphe nucleus; mlf, medial longitudinal fasciculus; VMH, ventromedial hypothal-
amus. Scale bar: (A, B, E-H), 100 �m; (C–D), 50 �m.

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DR (Fig. 10D–F). In addition, a significant proportion ofTrpOH-positive neurons also expressed CRF2 receptors,(32.5–58.1%, depending on the subdivision) (Fig. 10G–I,Table 1), which is consistent with previous studies describ-ing co-localization of CRF2 receptor and serotonin trans-orter mRNA expression (Day et al., 2004).

DISCUSSION

CRF2 receptor-ir cells were distributed throughout the ros-trocaudal extent of the DR, with clear regional differencesin the number and density of cells. There were low tomoderate numbers of CRF2 receptor-ir cells within therostral and caudal DR. The highest densities and the great-est numbers of CRF2 receptor-ir cells were observed in themid-rostrocaudal DR. At this anatomical level, CRF2 re-eptor-ir cells were observed in the dorsal, ventral, and, to

lesser extent, the ventrolateral parts of the DR. Theighest densities of CRF2 receptor-ir cells within the DRD,

DRV, and DRI corresponded to the location of the highestdensities of serotonergic neurons in these regions (Stein-busch, 1981). In agreement with previous studies of CRF2

and serotonin transporter mRNA expression (Day et al.,2004), CRF2 receptor immunofluorescence was co-local-zed with TrpOH-ir neurons. These data show that CRF2

receptor-ir cells are expressed throughout the DR, but thehighest concentrations seem to be restricted to specificsubdivisions of the mid-rostrocaudal DR and in somecases specific parts of these subdivisions. These dataindicate that CRF2 receptor mRNA expression previouslyescribed in the DR is associated with CRF2 receptor

mmunostaining of perikarya, and that the topographical

Fig. 6. Western blot analysis using the anti-CRF2 receptor antibody(sc-1826) in microdissected rat brain tissue. In all brain regions ana-lyzed, the anti-CRF2 receptor antibody recognized one major band atbetween 37 and 50 kDA, which corresponds to the predicted molecularweight of the full-length CRF2 receptor. The density of the bands indifferent brain regions corresponded with the density of CRF2 receptormRNA expression previously described in rat brain. Band density washighest in the lateral septum (LS), whereas it was lower in corticalregions such as the entorhinal cortex (En) and motor cortex (M1).There were no differences in actin levels among the different regions.Results are representative of three replicate western blots. Abbrevia-tions: DR, dorsal raphe nucleus; En, entorhinal cortex; LS, ventral partof the lateral septum; M1, motor cortex; MnR, median raphe nucleus;MWM, molecular weight markers; VMH, ventromedial hypothalamus.

istributions of the CRF2 receptor mRNA and protein ex-

pression are similar, including expression in serotonergicneurons.

Technical considerations

Immunostaining resulted in clear CRF2 receptor immuno-taining of perikarya within the DR and other brain regionsith high levels of CRF2 receptor mRNA expression. Re-

cent studies by Waselus et al. (2009) using electron mi-roscopy and immunogold labeling have demonstratedhat CRF2 receptor immunoreactivity is predominantlyytoplasmic in DR neurons under basal unstressed condi-ions (with a ratio of cytoplasmic to total immunogold par-icles of 0.85�0.01), and shifts toward a greater expres-ion at the plasma membrane following stress exposurewith a ratio of cytoplasmic to total immunogold particles of.56�0.03). Although we cannot distinguish plasma mem-rane-associated from cytoplasmic CRF2 receptor in ourtudies, our data are suggestive of expression of CRF2

receptor in both cellular compartments in unstressed rats,consistent with findings by Waselus et al. (2009). Interest-ingly, the anti-CRF2 receptor antibodies used in the studiesy Waselus et al. (2009) and in our studies were both

directed at the N-terminus of the CRF2 receptor (humanCRF2 receptor; Novus Biologicals, personal communica-ion), and both identified a single major band between 37nd 50 kDa, as determined using western blot (Wang etl., 2007).

