Cortagine infused into the medial prefrontal cortex attenuates predator-induced defensive behaviors and Fos protein production in selective nuclei of the amygdala in male CD1 mice

    Cortagine infused into the medial prefrontal cortex attenuates predatorinduced defensive behaviors and Fos protein production in selective nuclei of the amygdala in male CD1 mice Nathan S. Pentkowski, Philip Tovote, Arturo R. Zavala, Yoav Litvin, D. Caroline Blanchard, Joachim Spiess, Robert J. Blanchard PII: DOI: Reference:

S0018-506X(13)00137-2 doi: 10.1016/j.yhbeh.2013.06.008 YHBEH 3587

To appear in:

Hormones and Behavior

Received date: Revised date: Accepted date:

4 February 2013 21 June 2013 29 June 2013

Please cite this article as: Pentkowski, Nathan S., Tovote, Philip, Zavala, Arturo R., Litvin, Yoav, Blanchard, D. Caroline, Spiess, Joachim, Blanchard, Robert J., Cortagine infused into the medial prefrontal cortex attenuates predator-induced defensive behaviors and Fos protein production in selective nuclei of the amygdala in male CD1 mice, Hormones and Behavior (2013), doi: 10.1016/j.yhbeh.2013.06.008

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ACCEPTED MANUSCRIPT Title Page Title: Cortagine infused into the medial prefrontal cortex attenuates predator-induced defensive

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behaviors and Fos protein production in selective nuclei of the amygdala in male CD1 mice. Authors:

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Nathan S. Pentkowski1, 2, 4, Philip Tovote3, 4, Arturo R. Zavala5, Yoav Litvin1, 2, 4, D. Caroline

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Blanchard2, 3, 4, Joachim Spiess3, 4 and Robert J. Blanchard1, 2, 4

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Affiliations:

Department of Psychology, University of Hawaii, Honolulu, Hawaii

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Pacific Biomedical Research Center, University of Hawaii, Honolulu, Hawaii

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John A. Burns School of Medicine, University of Hawaii, Honolulu, Hawaii Specialized Neuroscience Research Program, University of Hawaii, Honolulu, Hawaii

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Department of Psychology, California State University, Long Beach, Long Beach, California

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Communicating author/ current address: Nathan S. Pentkowski

Arizona State University

School of Life Sciences, ISTB1 429 Tempe, AZ 85287-4501

Email: [email protected]

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ACCEPTED MANUSCRIPT Abstract Corticotropin-releasing factor (CRF) plays an essential role in coordinating the autonomic,

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endocrine and behavioral responses to stressors. In this study, we investigated the role of CRF

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within the medial prefrontal cortex (mPFC) in modulating unconditioned defensive behaviors, by examining the effects of microinfusing cortagine a selective type-1 CRF receptor (CRF1)

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agonist, or acidic-astressin a preferential CRF1 antagonist, into the mPFC in male CD-1 mice

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exposed to a live predator (rat exposure test-RET). Cortagine microinfusions significantly reduced several indices of defense, including avoidance and freezing, suggesting a specific role

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for CRF1 within the infralimbic and prelimbic regions of the mPFC in modulating unconditioned behavioral responsivity to a predator. In contrast, microinfusions of acidic-astressin failed to

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alter defensive behaviors during predator exposure in the RET. Cortagine microinfusions also reduced Fos protein production in the medial, central and basomedial, but not basolateral

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subnuclei of the amygdala in mice exposed to the rat predatory threat stimulus. These results suggest that CRF1 activation within the mPFC attenuates predator-induced unconditioned anxiety-like defensive behaviors, likely via inhibition of specific amygdalar nuclei. Furthermore, the present findings suggest that the mPFC represents a unique neural region whereby activation of CRF1 produces behavioral effects that contrast with those elicited following systemic administration of CRF1 agonists. Key words: corticotropin-releasing factor; CRF; anxiety; fear; PFC; emotion; defense.

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ACCEPTED MANUSCRIPT Introduction Prolonged exposure to excessive or uncontrollable stressors represents a common risk

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factor for a variety of psychiatric illnesses including depression, schizophrenia and anxiety

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disorders (Bale, 2005; Korte et al., 2005; Nemeroff et al., 1984). Corticotropin-releasing factor (CRF) plays an essential role in coordinating the autonomic, endocrine and behavioral responses

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to stressors (Carrasco and Van de Kar, 2003; Spiess et al., 1981; Vale et al., 1981). CRF-related

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neuropeptides produce their biological effects by binding to two G-protein coupled receptorsCRF1 and CRF2 (Eckart et al., 2002; Hauger et al., 2003; Hillhouse and Grammatopoulos,

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2006). CRF1 controls activation of the hypothalamic-pituitary-adrenal axis (Bale et al., 2000), and mediates many of the behavioral effects of CRF (Bale and Vale, 2004). In particular, gene

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deletion experiments indicate that anxiety-like defensive behaviors are potentiated principally through CRF1 (Smith et al., 1998; Timpl et al., 1998) and reduced via CRF2 (Bale et al., 2000;

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Kishimoto et al., 2000).

