UNIVERSITY OF CALIFORNIA, SAN DIEGO

The Effect of Lateral Septum CRF 2 Receptor Activation on is Modulated by

Stress

A Dissertation submitted in partial satisfaction

of the requirements for the degree Doctor of Philosophy

in

Neurosciences

by

Brook Lewis Henry

Committee in charge:

Professor Athina Markou, Chair Professor Wylie Vale, Co-Chair Professor Mark Geyer Professor Richard Hauger Professor Gerhard Schulteis

2006

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DEDICATION

I wish to dedicate this text to my mother, Bonnie Henry, for her unconditional love and support during the last 32 years.

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EPIGRAPH

Experience: that most brutal of teachers. But you learn. My God do you learn.

C.S. Lewis

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TABLE OF CONTENTS

Signature Page...... iii

Dedication...... iv

Epigraph...... v

Table of Contents...... vi

List of Abbreviations...... vii

List of Figures...... viii

Acknowledgement...... ix

Vita...... x

Abstract of the Dissertation...... xi

Chapter 1: General Introduction...... …..1

Chapter 2: General Methods and Materials...... 28

Chapter 3: The Effect of Septal 2 and Stress on Anxiety-Like Behaviors...... …...35

Chapter 4: The Effect of CRF 1 and CRF 2 Receptor Activation on Anxiety-Like Behaviors Mediated by the Lateral Septum...... …....67

Chapter 5: The Role of the Edinger-Westphal Nucleus...... 95

Chapter 6: Summary and Conclusions...... …....115

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LIST OF ABBREVIATIONS

ACTH...... adrenocorticotropin CORT...... corticosterone CREB...... cyclic adenosine monophosphate response element binding protein CRF...... corticotropin releasing factor EST...... expressed sequence tag EW...... Edinger-Westphal GPCR...... G-protein coupled receptor HPA...... hypothalamic-pituitary-adrenal ICV...... intracerebroventricular mA...... milliamp MAPK...... mitogen-activated protein kinase µg...... microgram µl...... microliter ng...... nanogram nM...... nanomolar nmol...... nanomoles

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LIST OF FIGURES

Figure 1: Septal urocortin 2 increased anxiety-like behavior...... …....52

Figure 2: ICV urocortin 2 decreased motor activity at high dose...... …...64

Figure 3: Effect of septal Urocortin 2 on anxiety-like behavior is dependent on stress...... ….66

Figure 4: Septal urocortin 2 increased anxiety-like behavior when combined with stress in CRF 2 receptor wildtype mice on a mixed C57BL/6 x 129 background...... 58

Figure 5: Septal urocortin 2 did not have a significant effect on plasma corticosterone...... 60

Figure 6: No effect of septal urocortin 2 in CRF 2 receptor KO mice...... …...... 83

Figure 7: CRF 2 antagonist astressin-2B blocked effect of urocortin 2 and stress on anxiety...... 85

Figure 8: Septal administration of the CRF 1 antalarmin does not block the effect of urocortin 2...... 87

Figure 9: Administration of the CRF 1 antagonist antalarmin into the central amygdala reduced the anxiogenic effect of restraint stress...... …...... 89

Figure 10: Effect of a quinolinic acid lesion on Urocortin 1 positive neurons in the Edinger-Westphal nucleus in ICR mice...... 108

Figure 11: A quinolinic acid lesion of the Edinger-Westphal nucleus reduces the anxiogenic-like effects of septal urocortin 2, but does not alter anxiety-like behavior under baseline conditions in the absence of stress...... 109

Figure 12: Model for the role of the septal CRF 2 receptor in anxiety...... 128

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ACKNOWLEDGEMENT

I would like to acknowledge Professor Athina Markou for her invaluable assistance and guidance over the past four years.

I would also like to acknowledge Professor Wylie Vale for supporting my research and providing a surfeit of wit and wisdom.

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VITA

1995-1996 Research Assistant, Institute For Mathematical Behavioral Science University of California, Irvine

1997 Bachelor of Arts in Psychology, University of California, Irvine Magna Cum Laude

1997-2000 Research Assistant, Department of Neurobiology and Behavior University of California, Irvine

2003 Master of Science in Neurosciences, University of California, San Diego.

2006 Doctor of Philosophy in Neurosciences, University of California, San Diego

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ABSTRACT OF THE DISSERTATION

The Effect of Lateral Septum CRF 2 Receptor Activation on Anxiety is Modulated by Stress

by

Brook Lewis Henry

Doctor of Philosophy in Neurosciences

University of California, San Diego, 2006

Professor Athina Markou, Chair

Professor Wylie Vale, Co-Chair

Corticotropin-releasing factor (CRF), a 41 amino acid , mediates endocrine, autonomic, and behavioral responses to stress. While the CRF 1 receptor appears to contribute to anxiety associated with stress, the role of the CRF 2 receptor remains unclear and may depend on drug dose, brain location, or testing environment.

Results involving treatments with selective CRF 2 receptor or antagonists, and

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the behavior of CRF 2 receptor knockout (KO) mice suggest both anxiogenic and effects of CRF 2 receptor activation.

This dissertation project examined the hypothesis that the effect of CRF 2 receptor activation on anxiety depends on the stress level of the animal. The selective

CRF 2 receptor urocortin 2 was infused into the mouse lateral septum, a region with high CRF 2 receptor expression. Mice were tested in low or high stress (30 minutes of restraint) conditions, and behavior in the light-dark box, open field, and novel object test was assessed. In the low stress environment, the high dose of 500 ng of septal urocortin 2 increased anxiety-like behavior, but lower doses (1-100 ng) did not have consistent effects. However, in the high stress condition, 100 ng of septal urocortin 2 significantly increased anxiety-like behavior compared to vehicle.

Urocortin 2 had no effect on anxiety-like behavior in CRF 2 receptor knockout mice and septal administration of the relatively selective CRF 2 receptor antagonist astressin-

2B, but not the CRF 1 receptor selective antagonist antalarmin, blocked the anxiogenic effects of urocortin 2. Urocortin 2 infusion into the medial septum or lateral ventricle did not affect measures of anxiety-like behaviors. A lesion of the Edinger-Westphal nucleus, a source of urocortin 1 input to the lateral septum, decreased the anxiogenic effect of septal urocortin 2 during stress. These results indicate that the effect of septal

CRF 2 receptor activation on anxiety-like behavior is dependent on the level of stress in the environment and may involve the release of urocortin 1 from the Edinger-

Westphal nucleus. Under low stress conditions, activation of the CRF 2 receptor may have limited effect on anxiety-like behavior, but in a high stress environment,

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activation of both the CRF 1 receptor and the CRF 2 receptor will increase the anxiety response.

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Chapter 1:

GENERAL INTRODUCTION

A. CRF and the CRF 1 receptor

Corticotropin releasing factor (CRF), a 41 amino acid peptide, has been shown to mediate the endocrine, autonomic, and behavioral responses to stress. Vale and colleagues (1981), isolated CRF from the sheep hypothalamus and identified the peptide as the hypothalamic factor that activates the hypothalamic-pituitary-adrenal

(HPA) axis, initiating the release of adrenocorticotropin (ACTH) from the pituitary and the subsequent release of from the adrenal cortex. CRF plays a critical role in the "flight or fight" response to stress, increasing blood sugar, blood pressure, heart rate, and inhibiting immune and digestive function through the release of catecholamines such as epinephrine and norepinephrine and glucocorticoids that include cortisol and corticosterone (Dunn and Berridge, 1990). In addition to the hypothalamus, CRF is expressed in several areas of the brain; the highest expression is observed in the region referred to as the extended amygdala, including the central amygdala and the bed nucleus stria terminalis. CRF projections from the extended amygdala innervate the lateral septum, descend through the medial forebrain bundle to reach noreadrenergic neurons in the ventrolateral medulla, and project to areas

1 2 implicated in and anxiety, including the dorsal raphe and the region of the (Swanson et al.,1983).

A number of studies have indicated that CRF plays an important role in mediating anxiety. Central administration of CRF has consistently produced anxiogenic-like effects in several animal models of stress, including 1) decreased exploratory activity in a novel environment and increased exploration in a familiar environment (Sutton et al., 1982), 2) induced taste and place aversion (Heinrichs et al.,

1991), 3) increased stress-induced freezing (Sherman et al., 1988), 4) disruption of food intake (Arase et al, 1998), 5) enhancement of defensive burying (Korte et al,

1994), 6) increased acoustic startle (Swerdlow et al, 1986), and 8) decreased social interaction (Dunn and File, 1987). There is also evidence that CRF may play a role in depression. Studies have reported 1) higher CRF in cerebrospinal fluid and increased plasma cortisol in depressed individuals, 2) deficits in suppression of the HPA axis in depressed patients, and 3) decreased CRF binding sites in the prefrontal cortex of suicide victims (Arborelius et al, 1999).

The first CRF receptor discovered, CRF 1, was originally isolated from a human Cushing corticotropic cell tumor (Chen et al, 1993) and subsequently cloned from rat whole brain (Perrin et al, 1993) and rat cerebellar (Chang et al, 1993) and mouse corticotrope (Vita et al., 1993) cDNA libraries. The receptor was identified as a

7-transmembrane G-protein-coupled receptor (GPCR) related to the receptor family that includes releasing factor, , , vasoactive intestinal peptide, and . The CRF 1 receptor is coupled to G s and activates , stimulating the release of the intracellular messenger

3 cAMP and the phosphorylation of the cAMP response element binding protein

(CREB). There is also evidence that CRF 1 receptor stimulation can activate other signaling pathways such as the mitogen-activated protein kinase (MAPK) pathway

(Rossant et al, 1999). CRF 1 is expressed in many areas of the rodent brain, including the cortex, cerebellum, hippocampus, basolateral amygdala, bed nucleus of the stria terminalis, medial septum, raphe, pons, and sensory nuclei in the brainstem (van Pett et al., 2000). As expected, high levels of the CRF 1 receptor are found in the anterior and intermediate pituitary. Outside the brain, the receptor has been identified in skin, ovary, testis, leukemic mast cells, rat mammary carcinoma, and melanoma cells

(Perrin and Vale, 1999).

There is considerable evidence that CRF 1 receptor activation is involved in the anxiogenic effects of CRF. Smith and colleagues (1998) reported that CRF 1 receptor knockout mice exhibited decreased HPA response to stress and showed less anxiety- like behavior in the light-dark emergence task and the elevated plus maze. These CRF 1 receptor deficient mice had lower basal levels of corticosterone due to a decrease in the size of the zona fasciculata, the region of the adrenal gland where the glucocorticoid is produced. Corticosterone (CORT) replacement in the drinking water of the KO mice did not alter anxiety-like behavior in the elevated-plus maze, indicating that the anixolytic-like profile of these animals was not due to low CORT levels. Timpl and colleagues. (1998) generated a separate line of CRF 1 receptor KO mice and also observed a reduction of HPA activity and decreased anxiety-like behavior. A more recent study (Muller et al., 2003) generated a conditional CRF 1 receptor KO mouse controlled by a calcium/calmodulin-dependent kinase II α

4 promoter that removed the receptor from the amygdala, cortex, and hippocampus, leaving the pituitary CRF 1 and the HPA axis intact. These mice exhibited decreased anxiety-like behavior in the light-dark box and elevated plus maze; these results indicate that CRF 1 receptors in limbic brain regions are critical for mediating CRF- induced anxiety-like behaviors.