RF2 receptor isoforms

It is possible that the CRF2 receptor immunostaining de-scribed here represents immunostaining of CRF2�, CRF2�,

RF2�-tr, and/or soluble (s)CRF2� receptor isoforms in therat DR. The antibody that was used is likely to recognize allknown isoforms of the CRF2 receptor because the anti-ody recognizes a 12-amino acid sequence of the N-ter-inus that is nearly identical in all CRF2 receptor isoforms

hat have been characterized (Fig. 1). A recent study (Ev-ns and Seasholtz, 2009) has shown that the sCRF2�

receptor is found in the rat brain, and that the mRNA isefficiently translated. In addition Evans and Seasholtz(2009) showed that the sCRF2� receptor is not trafficked tohe membrane, which may account in part for the apparentbundance of cytoplasmic staining seen in this study andrevious studies (Waselus et al., 2009). Consistent withhese findings, recent studies by Schulz and colleagues2010) showed that CRF2� receptor contains an N-terminal

pseudo signal peptide that is unable to target the peptide tothe endoplasmic reticulum membrane, resulting in very lowcell surface expression (Schulz et al., 2010). Furthermore,Tian and colleagues (Tian et al., 2006) showed that thesc-1826 antibody recognizes the CRF2�-tr receptor isoformusing western blot analysis (band at 16–32 kDa) in theforebrain, olfactory bulb, and cerebellum of rats and mice.The sc-1826 antibody was also shown to recognize theCRF2�-tr receptor in HEK-293 cells expressing the CRF2�-tr

receptor isoform, using both immunocytochemistry andwestern blot analysis (Tian et al., 2006). Based on theimmunohistochemical and immunofluorescence results in
our study, together with in situ hybridization studies, recep-
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tor autoradiography studies, and functional studies usingdirect microinjections of selective CRF2 receptor agonists

Fig. 7. Photomicrographs illustrating the distribution of CRF2 recepmmunostaining in the rostral DR (�7.10 mm bregma) and surro

agnification and shown in panels (B, C). (B) Scattered CRF2 receptor-(DRD). (C) Scattered CRF2 receptor-ir cells were found within the midlinucleus (Pa4). (D) Few, faintly-stained CRF2 receptor-ir cells were

Abbreviations: Aq, cerebral aqueduct; bv, blood vessel; DR, dorsal raphventral part; mlf, medial longitudinal fasciculus; Pa4, paratrochlear nuc100 �m.

able 1. Expression of CRF2 receptors in serotonergic cells of the ra

Region Rostrocaudal level(mm bregma)

# CRF2R�neurons

# TrpOHneurons

RD �7.46 63.3�8.2 42.7�5.RV �7.46 51.8�8.4 40.8�5.RD �7.64 75.0�9.6 41.3�3.RV �7.64 98.4�4.8 61.0�7.RVL/VLPAG �7.64 83.9�2.9 29.8�4.RD �8.00 91.8�4.6 64.2�7.RV �8.00 104.2�3.9 87.4�1.RVL/VLPAG �8.00 78.8�7.7 59.4�5.RD �8.18 89.5�10.8 64.0�3.RV �8.18 86.2�7.6 62.0�6.RVL/VLPAG �8.18 68.8�7.5 39.3�3.RC �8.54 78.3�8.5 54.7�7.RI �8.54 28.2�4.9 29.4�3.RC �9.16 42.4�5.5 40.5�4.
nR �9.16 29.2�5.7 24.8�0.8 1
nd antagonists within the DR, it seems likely that at leastne high-affinity receptor for CRF2 receptor ligands is

s in the rostral DR. (A) Low magnification image of CRF2 receptorgions. The black boxes indicate regions photographed at higher

ere found throughout the rostral dorsal part of the dorsal raphe nucleuspart of the dorsal raphe nucleus (DRV) and the adjacent paratrochlear