Pharmacological experiments indicate reliable anxiolytic and anxiogenic-like effects following global CRF1 inhibition and activation [i.e., systemic and intracerebroventricular (i.c.v.) administration], respectively (Bale and Vale, 2004). Indeed, i.c.v. administration of CRF1 agonists enhance defensive behavior in the acoustic startle (Risbrough et al., 2004), defensive withdrawal (Heinrichs and Joppa, 2001), social interaction (Campbell et al., 2004) and rat exposure (RET; Tovote et al., 2010) tests, while inhibition by central administration of antisense oligodeoxynucleotides directed against CRF1 mRNA attenuates defensive behavior (Heinrichs et al., 1997; Liebsch et al., 1995; Skutella et al., 1998). In addition, systemic and i.c.v. injections of CRF1 non-peptidic antagonists (e.g., CP-145,526, antalarmin or DMP696) reduce defensive behaviors in the elevated plus-maze (EPM), light/dark test and mouse defensive

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ACCEPTED MANUSCRIPT test battery (for review, see Bale and Vale, 2004; Carrasco and Van de Kar, 2003). In contrast to systemic and i.c.v. administration, intra-septal CRF1 activation attenuates defensive behavior

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(Radulovic et al., 1999), while CRF1 knockdown in the globus pallidus potentiates defensive

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responding (Sztainberg et al., 2011). These behavioral differences likely involve CRF acting on different neural circuits mediating different ethologically relevant defensive behaviors (Lowry

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and Moore, 2006), and indicate the need for site-specific manipulations of CRF systems

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(Todorovic et al., 2005).

Two regions of the medial prefrontal cortex (mPFC), the prelimbic (PrL) and infralimbic

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(IL) cortices, exhibit dense CRF1 but not CRF2 expression (Chalmers et al., 1995; Van Pett et al., 2000) and have been implicated in mediating emotional and defensive responses to various

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threats (Deacon et al., 2003; Morgane et al., 2005; Quirk and Beer, 2006; Shah and Treit, 2003, 2004), although the results are complex. Specifically, deletion of all forebrain CRF1 reduced

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defensive behavior in the light/dark test (Refojo et al., 2011), whereas microinfusions of human/rat-CRF (20 ng/side) into the mPFC increased defensive behavior in the EPM (Jaferi and Bhatnagar, 2007). In contrast, higher doses of human/rat CRF (200 ng/side) infused into dorsal regions of the frontal cortex decreased defensive behavior in the EPM (Zieba et al., 2008). Similarly, Ohata and Shibasaki (2011) reported that microinfusions of ovine-CRF into the mPFC dose-dependently increased (50 ng/side) or decreased (1000 ng/side) defensive behavior in the EPM, suggesting that the effects of mPFC CRF1 on defensive behavior are dose- and/or regiondependent. In the present study we sought to help clarify the aforementioned differences in behavior following mPFC CRF1 agonist infusions by utilizing a model of defensive behavior elicited by a predatory threat stimulus. Specifically, we investigated the role of CRF1 within the mPFC in

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ACCEPTED MANUSCRIPT modulating predator-induced defensive behavior by microinfusing the highly selective CRF1 agonist cortagine (Tezval et al., 2004) or antagonist acidic-astressin ([Glu11,16]Ast; Eckart et

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al., 2001) into the mPFC 15 min prior to testing in the RET. Additionally, using Fos protein as a

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neural marker, we examined the effects of cortagine on predator-induced functional activation in mPFC targets within the amygdala, hypothalamus and periaqueductal gray (PAG), nuclei that

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have been implicated in modulating defensive behavior (for review see Canteras, 2002;

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Dielenberg et al., 2001; Takahashi et al., 2005). Materials and Methods

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All procedures conducted on animals in these experiments were reviewed and approved by the Institutional Animal Care and Use Committee at the University of Hawaii. Animal care

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and housing adhered to the conditions set forth in the “Guide for the Care and Use of Laboratory

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Animals” (Institute of Laboratory Animal Resources on Life Sciences, National Research

Subjects

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Council, 1996). All efforts were made to minimize animal pain and suffering.

Subjects were 12-14 week old adult male CD-1 mice (Charles River Laboratories, St. Louis, MO), weighing between 35 and 41 g at the time of surgery. Mice were individually housed in standard polypropylene cages in a temperature (20 ± 2 ◦C) and illumination (12 hr light/dark cycle; lights on at 06:00 a.m.) controlled room, with free access to food and water. Mice were allowed 1 week to acclimate to the vivarium prior to surgery, during which time they were randomly assigned to either an experimental CRF-drug group or to an artificial cerebrospinal fluid (aCSF) vehicle-control group. Surgery

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ACCEPTED MANUSCRIPT Mice were deeply anesthetized with Avertin anesthesia (330 mg/kg, i.p.; Sigma, St. Louis, MO, USA) and were mounted in a stereotaxic apparatus (David Kopf Instruments,

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Tujunga, CA). The scalp was incised and retracted, and the head was positioned to place

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Bregma and Lambda in the same horizontal plane. Two sets of small burr holes (1.0 mm in diameter) were drilled in the skull in order to implant stainless steel guide cannulae (26G,