Treatment with CRF 1 receptor antisense nucleotides has been reported to reduce anxiety-like behavior in rodents. Heinrichs and colleagues (1997) showed that intracerebroventricular administration of CRF 1 receptor antisense oligos reduced defensive withdrawal in Wistar rats, and Liebsch and colleagues (1995, 1999) reported that CRF 1 receptor antisense treatment into the lateral ventricle or central amygdala reduced anxiety-like behavior in social defeat and the elevated plus maze. A number of CRF 1 receptor selective antagonists have been reported to decrease anxiety-like behavior in rodents, including NBI27914 (Bakshi et al., 2002: blocked stress-induced freezing), SSR125543A (Griebel et al., 2002: decreased social defeat and maternal separation-induced vocalizations), CRA 1000 and 1001 (Okuyama et al., 1999: reduced stress and CRF-induced anxiety-like behavior in the light/dark box test and elevated-plus maze), DMP695 (Millan et al., 2001: increased social interaction and punished responding in a Vogel conflict test), DMP696 (He et al., 2000: decreased anxiety-like behavior in defensive withdrawal) and DMP904 (Gilligan et al., 2000: decreased anxiety-like behavior in defensive withdrawal). Finally, the CRF 1 receptor antagonist antalarmin reduced anxiety-like behaviors both in rodents (Zorrilla et al.,

2002) and primates (Habib et al., 2000).

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B. CRF 2 Receptor and the Urocortin Family

In 1995, the second CRF receptor was isolated from a mouse heart cDNA library using a probe from the CRF 1 receptor (Perrin et al., 1995). The CRF 2 receptor is also a 7 transmembrane domain GPCR, with 70% and 68% homology to the CRF 1 receptor at the amino acid and nucleotide level, respectively. Stimulation of the CRF 2 receptor activates cAMP and phosphorylates CREB, as does the CRF 1 receptor

(Rossant et al., 1999). There are two splice variants of receptor in rodent that differ in their N-terminal domains; CRF 2(a) is primarily expressed in the brain, while CRF 2(b) is found mostly in the periphery, including skeletal muscle, skin, and heart, although it is also expressed in the choroid plexus and the blood vessels throughout the brain.

Within the brain, CRF 2(a) is observed mainly in subcortical structures that include the lateral septum, bed nucleus stria terminalis, medial nucleus of the amygdala, ventromedial hypothalamus, dorsal raphe, and the nucleus of the solitary tract in the brainstem. Although the distribution of the CRF 1 and CRF 2 receptors are distinctly different, both are expressed in the bed nucleus, the dorsal raphe, and the medial and cortical regions of the amygdala (van Pett et al., 2000). A third splice variant, CRF 2(c) , has been identified in human brain (Kostich et al., 1998), with highest expression in the hippocampus and septum.

In conjunction with the discovery of the CRF 2 receptor, Vaughan and colleagues (1995) isolated Urocortin 1, a novel mammalian CRF-related peptide, by screening a rat midbrain cDNA library with a probe for urotensin, a CRF homologue

6 present in the suckerfish. Urocortin 1, a 40 amino acid peptide, is 63% homologous to urotensin and 45% homologous to CRF at the amino acid level. This peptide has a slightly higher affinity for the CRF 1 receptor compared to CRF and more than a 10- fold greater affinity for the CRF 2 receptor. Urocortin 1 stimulates an increase in cAMP through both CRF receptors, with a 10-fold and 20-fold greater potency at the CRF 2(a) and CRF 2(b) receptors respectively (Donaldson, C. et al, 1996). The primary site of cellular urocortin expression in the brain is the Edinger-Westphal nucleus, but it is also expressed in the lateral superior olive, the supraoptic nuclei of the hypothalamus, and several motor nuclei in the brainstem. Most Urocortin 1 projections in the brain originate from the Edinger-Westphal nucleus or lateral superior olive and predominately project to the hindbrain (Bittencourt et al., 1999). Projections to the forebrain are generally weak and sparse, except for a strong projection to the lateral septum.

While urocortin 1 projects to several CRF 2 receptor sites in the brain, efferent fibers to some areas in the forebrain that contain the CRF 2 receptor are weak or nonexistent, implying the presence of additional that bind the CRF 2 receptor.

Urocortin 2 was identified through a search of the public human genome database

(Reyes et al., 2001) and the 38 amino acid peptide was found to have a 34% and 42% homology (at the amino acid level) to CRF and urocortin 1, respectively; the peptide has been cloned in rat, mouse, and human. Urocortin 2 is at least 1000-fold less effective at binding the CRF 1 receptor compared to Urocortin 1, but equipotent at binding and activating cAMP at the CRF 2 receptor, indicating the peptide is very selective for the second receptor. Urocortin 2 mRNA is expressed in distinct areas of

7 the brain, including the paraventricular, supraoptic, and arcute nuclei of the hypothalamus, the locus coeruleus, and motor nuclei in the brainstem (trigeminal, facial, hypoglossal). Intracerebroventricular (icv) injection of 1 µg of the peptide significantly increased fos expression in the bed nucleus of the stria terminalis, the central amygdala, the paraventricular nucleus of the hypothalamus and nucleus of the solitary tract. A more recent study using RT-PCR (Chen et al., 2003) confirmed the presence of urocortin 2 in the hypothalamus, brainstem, olfactory bulb and pituitary.

A third peptide, Urocortin 3, was found by screening the human genome EST

(Expressed Sequence Tag) database in Genbank with a probe containing a sequence in pufferfish related to urocortin (Lewis et al., 2001). This peptide has been identified in mouse, human, and rat, and is more closely related to urocortin 2 (40% homology) than Urocortin (18% in mouse) and CRF (26% in mouse). Urocortin 3 is very selective for the CRF 2 receptor, but has less affinity for the receptor (K i, 5 nM, mouse and 21.7 nM, human) compared to urocortin 1 (K i, 2.2 nM, rat) or urocortin 2 (K i, 2.1 nM, mouse and 1.7 nM, human). At high doses (> 1 µg) urocortin 2 increases cAMP through the CRF 1 receptor in cell culture, but urocortin 3 does not activate CRF 1 receptor-mediated cAMP even with a 10 µg dose, indicating that this peptide is the most selective for the CRF 2 receptor. A detailed study of the peptide (Li et al., 2002) revealed urocortin 3 mRNA and protein expression in the median preoptic nucleus in the hypothalamus, in the auditory complex in the brainstem (superior olivary nucleus), and throughout the medial extended amygdala (medial amygdala, perifornical region lateral to the hypothalamus, posterior/medial bed nucleus). Urocortin 3 immunoreactive fibers innervate the ventromedial hypothalamus, medial preoptic and

8 arcuate nuclei, as well as the perifornical region. Strong urocortin 3 projections were also evident in the posterior bed nucleus, the lateral septum, and medial amygdala, all sites of CRF 2 receptor expression.

C. CRF 2 Receptor and Anxiety

A Summary of the Evidence that CRF 2 Receptor Activation Decreases Anxiety

1. CRF 2 Receptor KO mice exhibit increased anxiety-like behaviors

Bale and colleagues (2000) developed a CRF 2 receptor knockout mouse on a

C57Bl/6 x 129 SvJae background. Under baseline conditions, these mice showed normal HPA axis activity, feeding and weight gain compared to wildtype littermates.

When subject to restraint stress, CRF 2 receptor mutant mice showed higher ACTH and

CORT levels after 2 to 10 minutes of restraint and exhibited decreased feeding after

24 hour food deprivation when compared to wildtype. Both male and female CRF 2 receptor KO mice demonstrated increased anxiety-like behavior in the elevated plus maze, spending less time and making fewer entries to the open arms, without alteration in overall locomotor activity. The KO mice also spent significantly less time in the center of the open field compared to wildtype littermates. These mutant mice also exhibited increased urocortin 1 mRNA expression in the Edinger-Westphal nucleus and increased CRF mRNA in the central amygdala, but no change in the CRF levels in the paraventricular nucleus of the hypothalamus.

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Two other groups have also created and examined separate lines of CRF 2 receptor KO mice. Coste and colleagues (2001) developed mice on a C57BL/6 x SvJ background. Although restraint stress significantly elevated HPA axis activity in these mutant mice compared to wildtype, there were no differences in anxiety-like behavior observed in the elevated plus maze. The CRF 2 receptor KO mice did show a slight decrease in time spent in the center of an open field. Administration of urocortin 1

(icv) reduced locomotion regardless of genotype, but suppressed feeding for 10 hours in wildtype mice compared to only 6 hours in the CRF 2 receptor mutants, suggesting that the CRF 2 receptor is involved in the late but not the early phase of CRF- suppressed feeding. The third strain of CRF 2 receptor KO mice (Kishimoto et al.,

2000) exhibited significantly increased anxiety-like behavior in the elevated plus maze and in the light/dark box test (in males).

2. CRF 2 Receptor Agonists decrease anxiety-like behavior in rat and mouse

Valdez and colleagues (2002) assessed the effect of a relatively selective CRF 2 receptor agonist on anxiety-like behavior in rat. They reported that that icv mouse urocortin 2 (100 ng, 1 µg, 10 µg) had no effect on anxiety-like behavior in Wistar rats when given 10 minutes before testing in the elevated-plus maze. To examine any delayed effects of the peptide, rats were given 1 µg of urocortin 2 (icv) and behavior was assessed 1, 4, and 6 hours later. There was no effect of urocortin 2 on anxiety-like behaviors 1 and 6 hours after administration, but an anxiolytic-like effect was

10 observed in the elevated plus maze after 4 hours, reflected in increased time spent in the open arms of the maze.

In a second study, Valdez and colleagues (2003) reported that icv mouse urocortin 3 (100 ng, 1 µg, 10 µg) decreased anxiety-like behavior in the elevated plus maze in group-housed Wistar rats after a 10 minute interval. The two lower doses significantly increased time spent in the open arms of the maze, while the 10 µg dose increased open arm entries. There was no effect of urocortin 3 (1 µg ) on anxiety-like behavior in the elevated-plus maze after a 30 or 60 minute delay. A third paper by this group (Valdez et al, 2004) reports that icv urocortin 3 (1 µg, 10 µg) reduced anxiety- like behavior in the elevated plus maze in rats 2 hours after cessation of chronic ethanol administration (i.e., ethanol withdrawal), but did not alter anxiety-like behaviors compared to vehicle-treated control rats (ethanol-free) rats.

Venihaki and colleagues (2004) tested the effect of icv urocortin 3 on anxiety- like behavior in C57BL/6 mice. In contrast to the data reported by Valdez and colleagues (2003), they did not observe any significant effects of 100 ng of the peptide on anxiety-like behaviors in the elevated plus maze or the open-field test after a 10 minute interval. They did find that 20 ng of urocortin 3 reduced the latency to enter the light side in the light-dark box test, but did not measure time spent in the light side or transitions between the light and dark compartments.

3. Blockade of the CRF 2 Receptor increases anxiety-like behaviors

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In order to evaluate the effect of CRF 2 receptor activation on anxiety, some studies have utilized antisense oligonucleotides to reduce CRF 2 receptor expression.

Isogawa and colleagues (2003) administered a CRF 2 receptor antisense oligonucleotide into the lateral ventricle of Wistar rats for three days. They report anxiogenic-like effects of this treatment in the elevated plus maze, light-dark box test, and conditioned fear test compared with vehicle or missense-oligonucleotide treated animals. Rats treated with the antisense oligonucleotide spent less time in the open arms of the elevated plus maze, less time in the light area of the light-dark box, and showed increased freezing behavior when placed in a shock chamber 24 hours after receiving 5 minutes of inescapable electric footshock. Liebsch and colleagues (1999) treated Wistar rats with 6 days of icv CRF 1 or CRF 2 receptor antisense 15-mer oligonucleotides (120 µg/day). They observed that CRF 2 receptor antisense treatment increased immobility in the Porsolt swim test compared to vehicle, but CRF 1 receptor antisense treatment had no effect.