thin the dorsolateral periaqueductal gray (dlPAG) dorsal to the DR.s; DRD, dorsal raphe nucleus, dorsal part; DRV, dorsal raphe nucleus,AG, dorsolateral periaqueductal gray. Scale bar: (A), 500 �m; (B–D),

ermined by dual immunofluorescence

double-labeledeurons

% TrpOH� neuronsthat were CRF2R�

% CRF2R� neuronsthat were TrpOH�

3.8�1.8 35.2�6.7 23.1�3.66.3�2.9 40.0�6.6 31.7�4.83.8�2.5 32.6�5.4 20.3�3.82.5�6.7 50.5�7.9 38.0�3.20.8�3.2 32.9�5.2 14.5�4.29.5�5.0 44.6�3.1 31.6�4.84.3�3.2 47.9�2.5 42.0�3.38.8�3.5 32.5�6.7 26.7�6.44.8�0.9 54.4�2.3 36.5�2.46.0�6.2 58.1�8.3 41.2�5.07.8�2.4 46.3�6.3 26.0�2.13.3�4.4 43.3�1.6 30.4�6.02.4�3.0 39.7�7.6 44.9�9.15.0�1.8 38.5�4.9� 35.6�5.4

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present in the DR, and perhaps more. Future work usingspecific antibodies or probes will be required to determinewhich isoform(s) of the CRF2 receptor are expressed in the

R, their relative abundance, and functional properties.

opography of the distribution of CRF2 receptor-irells in the DR

he dorsal and ventral portions of the mid-rostrocaudalR, where we observed the greatest concentrations ofRF2 receptor-ir cells, have unique patterns of anatomicalrojections to and from forebrain limbic structures. Theid-rostrocaudal DRD (relative to other subdivisions of theR) is known to receive strong projections from specific

orebrain structures including the prelimbic cortex, centralmygdaloid nucleus, bed nucleus of the stria terminalis,edial and lateral preoptic area, paraventricular nucleus of

he hypothalamus, and dorsal hypothalamic area (Peyront al., 1998; Vertes, 2004). The mid-rostrocaudal DRD isnown to project to a number of forebrain regions involvedn regulation of emotional behavior, including the medial

Fig. 8. Photomicrographs illustrating the distribution of CRF2 receptreceptor immunostaining in the mid-rostrocaudal DR (�8.00 mm bregat higher magnification and shown in panels (B–D). (B) CRF2 receptucleus (DRD) core (DRDc) and DRD shell (DRDSh) regions, with denells in the DRDSh. (C) Numerous and densely packed CRF2 recep

ventral part (DRV). (D) Numerous CRF2 receptor-ir cells were found inqueduct; bv, blood vessel; DR, dorsal raphe nucleus; DRD, dorsal raRDSh, dorsal raphe nucleus, dorsal part, shell region; DRV, dorsal raedial longitudinal fasciculus. Scale bar: (A), 500 �m; (B–D), 100 �m

refrontal cortex, basolateral amygdaloid nucleus, central f

mygdaloid nucleus, nucleus accumbens, dorsal hypotha-amic nucleus, and bed nucleus of the stria terminalis (Imait al., 1986; Van Bockstaele et al., 1993; Petrov et al.,994; Petit et al., 1995; Commons et al., 2003; Abrams etl., 2005). This pattern of projections suggests that thisegion provides a major contribution to the dorsal rapheorebrain tract, one of six major serotonergic tracts inner-ating the forebrain (Azmitia and Segal, 1978; Azmitia,981; Lowry et al., 2008). The mid-rostrocaudal DRV alsoontains large numbers of neurons projecting to the baso-

ateral amygdaloid nucleus (Abrams et al., 2005) as well asarge numbers of neurons projecting to the caudate puta-