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Plastics One, Roanoke, VA) bilaterally into the mPFC; guide cannulae were implanted 0.5 mm

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dorsal to the desired injection site. Cannulae were implanted using a David Kopf micromanipulator aimed at intermediate regions of the mPFC using coordinates obtained from

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the Mouse Brain in Stereotaxic Coordinates (Franklin and Paxinos, 2007); +1.78 mm anterior to bregma, +/- 0.30 mm from the midline and -2.0 mm ventral from the skull surface. Guide

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cannulae were secured to the skull using dental cement, and following implantation, dummy cannulae were inserted into each guide in order to prevent blockage and/or infection. Following

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surgery, animals received 1 ml of saline (0.9%, s.c.) to prevent dehydration and were returned to their home cage for a 5-day recovery period prior to the start of behavioral testing. Peptides

Cortagine and [Glu11,16]Ast were diluted in aCSF, pH = 7.4, until the final doses were obtained; 50 and 100 ng of cortagine, and 100 and 200 ng of [Glu11,16]Ast. Drug doses were selected based on previous research demonstrating enhanced defensive behavior following intracranial (Litvin et al., 2007) and i.c.v. (Tovote et al., 2010) cortagine administration. Control mice received infusions of the aCSF vehicle solution. Peptide Infusions Drug microinfusions for both experiments occurred 15 min prior to the start of behavioral testing. Prior to the injection, mice were lightly anesthetized with isoflurane, the cap and dummy

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ACCEPTED MANUSCRIPT were removed, and 0.2 l/side of the appropriate compound (cortagine, [Glu11,16]Ast or aCSF) was delivered over a 30-sec period. The injectors were connected via polyethylene-20 tubing

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(Plastics One) to 25 l Hamilton microsyringes mounted in an infusion pump

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(CMA/Microdialysis, Sweden) in order to control the rate of infusion. The injectors extended

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0.5 mm below the end of the guide cannulae to insure accurate peptide delivery. Prior to removal, the injectors remained in place for an additional 30 sec to allow for drug diffusion.

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Behavioral Testing in the Rat Exposure Test (RET)

The RET is a mouse model of predator-induced unconditioned defensive behaviors, the

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structure of which makes it particularly useful for observing avoidance, risk assessment, freezing and defensive burying (Blanchard et al., 2005a; Yang et al., 2004). All behavioral testing

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occurred between 10:00 and 13:00 hr. The test apparatus consisted of two rectangular Plexiglas chambers (home and exposure chambers) connected via a clear Plexiglas tube (4.5 cm in

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diameter, 13 cm in length, and elevated 1.5 cm from the floor). The home (7 × 7 × 12 cm) and exposure (46 × 24 × 21 cm) compartments were made of black Plexiglas on three sides and clear Plexiglas on one side to facilitate recording. A wire mesh screen divided the exposure chamber into two equal sized compartments in order to separate the mouse test subject (surface area) from the rat threat source. Adult male Long-Evans hooded rats weighing between 450 and 500 g from the breeding colony maintained at the University of Hawaii were used as the predatory threat stimulus. In order to keep the stimulus rats uniformly active during and across test sessions, and thus maintaining a consistent predatory threat between test trials, systemic injections of damphetamine (5.0 mg/kg, i.p.) were administered 20 min prior to testing (Blanchard et al., 2005a; Yang et al., 2004); if cessation of movement/stereotypy was observed rats received an

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ACCEPTED MANUSCRIPT additional dose of d-amphetamine (1.0 mg/kg, i.p.). Between each test trial the apparatus was thoroughly cleaned using an aqueous 10% ethanol solution.

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All test trials were conducted under white light to ensure the rat threat stimulus was as

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unambiguous as possible. Prior to the start of each trial, home cage bedding from the mouse test subject was placed on the floor of the home chamber and surface area. The testing procedure

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consisted of two phases. During the first phase (habituation), mice were individually placed in

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the center of the surface area, and were allowed to explore freely for 10 min without a rat present; 3 habituation sessions occurred over 3 consecutive days. On the fourth day (test), an

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amphetamine treated rat was introduced behind the wire mesh prior to placing the test mouse

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Behavioral Measures and Analysis

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into the apparatus.

Test trials were recorded and analyzed using the ethological behavioral analysis software

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Observer 5.0 (Noldus Information Technology, Wageningen, The Netherlands) by a highly trained observer blind to drug conditions. Use of this software allowed for a detailed, frame-byframe analysis. Dependent measures consisted of both proximal and behavioral measures. The proximal measures, assessed relative to the location of the stimulus rat, included the duration of time spent in the home chamber, tunnel and on the surface area; locomotor activity consisted of the number of transits between these locations. Behavioral measures included durations of stretch attend (risk assessment)-standing on all four paws with flat back and stretched neck orientated toward the threat source; freezing-complete cessation of movement except respiration; grooming-movement of forepaws or tongue over the body; defensive burying-pushing bedding from the home chamber into the tunnel opening; contact-duration of time spent in contact with

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ACCEPTED MANUSCRIPT the wire-mesh, measured as direct paw or head contact; and climbing-all forepaws simultaneously contacting the wire-mesh divider.