One limitation of using the antisense oligonucleotide treatment is that the

CRF 2 receptor expression is decreased, but not completely eliminated. As an alternative, several studies have tested the effect of anti--30 (ASV-30) on anxiety-like behavior. ASV-30 is a putative CRF 2 receptor selective antagonist that exhibits a high affinity for the CRF 2(a) (K i = 0.8 nM) and CRF 2(b) (K i = 1.4 nM) receptors (Higelin et al., 2001). Estimates of the ligand selectivity for the CRF 2 receptor compared to the CRF 1 receptor have varied; Ruhmann and colleagues (1998) reported a 110-fold preference for CRF 2(b) over the CRF 1 receptor in a binding assay displacing iodinated sauvagine in HEK293 cells transfected with CRF 1 or CRF 2

12 receptors. Lawerence and colleagues (2002) reported an 882-fold and 825 fold selectivity for the CRF 2(a) and CRF 2(b) receptors, respectively, using the same assay.

Brauns and colleagues (2001) reported yet a third estimate, with ASV-30 exhibiting a

340-fold preference for CRF 2(b) .

Kishimoto and colleagues (2000) reported that icv ASV-30 (400 ng) induced anxiety-like behavior in the elevated plus maze after 30 minutes in C57BL/6 mice.

The animals made significantly fewer entries and spent less time in the open arms of the maze without a difference in overall locomotion.

A Summary of the Evidence that CRF 2 Receptor Activation Increases Anxiety

1. CRF 2 Receptor Agonists increase anxiety-like behaviors in rodents

A number of papers have reported anxiogenic-like effects of CRF 2 receptor selective agonists. Pelleymounter and colleagues (2002) observed that three doses of icv urocortin 2 (0.03 nmol, 0.3 nmol, 3 nmol) induced anxiety-like behavior 30 minutes later in the elevated plus maze in Balb/c mice, reflected in significantly decreased time spent in the open arms of the maze. In a later study, Pelleymounter and colleagues (2004) compared the effect of mouse urocortin 2 and human urocortin 3

(icv) on anxiety-like behavior in the elevated plus maze in the same strain of mice.

They found no effect of urocortin 3 at any dose tested (0.03 – 3.0 nmol), but again observed anxiogenic-like effects with higher doses of urocortin 2 (0.3 nmol and 3

13 nmol). No change in anxiety-like behavior was observed with lower doses of urocortin

2 (0.003 – 0.03 nmol) in this experiment.

Risbrough and colleagues (2003) tested the effect of urocortin 2 on acoustic startle in group housed C57BL/6 mice. The startle reflex occurs when an intense auditory or tactile stimulus induces an automatic contraction of the skeletal musculature. Startle is increased during stress and significantly increased in rats treated with CRF (Swerdlow et al., 1986). In this procedure, the mice were placed into a small restraining cylinder mounted above an accelerometer that measures movement, allowed to acclimate for 5 minutes, then presented 48 acoustic pulses (90, 105 or 120 decibels) for the next 12 minutes. Mice treated with icv urocortin 2 (1-2 nmol) showed increased acoustic startle to the 120 decibel pulse when tested 1 hour after peptide infusion. There was no significant difference in startle with a lower (0.2 nmol) or higher (6 nmol) dose of the peptide. CRF, but not urocortin 2, increased startle when the mice were presented with the 90 and 105 decibel pulses, stimuli that induce a relatively a weaker startle response compared to the 120 decibel stimulus.

Hammack and colleagues (2003) reported that injection of urocortin 2 (8.7 - 87 ng) directly into the dorsal raphe increased conditioned fear and learned helplessness

24 hours later in Sprague-Dawley rats. Rats were housed alone and tested during the light cycle. In this experiment, rats were confined in plastic tubes and subjected to inescapable shock (100 1.0 milliamp (mA) shocks on a 1 minute variable-interval schedule) and kept in their home cages for 24 hours before testing fear and learned helplessness. A separate group of rats was injected with urocortin 2 without any shock and tested for helplessness 24 hours later. During the testing procedure, rats were

14 placed into a shuttle box (46 cm x 20 cm x 20 cm) with a stainless steel grid floor that delivered 0.5 mA footshocks. Freezing behavior was observed every 8 seconds for 5 minutes, then the rats were given two trials (fixed ratio-1, FR-1) where they were required to cross the shuttle box to terminate a footshock. The animals were observed for freezing behavior for the next 20 minutes, then presented with 3 more FR-1 trials.

Finally, the rats were tested with 25 FR-2 trials that required the animals to cross to the other side and come back to terminate the shock. Both inescapable shock and urocortin 2 treatment increased the freezing response following the footshock and increased the escape latency during the learned helplessness procedure.

2. CRF 2 Receptor Antagonists decrease anxiety-like behaviors

Ho and colleagues (2001) studied the effect of a CRF 2 receptor antisense oligonucleotide on the anxiety-like response (freezing) to electric shock, on conditioned fear to context associated with shock, and re-exposure to shock stimulus.

The antisense sequence was chosen after screening oligonucleotide libraries against

CRF 2 receptor mRNA and selecting the sequence most effective at reducing receptor expression in CRF 2 receptor transfected Chinese Hamster ovary (CHO) cells. Sprague-

Dawley rats were bilaterally cannulated in the lateral septum, and 2.5 nmol of the antisense oligo infused once per day for 7 days. To determine the extent of CRF 2 receptor antisense inhibition, rats were sacrificed 4 hours after a fluorescein-labeled oligonucleotide infusion. Fluorescence microscopy revealed that icv antisense treatment had penetrated through the lateral septum and regions near the ventricles

15 such as the ventromedial hypothalamus and central gray, but could not be detected in other areas of CRF2 receptor expression such as the bed nucleus and medial amygdala.

Seven days of antisense CRF 2 receptor treatment significantly decreased shock- induced freezing in naïve rats, decreased conditioned fear to context (rats were exposed to shock were placed back in the container where they received the shock), and reduced the freezing response to a second shock in conditioned rats.

Administration of the CRF 1 receptor antagonist DPC 904 augmented the anxiolytic effects of the CRF 2 receptor antisense treatment.

The selective CRF 2 receptor antagonist ASV-30 has also been reported to reduce anxiety-like behavior when injected into the ventricle. Takahashi and colleagues. (2001) studied the effect of ASV-30 on footshock-induced conditioned anxiety-like behavior and behavior in the elevated plus maze and defensive withdrawal in Sprague-Dawley rats. For the conditioned anxiety test, rats were given 3 footshocks during 1 minute in the test apparatus. 24 hours later, the animals were given 0 – 20 µg of icv ASV-30 twenty minutes before being placed back into the same container, where freezing was measured for 10 minutes. ASV-30 (2 – 20 µg) significantly reduced conditioned freezing, decreased anxiety-like behavior in the elevated plus maze (1- 10 µg) and increased the time spent in the light area during the defensive withdrawal test (5-10 µg). Pelleymounter and colleagues (2002) examined the effect of ASV-30 on the marble burying test, open field behavior, the elevated plus maze, and the HPA response to restraint stress in Balb/c mice. The mice were injected icv 30 minutes before each behavioral test. In this study, ASV-30 significantly decreased the number of marbles being buried (3-10 nmol), increased the time spent in

16 the center of the open field (3-10 nmol), and increased the time spent on the open arms of the elevated plus maze (0.1-10 nmol). Astressin, a non-selective CRF antagonist, but not ASV-30, decreased ACTH levels in response to stress. A third study,

Risbrough and colleagues (2003), found that ASV-30 (3 nmol) significantly blocked

CRF-induced increases in acoustic startle.

Bakshi and colleagues (2002) evaluated the effect of CRF receptor antagonists on footshock induced freezing in Sprague-Dawley rats. Animals were housed in pairs, tested during the light cycle, and all drugs were given immediately before behavioral testing. Rats were presented with 3 footshocks and latency to freeze and the time spent freezing was measured for 15 minutes after the shocks. Bilateral injection of the non- selective CRF antagonist αhCRF (100 ng) into the lateral septum decreased shock- induced freezing behavior. There was no significant effect of lower (30 ng) or higher

(300 ng) doses of the drug. A second non-selective CRF antagonist, D-Phe-CRH (12-41)

(100 ng) also decreased freezing behavior throughout the 15 minute test. These antagonists had no effect on locomotor activity or pain sensitivity. Injection of αhCRF into the medial septum/nucleus of the diagonal band, a region adjacent to the lateral septum that expresses CRF 1 receptors, or into the lateral ventricle, had no effect on freezing. Injection of the CRF 1 selective antagonist (NBI27914) into the lateral septum had no effect, but did decrease freezing when injected into the central amygdala.

Autoradiography showed that αhCRF (but not NBI27914) displaced [ 125 I]-sauvagine binding in the lateral septum, suggesting that the anxiolytic effect of the non-selective antagonists in the lateral septum was due to blocking CRF 2 and not the CRF 1 receptor.

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In a related study, Radulovic and colleagues (1999) observed that bilateral injection of h/rCRF (500 ng) into the lateral septum of Balb/c mice increased anxiety- like behavior in the elevated plus maze after 30 minutes. Septal infusion of both astressin and ASV-30 antagonized the CRF-induced anxiety-like behavior. This paper also reported that 1 hour of restraint stress induced anxiety-like behavior in the elevated plus maze. Both astressin and ASV-30 blocked the anxiogenic-like effect of the restraint stress when infused into the septum.

D. Dissertation Hypotheses

Considerable evidence indicates that CRF 1 receptor activation mediates the anxiogenic effects of CRF (Smith et al., 1998; Zorilla et al., 2002), but the role of the

CRF 2 receptor in anxiety remains controversial. CRF 2 receptor knockout mice exhibited greater anxiety-like behavior in the elevated plus maze and the light-dark box compared to wildtype mice in two out of three mouse lines tested (Bale et al.,

2000; Kishimoto et al., 2000, Coste et al., 2000), suggesting that activation of CRF 2 receptors reduces anxiety. Selective CRF 2 receptor agonists (urocortin 2 and 3) also decreased anxiety-like behavior in the elevated plus maze in rats (Valdez et al., 2002,

2003), and the open field and light-dark box in C57BL/6 mice (Venihaki et al., 2004).

In contrast, other studies report anxiogenic-like effects of CRF 2 receptor agonists in the elevated plus maze and the acoustic startle test in mice (Pelleymounter et al., 2002,

2004; Risbrough et al., 2003, 2004). Further, antagonism of CRF 2 receptors in the

18 lateral septum also decreases anxiety-like behavior (Bakshi et al., 2002, Radulovic et al., 1999).

Thus, the CRF 2 receptor appears to play a complex role in anxiety that may depend on a number of factors, including drug dose, the targeted region of the brain, and the environmental conditions during the test. While it appears difficult to reconcile all of the various, and sometimes, conflicting data about this receptor, it is relevant to note that a number of studies reporting anxiolytic-like effects of CRF 2 receptor agonists (Valdez et al., 2002, 2003; Venihaki et al., 2004) tested animals under relatively low-stress conditions, where the animals were handled by the experimenter and habituated to the testing environment (see above) In contrast, reports of anxiogenic-like effects of CRF 2 receptor activation appear to be most commonly observed in subjects tested in high stress environments (e.g., during restraint in the acoustic startle chamber while exposed to loud noises, Risbrough et al.,

2003, 2004; shock-induced freezing, Bakshi et al., 2002; Ho et al., or immobilization stress, Radulovic et al., 1999).