en and horizontal limb of the diagonal band of BrocaSteinbusch et al., 1980; Steinbusch, 1981; Semba et al.,988). The mid-rostrocaudal DRV (relative to other subdi-isions of the DR) receives strong projections from the

ateral orbital cortex and medial and lateral preoptic areasPeyron et al., 1998). Some of the forebrain regions thatroject to the mid-rostrocaudal DR contain neurons thatxpress Ucn 2 and Ucn 3, both of which have high affinity

in the mid-rostrocaudal DR. (A) Low magnification image of CRF2

surrounding regions. The black boxes indicate regions photographedwere differentially distributed in the dorsal part of the dorsal raphe

s of CRF2 receptor-ir cells in the DRDc and scattered CRF2 receptor-irs were found throughout the mid-rostrocaudal dorsal raphe nucleus,l raphe nucleus, ventrolateral part (DRVL). Abbreviations: Aq, cerebralus, dorsal part; DRDc, dorsal raphe nucleus, dorsal part, core region;leus, ventral part; DRVL, dorsal raphe nucleus, ventrolateral part; mlf,

or-ir cellsma) andor-ir cellsse clustertor-ir cellthe dorsaphe nucle

or CRF2 receptors and therefore could be sources of

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endogenous ligand(s) acting on full-length CRF2 receptorswithin the DR (reviewed by Reul and Holsboer, 2002). Forexample, neurons within the paraventricular nucleus of thehypothalamus express Ucn 2 mRNA while neurons withinthe bed nucleus of the stria terminalis and medial andlateral preoptic areas express Ucn 3 mRNA (Peyron et al.,1998; Lewis et al., 2001; Reyes et al., 2001; Li et al., 2002).Consistent with the hypothesis that urocortins play an im-portant role in regulation of serotonergic systems and be-havior, Ucn 1/Ucn 2 knockout mice (Neufeld-Cohen et al.,2010a) as well as Ucn 1/Ucn 2/Ucn 3 triple knockout micehave altered baseline serotonergic activity in neural sys-tems regulating anxiety states and altered anxiety-like be-havior (Neufeld-Cohen et al., 2010b). It is known that Ucn1-ir neurons from the Edinger-Westphal nucleus also proj-ect to the DR (Bittencourt et al., 1999), but it is currentlyunclear if the DR is also innervated by Ucn 2-, or Ucn3-containing fibers. As the DR is one of the few brainstructures where CRF2 receptor mRNA expression isigher than CRF1 receptor mRNA expression, future stud-

Fig. 9. Photomicrographs illustrating the distribution of CRF2 receptorf CRF2 receptor immunostaining in the caudal DR (�8.90 mm bregm

higher magnification and shown in panels (B–D). (B) Densely immunonucleus, caudal part, dorsal part (dDRC) and in bilateral clusters vennterfascicular part of the dorsal raphe nucleus (DRI), showing a vertichenotype of serotonergic neurons in the region. (D) Scattered CRF2

the lateral (LDTg) and pericentral parts (DTgP). Abbreviations: Aq, cerenucleus, caudal part, dorsal part; DRI, dorsal raphe nucleus, interfategmental nucleus, lateral part; mlf, medial longitudinal fasciculus. Sc

ies should examine the innervation of the DR by high- 1

affinity CRF2 receptor ligands including Ucn 2 and Ucn 3and the sources of fibers containing these CRF-relatedneuropeptides.

Electrophysiological responses to CRF2 receptorligands in the DR

The current study, describing high numbers of CRF2 re-ceptor-ir cells in the mid-rostrocaudal DR, is in agreementwith a number of electrophysiological studies investigatingthe effects of CRF and CRF-related neuropeptides onserotonergic neuronal firing rates. Electrophysiologicalstudies provide strong evidence that functional CRF2 re-eptors are expressed within the mid-rostrocaudal DR.ow concentrations of ovine CRF, concentrations that are

ikely to bind to CRF1 receptors (Primus et al., 1997),microinjected into the DR decrease the in vivo neuronalfiring rates of DR serotonergic neurons (Kirby et al., 2000),while high concentrations of ovine CRF, doses that arelikely to bind to CRF1 and CRF2 receptors (Primus et al.,

n the caudal dorsal raphe nucleus (DR). (A) Low magnification imagerrounding regions. The black boxes indicate regions photographed at