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Histology One hour following behavioral testing mice received bilateral microinfusions of

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methylene blue (0.2 l/side), and were immediately overdosed with Avertin anesthesia (500 mg/kg, i.p.) and perfused transcardially with 150 ml of ice-cold 0.9% phosphate-buffered saline

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(PBS) followed by 150 ml of 4% paraformaldehyde in PBS (0.1M), pH 7.4. Following extraction from the skull, brains were soaked in 4% paraformaldehyde for 48 h at 4°C and then

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cryoprotected by transferring them sequentially to 10, 20 and 30% sucrose solutions in phosphate buffer (PB; 0.1M), pH 7.4, each for 24 h. A series of coronal sections (25 m thick) taken

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throughout the entire rostral–caudal extent of each cannulae placement were collected. After drying (24 h), the sections were thionin-stained and analyzed under a microscope (Leica, USA)

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in order to verify cannulae tip placement by an observer blind to drug conditions. Fos Protein Immunohistochemistry A separate cohort of mice was used to examine the effects of cortagine (100 ng/side) microinfused into the mPFC on predator induced-Fos protein production in subnuclei throughout the amygdala, hypothalamus and PAG. The effects of cortagine on freezing behavior were also analyzed to insure that the behavioral effects of cortagine were repeatable between experimental cohorts. Ninety minutes after testing in the RET, mice were perfused and their brains were cryoprotected as described above except that mice in the Fos experiment did not receive infusions of methylene blue. The brains were frozen and a series of 25 µm sections were cut across the entire rostral-caudal extent of the areas of interest using a cryostat in the frontal plane. Free-floating sections were then processed for Fos protein immunohistochemistry as described

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ACCEPTED MANUSCRIPT previously (Dielenberg et al., 2001). Briefly, endogenous peroxidase activity was eliminated by incubating the tissue in 1% H2O2 in methanol for 15 min. Sections were then saturated with 5%

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goat serum and 0.3% Triton X-100 in 0.01 M PBS (PBG), and then incubated in rabbit anti-c-

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Fos antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA; diluted 1:20,000 in PBG) for 48 h at 4°C. Subsequently, the sections were washed and incubated for 1 h at room temperature in

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biotinylated goat anti-rabbit IgG antibody (diluted 1:400 in PBG) followed by ExtraAvidin-

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horseradish peroxidase (Sigma; diluted 1:1000 in PBG). Horseradish peroxidase activity was then visualized using the nickel diaminobenzidine and glucose oxidase reaction, which was

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stopped after 10 min by extensive washing in PBS. Sections were mounted on gelatin-coated

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Fos Immunoreactivity Analyses

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slides, and then dehydrated and cover-slipped with Eukitt (Sigma).

Fos immunoreactivity was examined using a Olympus BX40 (Olympus Imaging America

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Inc., Center Valley, PA) microscope set at 20X magnification and counted by an observer blind to drug conditions using ImageJ (National Institutes of Health). The anatomical locations and boundaries of each region were determined using a mouse brain atlas (Franklin and Paxinos, 2007). Sections taken at -1.58 mm from bregma included the anterior basolateral (BLA), anterior central, (CeA), anterior basomedial (BMA) and posterior medial (MeP) subnuclei of the amygdala, and the dorsomedial (DMH) and ventromedial (VMH) subnuclei of the hypothalamus. Sections taken at -4.48 mm from bregma included the dorsolateral PAG (PAGdl). Sample areas (0.0825 mm2) within each region of interest were analyzed and counted for Fos immunoreactive cells, which were identified by a brown-black oval-shaped nucleus. The final counts from the sample areas were averaged and scaled to provide a mean number of Fos-positive cells per mm2. Statistical Analyses

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ACCEPTED MANUSCRIPT Dependent measures were analyzed using separate one-way analysis of variance (ANOVA). Significant effects were followed by Newman-Keuls post-hoc tests in order to

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compare CRF drug and vehicle control means. Separate t-tests for independent samples were

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used to analyze Fos protein production for all regions of interest; α was set at 0.05 for all comparisons.

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Results

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Histology

Figure 1A presents a representative photomicrograph (right) and a schematic of injector

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tip placements (left) within the mPFC (shaded area) for mice included in the experiments. Only mice with correct injector tip placements located in the mPFC (encompassing both the PrL and

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IL cortices) within the anterior–posterior range defined as +1.54 to +1.98 mm anterior to bregma, the medial–lateral range defined as +0.1 to 0.6 mm lateral to the midsagittal suture, and the

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dorsal–ventral range defined as -2.2 to -3.0 mm ventral to the skull surface were included in the analysis (Franklin and Paxinos, 2007). Eight mice from the cortagine vehicle, seven from the 50 ng and seven from the 100 ng groups were excluded due to placements outside the circumscribed region. In all, 38 mice from the cortagine experiment with cannulae tips located within the mPFC were included in the analyses; vehicle (n=12), 50 ng (n=8) and 100 ng (n=18). Five mice from the 100 ng cortagine group with cannulae tips located within the medial orbital (MO) cortex were included as anatomical controls. Two subjects from the [Glu11,16]Ast vehicle, three from the 100 ng and four from the 200 ng groups were excluded due to misplaced cannulae. Thus, 31 mice from the [Glu11,16]Ast groups were included in the analyses; vehicle (n=8), 100 ng (n=11) and 200 ng (n=12). Two mice from the cortagine Fos experiment were excluded due to

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ACCEPTED MANUSCRIPT tissue damage sustained during histology. Thus, 12 mice from the Fos experiment were included in the behavioral and immunohistochemical analyses; vehicle (n=6) and 100 ng cortagine (n=6).