Based on the above observations, I propose that the effect of CRF 2 receptor activation on anxiety may depend on the level of stress in the environment, a variable that may alter the degree of receptor activation in specific brain regions or alter interactions between the CRF 1 and CRF 2 receptors. This dissertation project has focused on the role of the CRF 2 receptor in the lateral septum, a region with high

CRF 2 receptor expression that innervates several brain areas that play an important role in regulating affect and anxiety, including the amygdala, hypothalamus, bed nucleus of the stria terminalis, and the dorsal raphe (Sheehan et al., 2004). In the initial

19

experiments, as described in Chapter 3, I infused the CRF 2 receptor agonist urocortin

2 into the mouse lateral septum, medial septum and lateral ventricle and examined measures of anxiety-like behavior in the light-dark box, open field and novel object test. The next group of experiments examined the effect of an environmental stressor, restraint stress, on the effect of urocortin 2 infused into the septum and the effect of septal urocortin 2 on HPA axis activity. Chapter 4 describes the experiments that examined the role of CRF 1 and CRF 2 receptor activation in septal-mediated anxiety- like behaviors, including the effect of selective receptor antagonists administered into the septum, and the effect of urocortin 2 tested in CRF 2 receptor KO mice. The final study describes the effect of a quinolinic acid lesion of the Edinger-Westphal nucleus on anxiety-like behaviors in mice treated with septal urocortin 2. These results are presented and discussed in Chapter 5.

20

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25

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Chapter 2:

GENERAL METHODS AND MATERIALS

Animals

Male ICR mice were purchased from Harlan (Indianapolis, IN). CRF 2 receptor wildtype and knockout mice on a mixed C57BL/6 x 129 background were generated as previously described (Bale et al., 2000) and bred mating heterozygous mice.

Animals were group housed under a 12 hour light/dark cycle. All experiments were conducted during the dark cycle and all procedures were approved by the Institutional

Animal Care and Use Committee of The Salk Institute and The Scripps Research

Institute. All experimental protocols and animal facilities were in accordance with the

Association for the Assessment and Accreditation of Laboratory Animal Care and the

National Institutes of Health guidelines.

Drugs

Urocortin 2 and astressin-2B were synthesized under the direction of Dr. Jean

Rivier of the Clayton Foundation Laboratories for Peptide Biology, Salk Institute.

Urocortin 2 was dissolved in sterile physiological saline containing 0.1M bovine serum albumin, pH 7.4. Astressin-2B was initially dissolved in a small amount of acetic acid, and then diluted with sterile physiological saline containing 0.1M bovine serum albumin to a final pH of 6.5. Antalarmin was purchased from Sigma and

28 29 dissolved in a solution of 10% Cremaphor (Sigma), 5% ethanol, and 85% sterile saline with a final pH of 6.0. Peptides were prepared immediately before use.

It is relevant to note that urocortin 1 and 3, but not urocortin 2, are known to project to the rodent lateral septum. Urocortin 1 exhibits a high affinity for both the

CRF 1 and CRF 2 receptor and was thus not the optimal choice for a study designed to examine the effect of selective CRF 2 receptor activation. Although both urocortin 2 and 3 exhibit low affinity for the CRF 1 receptor (K i > 100 nM), urocortin 1 and 2 have similar affinity for the mouse CRF 2 receptor (K i = 0.8 and 0.9 nM, respectively), a significantly higher affinity compared to urocortin 3 (14.2 nM; Chen et al., 2004).

Urocortin 2 was thus selected as an appropriate choice for infusion into the lateral septum.

Surgery, drug administration, and histology

Mice under anesthesia (injected with a subcutaneous rodent cocktail, consisting of (41 mg/kg), xylazine (8.25 mg/kg), and acepromazine (2 mg/kg) were prepared with 26 gauge bilateral cannulae aimed at the intermediate lateral septum (+0.30 mm anterior to bregma, 0.5 mm lateral from the midline, -3.2 ventral from skull surface; flat skull), the central nucleus of the amygdala (-1.4 mm posterior to bregma, 2.4 mm lateral to the midline, -4.6 mm ventral from skull surface; flat skull) or single cannula targeting the medial septum (+0.30 anterior to bregma, 0.5 mm lateral from the midline, -3.5 mm ventral from skull surface) or the lateral ventricle (-0.5 mm posterior to bregma, 1.0 mm lateral from the midline, -2.5 mm ventral from skull surface; flat skull; Franklin and Paxinos, 1997). Cannula were

30 attached to the skull using dental acrylic. Peptides were infused through 33 gauge injector cannula (Plastics One, Roanoke, VA) attached to 10 µl Hamilton microsyringes mounted on an automated pump (Harvard Apparatus, Holliston, MA).

Animals were allowed one week to recover from the surgery before drug administration. Peptides were infused over a period of 120 seconds (volume of 0.25 µl per side for the bilateral lateral septal cannula, 0.5 µ l in the medial septum and central amygdala, and 5 µl in the ventricle to allow for diffusion throughout the brain) and the injector cannula remained in the brain for 60 seconds after drug administration.

Following the completion of each experiment, methylene blue dye was infused into each cannula using the drug delivery volume, the brains were removed and frozen and sections cut on the cryostat to assess cannula placement. Mice with lateral ventricle placement were examined for dye in both lateral and the third and fourth ventricles; animals without dye in distal ventricles were removed from analysis. Mice with lateral or medial septal placement were examined for cannula placement within the appropriate regions. Animals that did not have correct cannula placement were excluded from the analyses.

Behavioral Testing

Mice were handled for 5 minutes per day for 7 days prior to drug administration and behavioral testing. On the test day, each animal was taken from the housing room and habituated to a dark testing room with a 65-decibel white noise background for 1 hour before drug administration. After drug infusion, mice were returned to their cage or restrained for 30 minutes by taping their limbs to a Plexiglass

31 surface (Radulovic et al., 1999). Preliminary work indicated that 30 minutes of restraint was sufficient to increase anxiety-like behavior in the behavioral tests used and was thus chosen as the high stress condition. After this 30 minute period, each mouse was tested in the light-dark box, open field, and novel object test. Mice were placed into the light-dark box for 10 minutes, then immediately removed from the light-dark box and placed into the open field for 10 minutes. At the end of that period, a novel object was placed into the center of the open field and behavior measured for a third 10 minute period. This procedure and testing order was used for all experiments.

Behavior in these tests was filmed, with the experimenter absent from the room during the test.

The light-dark box apparatus consisted of a small dark chamber 27 x 15 x 27 cm high connected by 10 x 10 cm opening to a larger white chamber 27 x 29 x 27 cm high. The light intensity in the light compartment was 1000 lux, compared to 10 – 50 lux in the dark compartment. The mouse was placed in the dark compartment facing away from the opening to the light side, and the latency to first enter the light compartment, number of transitions from the dark to the light compartment, and total time spent in the light compartment were measured for 10 minutes. Entry into either side of the light-dark box was defined as the placement of all four paws into that side.

Immediately following the light-dark test, mice were placed into a open field chamber divided into 9 regions: four corner regions, four side regions and a center region. The corner regions were approximately 7 x 7 cm, the side regions were 7 x 14 cm, and the center region was 14 x 14 cm. Locomotion was assessed as the total number of crossings between regions of the open field during the test. The time spent in the

32 center square and percent of entries into the center region were assessed as measures of anxiety-like behavior. After 10 minutes in the open field, a novel object (white plastic cup) 3 cm in diameter and 5 cm high was placed into the middle of the center square and secured to the floor with tape affixed to the inside of the cup. During the novel object test, locomotion was assessed as the total number of crossings between regions. The time spent in the center square with the novel object and percent of entries into the center region were quantified as measures of anxiety-like behavior.

Experimental Design

All experiments were conducted using a between-subjects design for drug administration and restraint stress. Each mouse received either vehicle or one dose of urocortin 2. In experiments that examined the effect of stress on behavior and/or tested the effect of CRF receptor antagonists, each drug dose and stress condition was tested in separate groups of mice.

Data Analyses

SPSS software was used to perform analyses of variance (ANOVA) of peptide dose, stress level, and genotype on measures of anxiety-like behavior and locomotion in the light-dark box, open field, and novel object tests. The effect of urocortin 2 infusion into the lateral septum, medial septum, and lateral ventricle described in

Chapter 3 was assessed with one-way between-subjects ANOVA. Two-way ANOVA

(stress level x peptide dose) was used to assess the interaction between urocortin 2 and restraint stress. Three-way ANOVAs were used to assess the data for the experiments

33

involving the knockout mice (stress x peptide x genotype) and the CRF 2 receptor antagonists (antagonist x agonist x stress), and two-way ANOVA assessed the effect of the CRF 1 receptor antagonist antalarmin. Dunnett’s t-test and the Tukey test were used for appropriate post-hoc comparisons. The correlation between locomotion (total squares entered) and anxiety-like behavior (percent time in the center square) in the open field and novel object test was assessed with Pearson’s correlation coefficient.

The level of significance for all statistical tests was set to 0.05.

34

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Bale, T. L., Contarino, A., Smith G.W., Chan R., Gold L.H., Sawchenko P.E., Koob G.F., Vale W.W., Lee K.F. (2000). "Mice deficient for corticotropin-releasing hormone receptor-2 display anxiety-like behavior and are hypersensitive to stress." Nat Genet 24(4): 410-4.

Chen, A., Perrin, M., Brar, B., Li, C., Jamieson, P., Digruccio, M., Lewis, K., Vale, W. (2004) “Mouse corticotropin-releasing factor receptor type 2alpha gene: isolation, distribution, pharmacological characterization and regulation by stress and glucocorticoids.” Mol Endocrinol 19:441-58.

Franklin, K., Paxinos, G. (1997) The mouse brain in stereotaxic coordinates. San Diego: Academic.

Radulovic, J., Ruhmann, A., Liepold, T., Spiess, J. (1999). "Modulation of learning and anxiety by corticotropin-releasing factor (CRF) and stress: differential roles of CRF receptors 1 and 2." J Neurosci 19(12): 5016-25.

Chapter 3:

The Effect of Septal Urocortin 2 and Stress on Anxiety-like Behaviors

ABSTRACT

Intracerebroventicular administration of CRF 2 receptor agonists has been reported to both increase and decrease anxiety in animal models of behavior, but the effect of these peptides targeted at specific brain regions has not been extensively examined. Treatment with CRF 2 receptor ligands has been reported to decrease anxiety-like behavior under relatively low stress conditions, but tend to increase anxiety-like behavior in animals placed in a high stress test, suggesting that the level of stress in the environment may interact with the effect of CRF 2 receptor activation on anxiety-like behavior. The present study examined the effect of administering the

CRF 2 receptor agonist urocortin 2 into the mouse lateral septum under low or high stress (30 minutes of restraint) testing conditions and measured anxiety-like behavior in the light-dark box, open field, and novel object test in two different strains of mice.

Subsequent experiments also tested the effect of urocortin 2 infused into the murine medial septum and lateral ventricle. Finally, the effect of septal urocortin 2 on HPA axis activation was assessed. In the low stress condition, 500 ng of septal urocortin 2 increased anxiety-like behavior, but lower doses (1, 10, 100 ng) did not have consistent effects. However, in combination with restraint stress, 100 ng of septal

35 36 urocortin 2 significantly increased anxiety-like behavior compared to control mice.