CRF2 receptor-ir cells were evident in the midline of the dorsal raphecerebral aqueduct. (C) CRF2 receptor-ir cells were found within the

tion and bipolar, fusiform shape, features that are consistent with their cells were found within the adjacent dorsal tegmental nucleus, bothduct; bv, blood vessel; DR, dorsal raphe nucleus; dDRC, dorsal rapheart; DTgP, dorsal tegmental nucleus, pericentral part; LDTg, dorsalA), 500 �m; (B–D), 100 �m.

-ir cells ia) and sustained

tral to theal orientareceptor-bral aque

997), increase both in vivo and in vitro firing rates of DR

tstjdlti

(S to color


serotonergic neurons (Lowry et al., 2000; Kirby et al.,2000). In vitro extracellular recording studies by Lowry andcolleagues (Lowry et al., 2000) revealed that the majorityof presumed serotonergic neurons activated by CRF werelocated in the mid-rostrocaudal DRV relative to the mid-rostrocaudal DRD. These in vitro extracellular recordingsare consistent with our current findings, as the highestdensity of CRF2 receptor-ir cells were found in the mid-rostrocaudal DRV region. Microinjections of low doses ofUcn 2 (mUcn 2; Valentino, personal communication) intothe mid-rostrocaudal DR of anesthetized rats inhibits thefiring rates of serotonergic neurons, while injections of highdoses of mUcn 2 into the same region increase the firingrates of serotonergic neurons (Pernar et al., 2004). Thus,the concentrations of both CRF and mUcn 2 appear to becritical for their effects on the neuronal activity of seroto-nergic neurons in the DR, with a possibility of both inhibi-tory and excitatory responses depending on the local con-centration of ligand. Future studies should take into ac-count the rostrocaudal and dorsoventral differences inthe distribution of CRF2 receptors as well as the possi-bility of the presence of multiple isoforms of CRF2 re-

Fig. 10. Dual CRF2 receptor and tryptophan hydroxylase (TrpOH) imm(panels A–C), CRF2 receptor (panels D–F), and CRF2 receptor/TrpOB, E, H), and caudal (C, F, I) levels of the DR. Arrows indicate double-cale bar: 100 �m, insets, 15 �m. For interpretation of the references

article.

ceptors in the DR.

Functional implications—anxiety-relatedphysiological and behavioral effects of CRF2

receptor ligands in the DR

Our current findings of high numbers of CRF2 receptor-ir cells inhe mid-rostrocaudal DR are in agreement with a number oftudies investigating neurochemical and behavioral responseso intra-DR injections of CRF2 receptor ligands. Direct microin-ections of mUcn 2 into the DR induce potentiation of fear con-itioning and escape deficits measured 24 h later in a model of

earned helplessness (Hammack et al., 2003a). This potentia-ion of fear conditioning and escape deficits is prevented by priornjections of ASV-30 but not the CRF1 receptor antagonist2-methyl-4-(N-propyl-N-cycloproanemethylamino)-5-chloro-6-(2,4,6-trichloranilino)pyrimidine (NBI27914) (Hammack et al.,2003b). Furthermore, the potentiation of fear conditioning andescape deficits was observed following injections of high dosesof CRF (doses likely to bind to CRF2 receptors (Primus et al.,1997)) into the caudal, but not the rostral, DR (Hammack et al.,2002). These functional studies suggest that activation of CRF2

receptors within the DR, particularly within the mid-rostrocaudaland caudal DR may play an important role in the regulation of

scence in the dorsal raphe nucleus (DR). Photomicrographs of TrpOHimmunofluorescence (G–I) at the rostral (A, D, G), mid-rostrocaudal

ells shown in insets at the lower right corner of each photomicrograph.in this figure legend, the reader is referred to the Web version of this

unofluoreH doublelabeled c

emotional behavior.