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Behavioral Effects of Cortagine in the RET

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The data for both the behavioral and proximal measures in the RET following mPFC cortagine microinfusions are summarized in table 1, and figure 1 (B and C). One-way ANOVA

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indicated that mPFC cortagine microinfusions produced significant effects on the duration of

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freezing [F(2, 35)=13.24, p<0.001]. Subsequent Newman-Keuls post-hoc analysis indicated that both the 50 ng and 100 ng doses reduced freezing compared to vehicle controls (p<0.001 in each

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case), however, there was no significant difference between the two drug doses. Cortagine microinfusions significantly altered the duration of contact with the wire-mesh divider

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[F(2,35)=11.81, p<0.001]. Subsequent post-hoc analysis revealed that both doses of cortagine significantly increased contact duration compared to vehicle controls (p<0.05 in each case); there

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were no significant differences between cortagine dose groups. Cortagine microinfusions produced a significant effect on the duration of climbing [F(2,35)=9.18, p<0.001]. Post-hoc analysis revealed that 100 ng of cortagine dose dependently increased climbing durations compared to both the vehicle and 50 ng groups (p<0.05 in each case); whereas 50 ng failed to alter climbing compared to controls. There was also a significant effect of cortagine on the duration of time spent in the home chamber [F(2,35)=7.92, p<0.005]. Post-hoc analysis revealed that both the 50 ng and 100 ng doses of cortagine significantly reduced home chamber duration compared to vehicle controls (p<0.05 in each case); there was no difference between drug doses. Lastly, the ANOVA revealed a significant effect of drug on the overall duration of time spent on the surface area near the rat predatory threat stimulus [F(2, 35)=6.37, p<0.005]. Post-hoc analysis demonstrated that 100 ng of cortagine significantly increased the duration of time spent

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ACCEPTED MANUSCRIPT on the surface area compared to controls (p<0.05) and that 50 ng infusions tended to increase surface-area time, but this effect was not reliable (p=0.06). Finally, cortagine infusions into the

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mPFC failed to produce a significant effect on the durations of stretch attend, defensive burying,

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grooming or the number of transits, (see table 1) although there was a trend toward an increase in transits (p=.09).

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Figure 1 illustrates that the effects of cortagine were region-specific. Mice microinfused

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with cortagine (100 ng) into the MO region of the PFC did not differ from controls in any of the behavioral (figure 1B) or proximal (figure 1C) measures during testing in the RET.

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Effects of [Glu11,16]Ast in the RET

The data for both the behavioral and proximal measures in the RET following mPFC

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[Glu11,16]Ast microinfusions are presented in table 2. Overall, the ANOVAs indicated that

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measures.

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[Glu11,16]Ast did not produce significant group differences on any of the behavioral or proximal

Effects of Cortagine on Fos Protein Production in the RET The data for both freezing behavior and Fos protein production following cortagine microinfusions are presented in figure 3. Figure 2 illustrates the anatomical locations and boundaries of each region analyzed (figure 2A), as well as Fos protein production throughout subnuclei of the amygdala (figure 2B, left), and in the MeP of mice receiving cortagine (figure 2B, right, bottom) or aCSF (figure 2B, right, top) infusions. Microinfusions of cortagine (100 ng) into the mPFC reduced freezing t(10)=2.27, p<0.05 and the number of Fos-positive nuclei in the MeP t(10)=2.64, p<0.05, CeA t(10)=2.61, p<0.05 and BMA t(10)=2.48, p<0.05, but not BLA regions of the amygdala in mice exposed to the rat predatory threat stimulus. Microinfusions of cortagine also failed to alter predator-induced Fos production in the PAGdl (aCSF: 48.49±11.64

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ACCEPTED MANUSCRIPT vs. cortagine: 71.25±10.96), DMH (aCSF: 75.55±24.19 vs. cortagine: 131.06±36.43) and the dorsomedial region of the VMH (aCSF: 53.16±15.90 vs. cortagine: 103.66±22.58), although

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there was a trend toward an increase in the VMH (p=0.09).