Urocortin 2 infusion into the medial septum or lateral ventricle did not affect measures of anxiety-like behaviors. Administration of 100 and 500 ng of urocortin 2 into the lateral septum had no significant effect on levels of corticosterone in the mouse blood plasma, indicating that the anxiogenic-like effects of the peptide were not mediated by activation of the HPA axis. In conclusion, the results indicate that the effect of septal

CRF 2 receptor activation on anxiety-like behavior is dependent on the level of stress in the environment.

37

INTRODUCTION

Intracerebral administration of CRF and related peptide ligands has been shown to have significant effects on anxiety and stress-mediated behavior. Many of the behavioral responses to CRF are similar to those observed during stress. In rats, icv CRF decreased locomotor activity in familiar environments within a dose range of

100 ng to 10 µg (Britton et al., 1986; Butler et al., 1990; Sherman et al. 1986; Sutton et al., 1982). In contrast, treatment with the peptide decreased activity in a novel environment in rodents (Britton et al., 1982; Britton et al., 1984, Imaki et al., 1987).

The administration of CRF has been shown to decrease food intake in both rats and mice (Arase et al., 1988; Gosnell et al., 1983; Krahn et al., 1986; Rosenthal et al.,

1989) and to produce taste aversion at high doses (Heinrichs et al., 1991). Rats treated with icv CRF showed decreased responding (fewer lever presses for food) in the

Geller-Seifter conflict test (Britton et al., 1985) and decreased social interaction (File,

1987). Anxiogenic effects of CRF administration have also been reported in approach/avoidance tasks, including the elevated plus maze (File et al., 1988), defensive withdrawal (Takahashi et al., 1989) and a novel object task in mice

(Berridge and Dunn, 1986). Finally, CRF treatment has also been demonstrated to augment stress-induced behavior responses, including acoustic startle (Swerdlow et al., 1986) and shock-induced freezing (Sherman and Kalin, 1988).

More recent studies have tested the effect of urocortin, a peptide with a high affinity for both the CRF 1 and CRF 2 receptors, on anxiety-like behavior. Jones and colleagues (1998) reported that rat urocortin significantly increased locomotion in a

38 familiar environment and decreased time spent in the open arms of an elevated plus maze, similar to the behavior observed with CRF treatment. Spina and colleagues

(2002) observed that urocortin treatment in rats consistently increased anxiety-like behavior, reducing time on the open arms of the elevated plus maze, increasing time in the dark chamber of the defensive withdrawal test, and decreasing responding for food in the Geller Seifter conflict test. Balb/c mice treated with icv urocortin also showed increased anxiety-like behavior in the light-dark box, with fewer entries and less time spent in the light area. Infusion of urocortin into the basolateral amygdala is also reported to decrease social interaction in rats (Spiga et al., 2006).

While central administration of CRF and urocortin appears to induce a consistent and replicable increase in anxiety-like behavior in many different models of animal behavior, the effect of the selective CRF 2 receptor agonists on anxiety-like behavior is controversial. As mentioned in the general introduction, Valdez and colleagues reported that icv urocortin 2 decreased anxiety-like behavior in rat in the elevated plus maze after a four hour delay (Valdez et al., 2002), while urocortin 3 decreased anxiety-like behavior 10 minutes after icv infusion (Valdez et al., 2003) and during a 2 hour period of ethanol withdrawal (Valdez et al, 2004). Venihaki and colleagues (2004) reported that a low dose of icv urocortin 3 (20 ng) decreased anxiety-like behavior in the light-dark box, but a higher dose (100 ng) had no effect in the elevated plus maze or open field in C57BL/6 mice.

In contrast, Pelleymounter and colleagues reported anxiogenic-like effects of icv urocortin 2 in mice in the elevated plus maze (Pelleymounter et al., 2002; 2004), but no effect of urocortin 3 on anxiety-like behavior. Risbrough and colleagues (2003,

39

2004) observed that icv urocortin 2 potentiated the acoustic startle response in mice, putatively increasing anxiety-related behavior in these animals during this test.

Infusion of urocortin 3 into the mouse lateral ventricle is also reported to decrease maternal aggression in lactating female mice. D’Anna and colleagues (2005) observed that female mice with newborn pups exhibited fewer attacks and spent less time attacking intruder males placed in their cages when treated with urocortin 3. Previous studies have shown that lactating female rodents are less sensitive to CRF (da Costa et al., 1997) and CRF synthesis is decreased during lactation (Walker et al., 2001), suggesting that that a reduction in fear and anxiety may correspond to increased aggressive behavior. The reduced aggression observed with icv urocortin 3 might thus be interpreted as an increase in anxiety.

While a number of studies have examined the behavioral effect of icv CRF 2 receptor selective agonists, the effect of these ligands on anxiety when infused into specific brain sites has not been examined extensively. Hammack and colleagues

(2003) have reported that urocortin 2 infused into the dorsal raphe increased the freezing response to footshock in a conditioned fear paradigm, but the effect of the

CRF 2 receptor agonists in other brain regions has not been well documented.

The lateral septum is one of the few regions of the brain with a high concentration of CRF 2 receptors. It is perhaps unique in that this region is innervated by two CRF 2 receptor agonists, receiving a prominent urocortin 1 projection from the

Edinger-Westphal nucleus and a urocortin 3 projection that may originate from a rostral perifornical region adjacent to the hypothalamus (Bittencourt et al., 1999; Li et al, 2002). The septum is positioned as an essential region for integrating cortical and

40 subcortical function. It receives input from the prefrontal and entorhinal cortex and projects to several regions involved in regulating emotional states, including the amygdala, bed nucleus of the stria terminalis, and the hypothalamus. The septum receives input from a number of neurotransmitter systems, including dopaminergic input from the ventral tegmental area, serotonergic input from the raphe, adrenergic input from the locus coruleus, and cholinergic input from the laterodorsal tegmentum

(Sheehan et al., 2004). It is primarily composed of GABAergic projection neurons that target regions throughout the brain and may generally function to control or inhibit several types of behavior. Lesions of the septum are reported to 1) increase anxiety- like behavior, increasing conditioned freezing to footshock (Vouimba et al., 1998), 2) increase aggressive behavior (the “septal rage” phenomena) in rats and mice (Albert et al., 1980; Brady et al., 1953), and 3) to increase sexual behavior in rodents and humans (Gorman et al., 1992; Tsukahara et al., 2001).

The purpose of the experiments described in this chapter was to examine the effect of urocortin 2 infusion in the mouse lateral septum on anxiety-like behavior in the light-dark box, open field, and novel object test. The effect of the peptide in the lateral septum, medial septum and lateral ventricle is described in Part A. Part B of this chapter describes the effect of urocortin 2 in the no stress (no restraint) and high stress (30 minutes of restraint) testing conditions, and the effect of septal urocortin 2 on HPA axis activity.

Part A: Effect of Septal Urocortin 2 on Anxiety-like Behaviors

41

METHODS

The methods for each of these experiments followed the procedures detailed in

Chapter 2 for animal housing, drug preparation, surgery and histology, behavioral testing and data analysis. Urocortin 2 was administered as previously described and each animal tested for anxiety-like behavior in the light-dark box, open-field test, and novel object test as delineated in the behavioral testing section in the second chapter.

Experiment 1: Urocortin 2 infused into the lateral septum.

ICR mice were prepared with bilateral cannula in the lateral septum and were infused with vehicle, 1 ng, 10 ng, 100 ng, or 500 ng of urocortin 2 per side. There was an n of 9-10 mice per group.

Experiment 2: Urocortin 2 infused into the medial septum .

ICR mice were prepared with a single cannula in the medial septum and infused with vehicle, 500 ng, or 1 µg of urocortin 2 per mouse, n = 6-7 per group.

Experiment 3 : Urocortin 2 infused in the lateral ventricle .

42

ICR mice were prepared with a cannula in the lateral ventricle and infused with vehicle, 100 ng, 500 ng, 1 µg, or 5 µg of urocortin 2 per mouse, n = 7-8 per group.

RESULTS

Experiment 1: Effects of Urocortin 2 in the lateral septum.

Septal administration of the CRF 2 agonist urocortin 2 increased anxiety-like behavior in the light-dark box and novel object test, with a trend towards an anxiogenic-like effect in the open field test (Figure 1). Statistical analyses revealed that the highest dose of septal urocortin 2 (500 ng) significantly decreased the time spent in the light side of the light-dark box (F (4,46) = 3.058, p < 0.05) (Fig. 2b), and the percent entries (F (4,46) = 4.223, p < 0.01) and percent time (F (4,46) = 7.288, p < 0.001)

(Fig 1f) spent in the center region with the novel object; with a trend towards fewer percent entries (p = 0.057) and less time (p = 0.065) (Fig. 1d) in the center of the open field compared to wildtype mice. Lower doses of the peptide had no effect in the light- dark box, except for a decrease in transitions observed with the 100 ng dose (F (4, 46) =

3.041, p < 0.05). None of the lower doses had any effect in the open field. In the novel object test, the 1 ng and 100 ng doses had no significant effect on anxiety-like behavior, while 10 ng of urocortin 2 decreased time spent in the center (p < 0.05).

Urocortin 2 had no effect on locomotion in the open field and the novel object test at any dose tested.

43

Experiment 2: Effects of Urocortin 2 in the medial septum .

Infusion of the peptide into the medial septum did not have a significant effect on any measure of behavior in the light-dark box, open field or novel object test.

Experiment 3: Effects of Urocortin 2 in the lateral ventricle.

Urocortin 2 infused into the lateral ventricle decreased locomotion at the highest dose tested (5 µg) (Fig. 2c,e), but did not affect locomotor or anxiety-like behavior at lower doses. Data analyses showed that 5 µg of the peptide increased latency to enter the light side of the light-dark box (F (4, 32) = 4.260, p < 0.05) (Fig. 2a), and decreased the percent time spent in the center of the open field (F (4, 32) = 2.914, p <

0.05) (Fig. 2d), suggesting an increase in anxiety-like behavior; however, this dose also significantly decreased locomotion in both the open field (F (4, 32) = 3.623, p <

0.05) and novel object test (F (4, 32) = 3.324, p < 0.05).

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Part B: Effect of Stress on the Behavioral Effect of Septal Urocortin 2

METHODS

The methods for the each of these experiments followed the procedures detailed in Chapter 2 for animal housing, drug preparation, surgery and histology, behavioral testing and data analysis. Urocortin 2 was administered as previously described and each animal tested for anxiety-like behavior in the light-dark box, open- field test, and novel object test as delineated in the behavioral testing section in the second chapter.

Experiment 1: Septal urocortin 2 combined with stress .

ICR mice were prepared with bilateral cannula in the lateral septum and infused with vehicle or 100 ng of urocortin 2 per side combined with no stress or 30 minutes of restraint stress, n = 8-9 per group.

Experiment 2: Multiple Doses of septal urocortin 2 were combined with stress in

CRF 2 receptor wildtype mice (C57BL/6 x 129 mixed background).

Mice were prepared with bilateral cannula in the lateral septum and infused with vehicle, 1 ng, 10 ng, or 100 ng of urocortin 2 per side combined with no stress or

30 minutes of restraint stress, n = 6-7 per group.