h

o(s

T

mo

(rri er is refe


Intra-DR microinjections of Ucn 1, which binds withigh affinity to both CRF1 and CRF2 receptors, have also

been shown to induce physiological and behavioral re-sponses. Intra-DR injections of Ucn 1 induce hypothermiaand decrease food and fluid consumption in mice (Turekand Ryabinin, 2005; Weitemier and Ryabinin, 2006). Fur-thermore, intra-DR injections of the CRF2 receptor antag-nist ASV-30 prevent ethanol-induced hyperthermiaTurek and Ryabinin, 2005). Together, these functionaltudies support the hypothesis that CRF2 receptors within

the DR are involved in the regulation of multiple physio-logical and behavioral responses, consistent with theirwidespread distribution in the DR.

CONCLUSION

In conclusion, this study describes for the first time thedistribution of CRF2 receptor-ir cells throughout the DR.

he highest numbers and the highest densities of CRF2

receptor-ir cells were found in the mid-rostrocaudal DR,particularly in the DRD and DRV subdivisions. Togetherwith previous studies, these findings are consistent withthe hypothesis that CRF2 receptor activation within themid-rostrocaudal and caudal DR is an important compo-nent of neural mechanisms regulating anxiety- or stress-related physiological and behavioral responses. Based onthe widespread distribution of CRF2 receptors in the DRand the functional heterogeneity of the DR, CRF2 recep-tors in this region are likely to modulate diverse physiolog-ical and behavioral responses.

Functionally integrated CRF and mesolimbocortical se-rotonergic systems arising from the mid-rostrocaudal and

Fig. 11. Photomicrographs of CRF2 receptor immunofluorescence anused for quantification. Photomicrographs of TrpOH immunofluorescenB��, D��, F��), and CRF2 receptor/TrpOH/DAPI triple immunofluoresepresents the dorsal raphe nucleus, dorsal part (DRD), row (C–D��epresents the dorsal raphe nucleus, ventrolateral part (DRVL). Scalenterpretation of the references to color in this figure legend, the read

caudal DR are likely to play an important role in stress-

related psychopathology including anxiety and mood dis-orders, as well as stress-induced relapse to drug abuse(Arborelius et al., 1999; Sarnyai et al., 2001; Heinrichs andKoob, 2004). CRF innervation of the human DR is greatestin the mid- to caudal levels (Austin et al., 1997), anddepressed patients have increased CRF immunoreactivityin the caudal subnucleus of the DR (Austin et al., 2003). Inaddition, recent studies of human depressed suicide pa-tients have revealed that the widely reported increases intryptophan hydroxylase 2 (tph2) mRNA and protein ex-pression in the DR (Underwood et al., 1999; Bonkale et al.,2006; Bach-Mizrachi et al., 2006) may be restricted to thecaudal subnucleus (Bach-Mizrachi et al., 2008) and itsprojection sites (Perroud et al., 2010). Consequently, dys-regulation of CRF2 receptor signaling within the caudal DR

ay have important implications for affective disorder andther stress-related neuropsychiatric disorders.

Acknowledgments—C.A. Lowry was supported by a WellcomeTrust Research Career Development Fellowship (RCDF 068558/Z/02/Z), and a 2007 NARSAD Young Investigator Award and iscurrently supported by an NSF CAREER Award (NSF-IOS#0845550). The project described was supported by Award Num-bers R01MH065702 (AS/CAL), R01MH086539 (CAL) and1F32MH084463 (JLL) from the NIMH. The content is solely theresponsibility of the authors and does not necessarily representthe official views of the National Institute of Mental Health or theNational Institutes of Health.

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(Accepted 22 March 2011)(Available online 29 March 2011)

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