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Discussion

Results from the present study are in agreement with the involvement of the PrL and IL

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subregions of the mPFC in modulating predator-induced unconditioned defensive behavior (Wall

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et al., 2004), and indicate a critical role for mPFC CRF1 in mediating these behavioral effects. Microinfusions of the selective CRF1 agonist cortagine into the mPFC reduced several indices of

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defense, including levels of freezing to, and avoidance of, the predatory threat stimulus (see figure 1B and 1C); effects consistent with those obtained in the EPM following high doses of the

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less selective agonists human/rat- and ovine-CRF (Jaferi and Bhatnagar, 2007; Ohata and Shibasaki, 2011). The attenuating effects of CRF1 activation were region-dependent as

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cortagine microinfused into the MO region of the frontal cortex failed to alter defensive behavior. Finally, the behavioral effects obtained in the present study were specific to measures of defense, as cortagine did not alter non-defensive behaviors (i.e., grooming). The behavioral effects of cortagine likely involved reduced amygdalar neuronal processing, as cortagine-induced reductions in defensiveness were associated with parallel reductions in levels of Fos protein production (see figure 3B) in amygdalar nuclei that receive projections from the mPFC, including the MeP, CeA and BMA (Vertes, 2004), regions that have been implicated in modulating predator-induced defensive behavior (Canteras, 2002; Dielenberg et al., 2001; Martinez et al., 2008; Takahashi et al., 2005). Reduced levels of Fos in the MeP may indicate that cortagine microinfusions reduced neuronal processing of predatory odors as lesions of the MeP have been shown to reduce predator odor-induced defensive behavior

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ACCEPTED MANUSCRIPT (Blanchard et al., 2005b; Li et al., 2004). In addition, reduced levels of Fos in the CeA may be particularly important in relation to cortagine-induced reductions in freezing as optogenetic

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stimulation of the CeA induces freezing behavior (Ciocchi et al., 2010).

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In contrast to CRF1 activation, antagonism of mPFC CRF1 following [Glu11,16]Ast microinfusions produced no changes in the defensive repertoire of experimental mice (see table

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2), suggesting that endogenous activation of CRF1 within these cortical regions do not play a

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major role in mediating defensive behavior in the RET, but influence defensive behavior in response to agonist activation. These null effects of CRF1 antagonism are consistent with a lack

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of effect following i.c.v. administration of [Glu11,16]Ast in the RET. Nonetheless, the null effects of mPFC CRF1 antagonism are somewhat surprising given that deletion of all forebrain CRF1

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reduced defensive behavior in the light/dark test (Refojo et al., 2011). These differences between studies suggest that mPFC CRF1 activation produces differential effects on defensive behavior

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compared to CRF1 located throughout the entire forebrain region, further suggesting that the effects of CRF are region dependent (Radulovic et al., 1999). The present results suggest that stimulation of CRF1 within the PrL and IL subregions of the mPFC shifts predator-induced defensive behavior from a strategy of active avoidance to threat investigation. Indeed, cortagine-infused mice displayed reduced levels of immobility (i.e., freezing) and predatory avoidance (i.e., chamber duration), while simultaneously exhibiting enhanced levels of threat/environmental investigation (i.e., surface duration, mesh contact, transits and mesh climbing). Similar reductions in freezing and avoidance have been reported in the RET following peripheral injections of the classic anxiolytics diazepam (Tovote, 2006) and buspirone (Blanchard et al., 2005a). Furthermore, when rats are exposed to feline predators in environments where escape is possible via tunnel/ burrow systems, conditions available to

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ACCEPTED MANUSCRIPT subjects in the present study, levels of freezing and avoidance of the surface area closest to the predator are reduced following treatment with the benzodiazepines diazepam and

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chlordiazepoxide (Blanchard et al., 1990). Collectively, these data suggest a role for mPFC

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CRF1 in mediating anxiety-like defensive behaviors.

The reductions in defensive behavior detected in the present study differs from those

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following i.c.v. cortagine administration (Farrokhi et al., 2007; Tezval et al., 2004; Tovote et al.,

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2010), suggesting that the mPFC, encompassing both the IL and PrL cortices, is a unique anatomical site for central actions of CRF1. In addition to the mPFC (Jaferi and Bhatnagar,

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2007; Ohata and Shibasaki, 2011), site-specific effects following CRF manipulations have also been detected in the lateral septum (Radulovic et al., 1999) and globus pallidus (Sztainberg et al.,

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2011). For instance, intra-septal microinfusions of human/rat-CRF reduced, rather than increased the expression of conditioned fear (Radulovic et al., 1999), and CRF1 knockdown in

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the globus pallidus enhanced defensive behavior in the EPM (Sztainberg et al., 2011). In contrast, stimulation of CRF1 within the ventral hippocampus (Pentkowski et al., 2009) and PAG (Litvin et al., 2007) enhances defensive behavior in the EPM and RET, respectively. These neuroanatomical specific effects of CRF action may provide anatomical explanations for studies that have shown conflicting results (antidepressant and anxiogenic) following i.c.v. cortagine microinfusions (Tezval et al., 2004). Collectively, these data suggest that the behavioral effects of central CRF activation are likely dependent not only on the specific receptor subtype, but also on the degree to which environmental threats activate specific brain systems, and the extent to which different CRF manipulations (i.e., i.c.v. vs. mPFC infusions) affect those specific systems. The present results in conjunction with previous reports suggest that the effects of mPFC CRF1 activation on defensive behavior are dose-dependent. Microinfusions of the CRF agonists

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ACCEPTED MANUSCRIPT human/rat- and ovine-CRF into the mPFC potentiated defensive behavior at low (20 – 50 ng/side) doses (Jaferi and Bhatnagar, 2007; Ohata and Shibasaki, 2011), while higher (200 –

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1000 ng/side) doses produced the opposite effect (Ohata and Shibasaki, 2011; Zieba et al., 2008).