45

Experiment 3: The effect of septal urocortin 2 on plasma corticosterone

Mice were prepared with bilateral cannula in the lateral septum. Before peptide administration, the animals were habituated to the testing room for 1 hour as previously described. They were subsequently infused with vehicle, 100 ng, or 500 ng of urocortin 2 per side and place back into the home cages for 30 minutes. After the 30 minute interval, the mice were decapitated and trunk blood collected and mixed with

10 µl of 0.5 M EDTA. The blood samples were placed on ice, centrifuged at 5000 rpm for 10 minutes. Plasma was aliquoted and stored at -20 °C until assayed.

Corticosterone levels in plasma were assessed using the radioimmunoassay kit obtained from ICN Biomedicals Inc. (Costa Mesa, CA). The mice were run in the middle of their dark cycle, during the same period of time as the previously reported behavioral experiments. There was an n of 11-13 per group.

RESULTS

Experiment 1: Effects of septal urocortin 2 combined with stress.

In this experiment, ICR mice were infused with 100 ng of septal urocortin 2 combined with 30 minutes of restraint stress or no stress. The purpose of this experiment was to determine if stimulation of the septal CRF 2 receptor would have a different effect on anxiety–like behavior when tested during low versus high stress

46 conditions. As reported in Part A, septal administration of 100 ng of urocortin 2 did not have a significant effect on anxiety. This dose was thus chosen in the current and subsequent experiments in order to determine if stress would interact with a dose of the CRF 2 agonist that was inactive in the no stress condition. The results indicated that this dose of urocortin 2 had no effect on anxiety-like behavior in the no stress condition, but significantly increased anxiety-like behavior in the stress condition compared to vehicle-treated mice (Figure 3). Analyses of the data (two–way ANOVA followed by Tukey post-hoc test) revealed significant interactions between urocortin 2 and stress on time spent in the light-side of the light dark box (F (1,29) = 7.901 , p <

0.01), the percent center entries (F (1, 29) = 4.498, p < 0.05) and percent time in the center (F (1, 29) = 4.338, p < 0.05) of the open field, and the percent center entries (F (1,29)

= 4.603, p < 0.05) and percent time spent in the center (F (1, 29) = 4.881, p < 0.05) with the novel object. Post-hoc testing showed that urocortin 2 significantly increased anxiety-like behavior compared to vehicle only in stressed mice, decreasing the time spent in the light side of the light-dark box (p < 0.01) (Fig. 3b), the percent center entries (p < 0.01) and percent time in the center (p < 0.01) (Fig. 3d) of the open field, and the percent center entries (p < 0.01) and percent time in the center (p < 0.001)

(Fig. 3f) with the novel object.

Restraint stress increased anxiety-related measures in the light-dark box, open field, and novel object test (increased latency to enter light side of light-dark box (F (1,

29) = 10.479, p < 0.01) (Fig. 3a); decreased time in the light side of the light-dark box

(F (1, 29) = 25. 02, p < 0.001) (Fig. 3b); decreased percent center entries ( F (1, 29) =

13.023, p < 0.01) and percent time (F (1, 29) = 29.551, p < 0.001) (Fig. 3d) in the center

47

of the open field; decreased percent center entries (F (1, 29) = 16 .05, p < 0.001) and percent time (F (1,29) = 22.48, p < 0.001) (Fig. 3f) in the center with the novel object), but also decreased overall locomotion in the open field (F (1,29) = 10.352, p < 0.01)

(Fig. 3c) and with the novel object (F (1,29) = 4.849, p < 0.05) (Fig. 3e). However, urocortin 2 had no significant effect on locomotor activity.

There was a non-significant trend towards a decrease in locomotion in urocortin 2-treated mice in the stress condition compared to the vehicle-treated mice in the novel object test. The correlation between total locomotion (squares entered) and anxiety-like behavior (percent time in the center square) was assessed for both the open field and novel object test. The analyses indicated that there was no significant correlation between locomotion and anxiety-like behavior in the no stress (r = 0.203, n.s.) or stress condition (r = 0.210, n.s.) in the open field test, or the no stress (r =

0.016, n.s.) or stress condition (r = 0.333, n.s.) in the novel object test. These data suggest that the effects of the peptide on anxiety-like behavior were not confounded by alterations in locomotor activity.

Experiment 5: Effects of multiple doses of septal urocortin 2 in CRF 2 receptor wildtype mice

The results from this experiment show a dose-dependent effect of urocortin 2 in the lateral septum, with the highest dose (100 ng) increasing anxiety-like behavior only in the stress condition, while lower doses had little effect, regardless of stress

(Figure 4). There was a significant interaction between urocortin 2 and stress on the

48

latency to enter the light side of the light-dark box (F (1, 44) = 30.909, p < 0.001), time spent in the light side (F (1,44) = 23.871, p < 0.001), and percent time in the center with a novel object (F (3,44) = 3.518, p < 0.05). Post-hoc testing showed that 100 ng of urocortin 2 significantly increased anxiety-like behavior compared to vehicle in stressed mice, increasing the latency to enter the light side of the light-dark box (p <

0.001) (Fig. 4a), decreasing time spent in the light side of the light-dark box (p <

0.001) (Fig. 4b), and decreasing the percent time in the center with the novel object (p

< 0.001) (Fig 4f), with a trend towards decreased percent time in the center of the open field (p < 0.068) (Fig. 4d). There were no significant effects of lower peptide doses, with the exception that 10 ng of urocortin 2 decreased percent time in the center with the novel object when compared to vehicle in the stress condition.

As in the previous experiment, restraint stress increased anxiety-related measures in the light-dark box, open field, and novel object test, but also decreased overall locomotion in the open field (F (1,44) = 4.775, p < 0.05) (Fig. 4c), and with the novel object (F (1,44) = 8.214, p < 0.01) (Fig. 4e). However, treatment with urocortin 2 did not affect locomotor activity in the stress or no stress condition, supporting the contention that the effects of the peptide are mediated by changes in anxiety-like behavior.

Experiment 3: The effect of septal urocortin 2 on plasma corticosterone

49

The septal infusion of 100 ng and 500 ng of urocortin 2 did not have a significant effect on plasma corticosterone compared to vehicle-treated mice (Figure

5).

DISCUSSION

In summary, the current studies demonstrated that the effect of septal urocortin

2 on anxiety-like behavior is influenced by the stress level of the mouse. Under low stress conditions, only the high dose of 500 ng of septal urocortin 2 consistently increased anxiety-like behavior in both the light-dark box and novel object test, with a strong trend towards anxiety-like behavior in the open field test. 100 ng of septal urocortin 2 had no significant effect on anxiety-like behavior under low stress conditions in ICR mice; however, when combined with 30 minutes of restraint stress, this dose significantly increased anxiety-like behavior in the light-dark box, open field and novel object test compared to the effects of restraint stress alone. This effect was replicated in mice on a C57BL/6 x 129 mixed background, where septal urocortin 2 increased anxiety-like behavior only when combined with restraint stress in the light dark box (100 ng) and novel object test (10 ng and 100 ng).

Infusion of urocortin 2 into the medial septum did not have a significant effect on anxiety measures, consistent with the hypothesis that the anxiogenic effects of the peptide were mediated by CRF 2 receptors in the lateral septum. The peptide had no effect when infused into the lateral ventricle, except at the highest dose of 5 µg. Mice treated with this dose showed increased latency to enter the light side of the light-dark

50 box and spent less time in the center of the open field, but also showed significant decreases in overall locomotion in both the open field and novel object test. Because of the decrease in motor activity, the effect of this dose on anxiety-like behavior in the light-dark box and open-field is not clear.

High doses of a CRF 2 receptor agonist delivered into the ventricle would be expected to occupy some CRF 2 receptors in the septum; however, infusion of the peptide into the ventricle may also activate CRF 2 receptors in other brain areas that may oppose the septal effects on anxiety. Urocortin 2 infused into the lateral septum did not alter locomotion in the open field or novel object tests in the low or high stress conditions, demonstrating that effects on anxiety measures were not confounded by changes in motor activity.

The third experiment in Part B examined the effect of septal urocortin 2 on

HPA axis activation. During stress, CRF is released from the paraventricular nucleus of the hypothalamus into the hypophyseal portal system, activating the release of adrenocorticotropin hormone (ACTH) from the anterior pituitary. ACTH is subsequently released into the bloodstream, where it stimulates the release of glucocorticoids from the adrenal cortex (Dunn and Berridge, 1990). Glucocorticoids, such as corticosterone, play a critical role in mediating the stress response, increasing blood pressure and cardiac output, suppressing immune function and inflammation, and increasing blood glucose concentration (Sapolsky, 2000). Glucocorticoids also appear to play a role in the anxiety and fear associated with stressors in the environment. Thompson and colleagues (2004) reported that subutaneous injections of corticosterone in the rat augmented the effects of fear conditioning, increasing the

51 freezing response to an environment associated with footshock. Lee and colleagues

(1994) observed that corticosterone injections enhanced CRF-induced acoustic startle.

When corticosterone was removed in adrenalectomized rat pups, they showed decreased anxiety (freezing) when exposed to a strange adult male (Takahashi et al.,

1993). Corticosterone treatment is also reported to increase anxiety-like behavior in the light-dark box in mice, increasing the latency to enter the light compartment

(Ardayfio et al., 2006).

While both CRF and urocortin activate the HPA axis (Asaba et al., 1998), the effect of relatively selective CRF 2 receptor agonists on glucocorticoid release has not been extensively examined. One recent study (Jamieson et al., 2006) report that 1-10

µg of icv urocortin 3 in rat increased plasma corticosterone and ACTH. In the current study, administration of septal urocortin 2 (100 ng and 500 ng) did not have any effect on plasma corticosterone in the mouse. These data would suggest that the anxiogenic- like effects of urocortin 2 in the septum were not mediated by peripheral release of glucocortiocoids, but were induced by the action of the CRF 2 receptor agonist within the brain.

52

Figure 1: Septal urocortin 2 increased anxiety-like behavior.

Effect of bilateral septal infusion of urocortin 2 (vehicle, 1 ng, 10 ng, 100 ng, 500 ng) into ICR mice on a) latency to enter light side of light-dark box (seconds), b) total time spent in light side of light-dark box (seconds), c) locomotion in open field (total square entries), d) percent time in center of open field, e) locomotion in open field with novel object (total square entries), and f) percent time spent in center with novel object. The high dose of 500 ng increased anxiety-like behavior in the light-dark box and novel object test compared to vehicle, with a trend towards increased anxiety- like behavior in the open field test, but did not alter locomotion. Lower doses of 1 and 100 ng did not significantly alter anxiety-like behavior. 10 ng of urocortin 2 increased anxiety-like behavior in the novel object test, but had no effect in the light-dark box or open field. Data presented as mean ± SEM. * p < 0.05, ** p < 0.01.

53

54

Figure 2: ICV urocortin 2 decreased motor activity at high dose .

Effect of urocortin 2 infusion into the lateral ventricle of ICR mice (vehicle, 100 ng, 500 ng, 1 µg, 5 µg) on a) latency to enter light side of light-dark box (seconds), b) total time spent in light side of light-dark box (seconds), c) locomotion in open field (total square entries), d) percent time in center of open field, e) locomotion in open field with novel object (total square entries), and f) percent time spent in center with novel object. The highest dose of icv urocortin 2 (5 µg) significantly reduced locomotion in the open field and novel object test compared to vehicle, but the peptide had no effect on anxiety-like behavior or locomotion at lower doses. Data presented as mean ± SEM. * p < 0.05.

55

56

Figure 3: Effect of septal Urocortin 2 on anxiety-like behavior is dependent on stress.

Septal urocortin 2 (100 ng) in ICR mice treated with no stress or 30 minutes of restraint stress on a) latency to enter light side of light-dark box (seconds), b) total time spent in light side of light-dark box (seconds), c) locomotion in open field (total square entries), d) percent time in center of open field, e) locomotion in open field with novel object (total square entries,, and f) percent time spent in center with novel object. This dose of urocortin 2 had no effect on anxiety-like behavior in the no stress condition, but significantly increased anxiety-like behavior compared to vehicle in the light-dark box, open field and novel object test when combined with 30 minutes of restraint stress. There was no difference in locomotion between vehicle- and peptide-treated mice in the stress condition. Data presented as mean ± SEM. ** p < 0.01,*** p < 0.001.

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58

Figure 4: Septal urocortin 2 increased anxiety-like behavior when combined with stress in CRF 2 receptor wildtype mice on a mixed C57BL/6 x 129 background.

Effect of septal urocortin 2 (vehicle, 1 ng, 10 ng, 100 ng) in CRF 2 receptor wildtype mice treated with no stress or 30 minutes of immobilization stress on a) latency to enter light side of light-dark box (seconds), b) total time spent in light side of light-dark box (seconds), c) locomotion in open field (total square entries), d) percent time in center of open field, e) locomotion in open field with novel object (total square entries), and f) percent time spent in center with novel object. Urocortin 2 did not affect anxiety-like behavior in the no stress condition at any dose tested. In the stress condition, 100 ng of urocortin 2 increased anxiety-like behavior in the light-dark box and novel object test; the 10 ng dose increased anxiety-like behavior in the novel object test in the stress condition but not the no stress condition. Data presented as mean ± SEM, *** p < 0.001.

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Figure 5 : Septal urocortin 2 did not have a significant effect on plasma corticosterone

Mice prepared with bilateral septal cannula were infused with vehicle, 100 ng, or 500 ng of urocortin 2 per side and place back into the home cages for 30 minutes. After the 30 minute interval, the mice were decapitated and trunk blood collected and assayed for plasma levels of corticosterone. Septal infusion of 100 ng and 500 ng of urocortin 2 did not have a significant effect on plasma corticosterone compared to vehicle-treated mice.

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Chapter 4:

The Effect of CRF 1 and CRF 2 Receptor Activation on Anxiety-like Behaviors Mediated by the Lateral Septum

ABSTRACT

Administration of CRF 1 receptor antagonists has been demonstrated to consistently reduce anxiety-like behavior, but the effect of selective CRF 2 receptor antagonists on anxiety-like behavior remains controversial. ICV infusion of CRF 2 antagonists in rat and mouse has been reported to both increase and decrease anxiety- like behavior, while the blockade of CRF 2 receptor function in specific brain sites such as the lateral septum and dorsal raphe appears to decrease anxiety-like behavior. The potential interaction between the both CRF receptors and the resulting effect on anxiety-like behavior is unknown. The purpose of the current studies was to determine if the anxiogenic effect of septal urocortin 2 reported in previous experiments is dependent on activation of the CRF 2 receptor and/or the activation of the CRF 1 receptor. Urocortin 2 (100 ng) was administered into the septum of CRF 2 receptor knockout and wildtype mice in combination with 30 minutes of restraint stress and anxiety-like behavior was assessed in the light-dark box, open field and novel object test. In a subsequent experiment, mice were pretreated with the CRF 2 receptor selective antagonist astressin-2B ten minutes before septal urocortin 2 administration.

To determine if CRF 1 receptor activation was involved in the anxiogenic effects of urocortin 2, the CRF 1 receptor selective antagonist antalarmin was infused

67 68 into the lateral septum 10 minutes before septal adminstration of urocortin 2. A subsequent study assessed the effect of bilateral infusion of antalarmin into the central amygdala on anxiety-like behavior induced by restraint stress. The results showed that septal urocortin 2 increased anxiety-like behavior in wildtype, but not CRF 2 receptor knockout mice in the light-dark box when combined with restraint stress. Lower doses of septal astressin-2B (50 and 200 ng) blocked the anxiogenic-like effect of urocortin

2, while the highest dose tested (400 ng) decreased anxiety-like behavior due to restraint stress in both vehicle- and peptide-treated mice. Septal administration of antalarmin (100 ng and 300 ng) had no effect on the anxiogenic effects of urocortin 2, but the higher dose of the CRF 1 antagonist (300 ng) reduced anxiety-like behavior when infused into the central amygdala. These data indicate that the anxiogenic effect of septal urocortin 2 is mediated by CRF 2 receptors in the lateral septum.

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INTRODUCTION

The two primary CRF receptor subtypes, CRF 1 and CRF 2, are both members of the class B 7-transmembrane G-protein-coupled receptor group. Both receptors stimulate the release of the intracellular messenger cAMP and the phosphorylation of the cAMP response element binding protein (CREB) via activation of the Gs G- protein, and also appear to signal through phospholipase C and the mitogen-activated protein kinase (MAPK) pathway (Steckler and Dautzenberg, 2006; Rossant et al.,

1999). While the CRF 1 receptor is expressed in the rodent cortex, cerebellum, hippocampus, basolateral amygdala, bed nucleus of the stria terminalis, medial septum, raphe, pons, and sensory brainstem nuclei, (van Pett et al., 2000), the CRF 2 receptor is observed mainly in subcortical structures that include the lateral septum, bed nucleus of the stria terminalis, the cortical and medial amygdala, ventromedial hypothalamus, and dorsal raphe. There are two membrane-bound splice variants of the

CRF 2 receptor in rodent that differ in their N-terminal domains; CRF 2(a) is primarily expressed in the brain, while CRF 2(b) is found mostly in the periphery, including skeletal muscle, skin, and heart. A soluble variant of the CRF 2(a) receptor has recently been identified in mouse (Chen et al., 2005), primarily located in regions that also express the CRF 1 receptor, including the cortex, olfactory bulb, and basolateral amygdala.

A number of studies report that reducing CRF 1 receptor expression or treatment with CRF 1 receptor antagonists decreases anxiety-like behavior. Heinrichs and colleagues (1997) treated rats with icv CRF 1 antisense oligonucleotides and

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reduced CRF 1 receptor expression in the amygdala and cortex by 10 – 12 %. Rats in this treatment group showed less anxiety-like behavior in the defensive withdrawal test compared to control animals. Liebsch and colleagues (1995, 1999) observed that

CRF 1 receptor antisense nucleotides infused into the lateral ventricle and central amygdala reduced anxiety-like behavior in social defeat and the elevated plus maze test in rats. The CRF selective antagonist antalarmin blocked the induction and expression of conditioned fear in rats exposed to footshock (Deak et al., 1999) and also decreased stress-induced behavior in rhesus monkeys (Habib et al., 2000). A second commonly used CRF 1 receptor antagonist (CP-154-526) has been demonstrated to also reduce conditioned fear (Hikichi et al., 2000), decrease anxiety- like behavior in the elevated plus maze (Lundkvist et al., 1996), and reduce ultrasonic vocalizations from isolated rat pups (Kehne, et al., 2000). CRF 1 antagonists have also been reported to decrease shock induced freezing (Bakshi et al., 2002) and reduce defensive behavior in social defeat (Robinson et al., 2004) when infused into the amygdala, but have no effect on anxiety-like behavior when infused into the lateral septum (Bakshi et al., 2002).

There have been conflicting reports about the effect of CRF 2 receptor antagonists on anxiety-like behavior. ICV infusion of a CRF 2 receptor antisense oligonucleotide into Wistar rats increased anxiety-like behavior in the elevated plus maze, light-dark box test, and conditioned fear test (Isogawa et al., 2003). ICV treatment with the CRF 2 receptor antagonist ASV-30 is also reported to induce anxiety-like behavior in the elevated plus maze in C57BL/6 mice (Kishimoto et al.,

2000). In contrast, Takahashi and colleagues (2001) reported that icv ASV-30 reduced

71 conditioned freezing and decreased anxiety-like behavior in the elevated plus maze and defensive withdrawal test in Sprague-Dawley rats. Pelleymounter and colleagues

(2002) report that icv ASV-30 decreased anxiety-like behavior in the marble burying test, open field, and elevated plus maze, while Risbrough and colleagues (2003) observed that ASV-30 blocked CRF-induced increases in acoustic startle.

The blockade of CRF 2 receptor function in specific brain sites such as the lateral septum and dorsal raphe appears to decrease anxiety-like behavior. Ho and colleagues (2001) observed that infusion of a CRF 2 receptor antisense oligonucleotide into the rat lateral septum for 7 days decreased shock-induced freezing. Bakshi and colleagues (2002) report that injection of the non-selective CRF receptor antagonist

αhCRF, but not the CRF 1 selective antagonist NBI27914, into the lateral septum also decreased shock-induced freezing in rat, an effect apparently mediated by septal CRF 2 receptors. Radulovic and colleagues (1999) reported that infusion of ASV-30 into the lateral septum reduced the anxiogenic-like effect of septal CRF and restraint stress.

When ASV-30 was infused into the dorsal raphe, the antagonist decreased conditioned fear and reduced learned helplessness (Hammack et al., 2003).

The deletion of the CRF 2 receptor in knockout mice has produced variable effects on anxiety-like behavior. Bale and colleagues (2000) reported that CRF 2 receptor knockout mouse on a C57Bl/6 x 129 SvJae background demonstrated hyperactivity of the HPA axis and showed increased anxiety-like behavior in the elevated plus maze and open field test. Coste and colleagues (2001) reported that a separately developed strain of CRF 2 receptor KO mice did not show increased anxiety-like behavior in the elevated plus maze, and exhibited a nonsignificant trend

72 towards increased anxiety-like behavior in the open field test. A third KO strain

(Kishimoto et al., 2000) did show increased anxiety-like behavior in the elevated plus maze and light-dark box, but less anxiety-like behavior in the open field.

The purpose of the experiments described in this chapter was to examine the effect of selective CRF 1 and CRF 2 receptor antagonists in the lateral septum on anxiety-like behavior following the administration of septal urocortin 2. The effect of septal urocortin 2 was also tested in CRF 2 receptor KO mice to assess the role of this receptor on behavioral changes mediated by the peptide treatment.

METHODS

The methods for each of these experiments followed the procedures detailed in

Chapter 2 for animal housing, drug preparation, surgery and histology, behavioral testing and data analysis. Urocortin 2, astressin-2B, and antalarmin were administered as previously described and each animal tested for anxiety-like behavior in the light- dark box, open-field test, and novel object test as delineated in the behavioral testing section in the second chapter.

Experiment 1: Septal urocortin 2 combined with stress in CRF 2 receptor knockout and wildtype mice.

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CRF 2 receptor wildtype and knockout mice were prepared with bilateral cannula in the lateral septum and infused with vehicle or 100 ng of urocortin 2 per side in combination with no stress or 30 minutes of restraint stress, n = 8-10 per group.

Experiment 2: Septal urocortin 2 + CRF 2 receptor antagonist.

CRF 2 receptor wildtype mice were prepared with bilateral cannula in the lateral septum and infused with vehicle, 50 ng, 200 ng, or 400 ng of the CRF 2 receptor antagonist astressin-2B per side. Ten minutes later, they were infused with vehicle or

100 ng of urocortin 2 in combination with no stress or 30 minutes of restraint stress, n

= 7-16 per group.

Experiment 3: Septal urocortin 2 + CRF 1 receptor antagonist .