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The present results with cortagine (50 – 100 ng/side) are congruent with higher doses of human/rat- and ovine-CRF, results likely due to cortagines pharmacological properties. Indeed,

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cortagine’s selectivity is more than two-fold higher than that of human/rat- and ovine-CRF on

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the basis of binding affinity and approximately five-fold higher than that of human/rat- and ovine-CRF on the basis of biological potency (Tezval et al., 2004). Mechanistically, lower doses

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of CRF may preferentially target CRF1 on mPFC glutamatergic projection neurons with higher doses leading to activation of CRF1 on 5-hydroxytryptamine (5-HT) neurons or glutamatergic

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collaterals within the mPFC (Vertes, 2004; Zhou et al., 2010). Indeed selective knockdown of all forebrain CRF1, which were predominantly located on glutamatergic neurons reduced

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defensive behavior (Refojo et al., 2011), while in-vitro CRF application in mPFC slices potentiated inhibitory postsynaptic potentials (IPSPs) via enhancement of 5-HT driven gammaaminobutyric acid (GABA) activity (Tan et al., 2004). This potentiation of mPFC GABAergic neuronal activity could reduce amygdala activation leading to reduced defensive behavior via either direct projections from the IL to the MeP, CeA, BMA or cortical nuclei of amygdala, or direct projections from the PrL to the capsular part of the CeA. Alternatively, mPFC projections to intercalated neurons in the BLA inhibit output from the CeA (Likhtik et al., 2005), suggesting that the present results may have occurred from enhanced mPFC output. Further research is needed to delineate the precise mechanisms by which mPFC CRF1 stimulation can potentiate (Jaferi and Bhatnagar, 2007; Ohata and Shibasaki, 2011) or attenuate (Ohata and Shibasaki, 2011; Zieba et al., 2008) defensive behavior.

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ACCEPTED MANUSCRIPT The hypothesis that mPFC CRF1 activation attenuates defensive behavior via inhibiting mPFC output is consistent with numerous studies investigating the neuroanatomical correlates of

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emotion in various tests of defense (Quirk and Beer, 2006). Transection of the mPFC produced

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anxiolytic-like effects in the EPM and social interaction test (Gonzalez et al., 2000), results that contrast with those obtained during behavioral testing in an open field and EPM following mPFC

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electrolytic lesions (Jinks and McGregor, 1997). Damage sustained in the latter study to fibers

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of passage may explain these discrepancies, as inactivation of the PrL and IL cortices using the GABA agonists midazolam (Shah and Treit, 2004) or muscimol (Shah et al., 2004), the AMPA

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antagonist CNQX (Bi et al., 2013), or excitotoxic lesions using ibotenic acid (Shah and Treit, 2003; Sullivan and Gratton, 2002), which all spare fibers of passage, all produced anxiolytic-like

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effects in the EPM, shock-probe burying and social interaction test. Furthermore, mice with excitotoxic mPFC lesions also demonstrated anxiolysis in the EPM and successive alley test

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(Deacon et al., 2003). In partial contrast, excitotoxic lesions restricted to the PrL and anterior cingulate cortices, produced anxiolytic-like effects in an open field test, while having no effect during the EPM (Lacroix et al., 1998), and pre-training lesions of IL increased conditioned freezing (Moscarello and LeDoux, 2013), suggesting that complete anxiolysis also requires inactivation of the IL cortex. Lacroix and colleagues (2000) provide support for this assertion as ibotenic acid lesions encompassing both the IL and PrL cortices decreased anxiety-like defensive responses in the EPM and open field test (Lacroix et al., 2000). Collectively, these results indicate that inactivation/inhibition of the mPFC, including both the PrL and IL cortices, attenuates the expression of innate defensive behaviors to various forms of threat (Davidson, 2003), and further suggest that the present effects resulted from CRF1-induced inhibition of the mPFC.

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ACCEPTED MANUSCRIPT Conclusion In conclusion, results from the present experiments confirm that CRF1 activation within

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the mPFC encompassing both the PrL and IL cortices attenuates defensive behaviors (Jaferi and

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Bhatnagar, 2007; Ohata and Shibasaki, 2011) and extend these behavioral effects to mouse models incorporating predator-induced threat (i.e., RET). The present results also indicate that

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the actions of forebrain CRF1 (Refojo et al., 2011) are site specific (i.e., mPFC) and are limited

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to phasic and not tonic effects. These results also suggest that the reductions in defensive behavior induced by mPFC CRF1 activation involve suppressed neuronal activation in discrete

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nuclei of the amygdala. These results provide further evidence that the effects of CRF are region dependent (Radulovic et al., 1999), and indicate the continued need for studies investigating the

mediate defensive behavior.

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Acknowledgements

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anatomical specificity of CRF system, particularly the mechanisms by which CRF1 in the PFC

The authors thank Lanikea B. King, Arturo D. Garcia, Amy Vasconcellos and Udo Schnitzbauer for their expert assistance. Arturo R. Zavala was supported by a grant from the National Center on Minority Health and Health Disparities (P20MD003942). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Center on Minority Health and Health Disparities. The authors have no conflicts of interest to report or any involvement to disclose, financial or otherwise, that may bias the conduct, interpretation, or presentation of this work. Abbreviations 5-HT: 5-hydroxytryptamine; [Glu11,16]Ast; acidic-astressin; ANOVA: analysis of variance; BMA: anterior basomedial amygdala; CeA: anterior central amygdala; aCSF: artificial

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ACCEPTED MANUSCRIPT cerebrospinal fluid; BLA: basolateral amygdala; CRF: corticotropin-releasing factor; EPM: elevated plus-maze; GABA: gamma-aminobutyric acid; IL: infralimbic cortex; IPSP: inhibitory

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postsynaptic potential; i.c.v.: intracerebroventricular; MO: medial orbital cortex; mPFC: medial

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prefrontal cortex; PB: phosphate buffer; PBG: phosphate-buffered goat serum; PBS: phosphatebuffered saline; MeP: posterior medial amygdala; PrL: prelimbic cortex; RET: rat exposure test.

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Figure Legends

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Figure 1. (A) Representative photomicrograph (right) and schematic depiction (left) of injector tip placements within the mPFC for mice included in the analyses. (B) Effects of cortagine

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(mean + SEM) microinfused into the mPFC or MO frontal cortex on behavioral measures, (C)

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and location preferences during testing in the RET. Differences for which p<0.001***,

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p<0.01**, p<0.05*, compared to aCSF vehicle controls, p<0.05# compared to all other groups. Figure 2. (A) Schematic representation of a coronal section of the mouse brain (-1.58 mm from Bregma) adapted from Franklin and Paxinos (2007) illustrating regions analyzed for Fos protein production. Numbers represent the regions analyzed as follows: (1) CeA-anterior central amygdala, (2) BLA-anterior basolateral amygdala, (3) BMA-anterior basomedial amygdala, (4) MeP-posterior medial amygdala, (5) DMH-dorsomedial hypothalamus and (6) VMHventromedial hypothalamus. (B) Representative photomicrographs of coronal sections illustrating Fos protein production throughout subnuclei of the amygdala (left: 1x magnification) and in the posterior MeP (right: 20x magnification) from animals receiving microinfusions of aCSF vehicle (top right) or 100 ng cortagine (bottom right) into the mPFC 15-min prior to testing in the RET. Scale bar = 100 µm; arrow = Fos positive cell.

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p<0.05* compared to aCSF vehicle controls. Abbreviations: BMA-anterior basomedial

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amygdala; BLA-anterior basolateral amygdala; CeA-anterior central amygdala; MeP-posterior

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medial amygdala.

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Figure 2

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Figure 3

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50 ng

Stretch Attend p=.17

5.08 + 1.91

1.56 + 0.60

5.89 + 1.27

Burying p=.53

9.00 + 3.81

8.88 + 4.72

14.58 + 4.00

Transits p=.09

38.17 + 6.16

Grooming p=.13

47.00 + 10.40

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aCSF

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56.88 + 7.14

73.44 + 21.03

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Behavioral Measures ANOVA p values

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Table 1. Effects of cortagine microinfused into the mPFC during testing in the RET; differences were not significant.

100 ng

60.44 + 7.75

39.58 + 6.36

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Data are presented as mean ± SEM. Behavioral measures are durations of events in seconds (or numbers of transits) in the 10-min observation period.

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99.59 + 33.31

68.21 + 27.34

1.75 + 0.77

1.23 + 0.65

1.88 + 0.55

Contact p=.31

167.25 + 28.74

Climbing p=.61

45.00 + 11.81

Burying p=.60 Grooming p=.19

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95.50 + 45.58

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Stretch Attend p=.74

100 ng

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Freezing p=.76

aCSF

207.68 + 34.53

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Behavioral Measures ANOVA p values

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Table 2. Effects of [Glu11,16]Ast microinfused into the mPFC during testing in the RET; differences were not significant.

200 ng

239.46 + 28.77

63.92 + 11.57

2.50 + 1.12

4.86 + 1.97

3.75 + 1.25

45.44 + 7.37

54.73 + 6.81

39.58 + 4.26

46.50 + 7.43

34.18 + 4.09

31.83 + 3.05

224.44 + 60.12

181.91 + 56.28

Tunnel p=.09

36.88 + 10.00

23.05 + 7.00

30.88 + 9.00

Surface p=.60

349.19 + 56.90

400.09 + 57.04

425.21 + 40.00

Chamber p=.63

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Transits p=.09

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53.32 + 14.75

51.04 + 40.27

Data are presented as mean ± SEM. Behavioral measures are durations of events in seconds (or numbers of transits) in the 10-min observation period.

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Highlights 1. We examined the effects of a CRF agonist in the PFC on predator-induced defensiveness. 2. Cortagine infused into the medial PFC reduced predator-induced defensive behaviors. 3. Cortagine reduced predator-induced Fos expression in specific amygdala nuclei.

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