ICR mice prepared with bilateral cannula in the lateral septum were treated with the CRF 1 receptor antagonist antalarmin prior to the septal infusion of urocortin

2. This experiment was conducted to determine if the effect of septal urocortin 2 was mediated by CRF 1 receptors in the lateral septum. The mice were infused with vehicle,

100 ng, or 300 ng of antalarmin, followed ten minutes later by an infusion of vehicle or 100 ng of urocortin 2. Following the second septal infusion, all mice were restrained for 30 minutes before testing in the light-dark box, open field, and novel object test, n = 7-9 per group. These doses of antalarmin were selected based on the

74 results of a previous study (Robison et al., 2004) reporting a decrease in stress-related behavior in mice treated with an equivalent dose of this drug.

Experiment 4: CRF 1 receptor antagonist infused into the central amygdala.

To determine if the doses of antalarmin infused into the lateral septum were sufficient to antagonize CRF 1 receptor-mediated behavior, ICR mice were prepared with bilateral cannula targeting the central nucleus of the amygdala. Mice were infused with vehicle, 100 ng, or 300 ng of antalarmin into the amygdala 10 minutes before 30 minutes of restraint stress, then tested in the light-dark box, open field, and novel object test, n = 7-9 per group.

RESULTS

Experiment 1: Effects of septal urocortin 2 combined with stress in CRF 2 receptor knockout and wildtype mice.

The data show that treatment with urocortin 2 increased anxiety-like behavior in wildtype but not knockout mice in the stress condition, and had no effect on either genotype in the no stress condition (Figure 6). Statistical analysis (3-way ANOVA, followed by the Tukey post-hoc test) revealed a three-way interaction between stress, genotype, and peptide treatment on the latency to enter the light side of the light-dark box (F (1,71) = 11.749, p < 0.01), and the percent time spent in the center of the open

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field ((F (1,71) = 4.014, p < 0.05). In wildtype mice in the stress condition, urocortin 2 significantly increased the latency to enter the light side of the light-dark box (p <

0.001) (Fig. 6a) and decreased time spent in the center of the open field (p < 0.01) (Fig

6d) compared to vehicle, while the peptide had no effect in knockout mice. A 2-way

ANOVA in wildtype mice (conducted according to our a priori hypothesis that urocortin 2 will affect behavior in this group of mice) showed a significant interaction between stress and peptide on the time spent in the light side of the light-dark box

(F (1,33) = 4.491, p < 0.05) and percent time spent in the center with the novel object

(F (1, 33) = 5.119, p < 0.05). Post-hoc tests revealed that urocortin 2 decreased time spent in the light side of the light-dark box (p < 0.05) (Fig. 6b) and decreased percent time in the center with the novel object (p < 0.05) (Fig. 6f) only in the stress condition when compared to vehicle. There were no significant effects of septal urocortin 2 on anxiety-like behavior in CRF 2 receptor knockout mice in any of the three behavior tests.

CRF 2 receptor knockout mice exhibited more anxiety-like behavior compared to wildtype, spending significantly less time in the center of both the open field (F (1,71)

= 13.517, p < 0.001) (Fig. 6d) and the novel object test (F (1,71) = 4.778, p < 0.05) (Fig.

6f), but did not demonstrate any differences in overall locomotion. Restraint stress increased anxiety-related measures in the light-dark box, open field, and novel object test and decreased locomotion in both the open field (F (1,33) = 36.662, p < 0.001) and the novel object test (F (1,33) = 6.319, p < 0.05) as previously demonstrated, but there was no effect of urocortin 2 on locomotion in the stress or no stress condition.

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Experiment 2: Effects of septal urocortin 2 combined with a CRF 2 receptor antagonist:

Septal administration of the CRF 2 receptor antagonist astressin-2B did not affect anxiety-like behavior in the no stress condition, but did reduce anxiety-like behavior in mice receiving urocortin 2 or vehicle in the stress condition (Figure 7).

There was a significant 3-way interaction between stress, urocortin 2, and astressin-2B on the latency to enter the light side of the light-dark box (F (3,148) = 7.216, p < 0.001) and the time spent in the light side of the light-dark box (F (3,148) = 2.835, p < 0.05), and a significant interaction between stress and astressin-2B on the percent time in the center of the open field (F (3,148) = 13.342, p < 0.001) and with the novel object (F (3,148)

= 12.512, p < 0.001).

Post-hoc tests showed that the highest dose of astressin–2B (400 ng) decreased anxiety-like behavior in the light-dark box in vehicle-treated mice in the stress condition, decreasing the latency to enter the light side (p < 0.001) (Fig. 7a) and increasing time spent in the light (p < 0.001) (Fig. 7b), but lower doses of the antagonist had no effect on anxiety-like behavior in these mice. In contrast, 50 ng and

200 ng of astressin-2B decreased anxiety-like behavior in urocortin 2-treated mice in the stress condition, significantly decreasing latency to enter the light side (50 ng, p <

0.01; 200 ng, p < 0.001) (Fig. 7a) and increasing time spent in the light (50 ng, p <

0.01; 200 ng , p < 0.001) (Fig. 7b) compared to control.

In the open field test, one-way ANOVA for vehicle-treated mice in the stress condition (conducted according to our a priori hypothesis that astressin-2B will have

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an effect in this group of mice) showed that the CRF 2 receptor antagonist significantly increased time in the center square (F (3,37) = 13.109, p < 0.001). Astressin-2B also reduced anxiety-like behavior (increased time in the center) in urocortin 2-treated mice in the stress condition. (F (3,37) = 40.467, p < 0.001) (Fig 7d). However, post-hoc tests showed that 50 ng of astressin-2B reduced anxiety-like behavior in mice treated with urocortin 2 (p < 0.05), but had no significant effect in vehicle-treated mice (Fig 7d); the higher doses of astressin-2B decreased anxiety-like behavior in both groups. In the novel object test, one-way ANOVA showed that astressin-2B decreased anxiety-like behavior in stressed mice treated with either vehicle (F (3,37) = 5,211, p < 0.01) or urocortin 2 (F (3,37) = 30.638, p < 0.001) (Fig 7f). Post-hoc tests showed that 50 and 100 ng of astressin-2B significantly decreased anxiety-like behavior in mice treated with urocortin 2 (50 ng, p < 0.01; 100 ng, p < 0.001) but these doses had no significant effect on vehicle-treated mice (Fig 7f). Only the highest dose of astressin-2B (400 ng) decreased anxiety-like behavior in both groups (vehicle, p < 0.01; urocortin 2, p <

0.001).

Restraint stress increased anxiety-related measures in the light-dark box, open field, and novel object test and decreased locomotion in the open field (F (3,148) = 6.010, p < 0.05) (Fig. 2c) and the novel object test (F (3,148) = 11.239, p < 0.001) (Fig. 7e), but neither urocortin 2 or astressin-2B had a significant effect on locomotion.

In summary, septal administration of lower doses of the CRF 2 receptor antagonist astressin-2B (50 and 200 ng) decreased the anxiogenic effect of urocortin 2 combined with stress in the light-dark box and novel object test, but had no effect on anxiety-like behavior in vehicle-treated mice. In the open field test, 50 ng of astressin-

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2B had no effect on vehicle-treated mice in the stress condition, but increased the time spent in the center for urocortin 2-treated mice, while the higher doses of the antagonist decreased anxiety-like behavior in both groups. The highest tested dose of urocortin 2 (400 ng) decreased anxiety-like behavior in the stress condition for both vehicle- and drug-treated mice in all three behavioral assays.

Experiment 3: Effects of septal urocortin 2 combined with a CRF 1 receptor antagonist:

Septal administration of the CRF 1 receptor antagonist antalarmin did not affect anxiety-like behavior in restraint-stressed mice treated with septal urocortin 2 (Figure

8). In this experiment, urocortin 2 significantly increased the latency to enter the light side of the light-dark box (F (1,42) = 52.369, p < 0.001) (Fig. 8a), decreased the time spent in the light side of the light-dark box (F (1,42) = 19.609, p < 0.001) (Fig. 8b), decreased percent center entries (F (1,42) = 11.083, p < 0.01) and percent time (F (1,42) =

39.893, p < 0.001) (Fig. 8d) in the center of the open field, and decreased percent entries (F (1,60) = 11.885, p < 0.01) and percent time (F (1,42) = 22.105, p < 0.001) (Fig.

8f) in the center with the novel object when compared to vehicle. There were no significant differences between mice treated with septal antalarmin or vehicle on any behavioral measure tested.

Experiment 4: CRF 1 receptor antagonist infused into the amygdala.

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Administration of antalarmin into the central nucleus of the amygdala decreased anxiety-like behavior in the light-dark box, decreasing the latency to enter the light side (F (2,42) = 5.461, p < 0.01) (Fig. 9a) and increasing time spent in the light

(F (2,42) = 4.942, p < 0.05) (Fig. 9b). Antalarmin also decreased anxiety-like behavior in the open field, significantly increasing time spent in the center (F (2,42) = 5.274, p <

0.01) (Fig. 9d). There was a significant interaction between stress and antalarmin on the latency to enter the light side of the light-dark box (F (2,42) = 8.055, p < 0.05), time spent in the light side (F (2,42) = 10.326, p < 0.001), and time spent in the center of the open field (F (2,42) = 4.568, p < 0.05). Post-hoc tests showed that antalarmin had no significant effect on anxiety-like behavior in the no stress condition for any test. In the stress condition, the highest dose of antalarmin (300 ng) significantly decreased the latency to enter the light side of the light-dark box (p < 0.01), increased time spent in the light side of the light-dark box (p < 0.001), and increased time spent in the center of the open field (p < 0.001), with a trend towards increased time in the center with the novel object (p = 0.052) (Fig 9f). The lower dose of antalarmin (100 ng) did not significantly affect anxiety-like behavior, and neither dose had a significant effect on locomotion.

DISCUSSION

The experiments in this chapter examined the role of CRF 1 and CRF 2 receptor activation in the behavioral effects of septal urocortin 2 administration. The first study

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tested the effect of septal urocortin 2 in wildtype and CRF 2 receptor KO mice. As previously reported, urocortin 2 (100 ng) did not have a significant effect on anxiety- like behavior in the no stress condition. In wildtype mice subject to restraint stress, septal urocortin 2 increased the latency to enter the light side of the light-dark box, decreased time in the light side of the light-dark box, and significantly reduced the time spent in the center of the open field and novel object test. There were no effects of septal urocortin 2 on anxiety-like behavior in CRF 2 receptor KO mice in the light- dark box, with both vehicle- and drug-treated mice spending 50 to 60 seconds in the light side in the stress condition. However, the stressed knockout mice spent very little time in the center of both the open field and novel object test (< 6% of the time) regardless of drug treatment, so it is difficult to determine if urocortin 2 had any effect in these behavioral assays. It is possible that the anxiety response reached a floor effect under these conditions, given the high level of anxiety-like behavior observed in the knockout mice during restraint stress.

Overall, the CRF 2 receptor KO mice showed increased anxiety-like behavior in the three behavioral tests compared to the wildtype controls; vehicle-treated KO mice spent less time in the light side of the light-dark box, less time in the center of the open field, and less time in the center with the novel object test compared to wildtype.

The increased anxiety-like behavior observed in the KO mice is in agreement with previous reports (Bale et al, 2000; Kishimoto et al., 2000, Bale and Vale, 2004). This finding is in apparent contradiction to the increased anxiety-like behavior reported with the administration of urocortin 2, a CRF 2 receptor agonist. It is possible that the anxiogenic phenotype observed in the knockout mice may result from other factors

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besides the absence of the CRF 2 receptor; Bale et al. (2000) reported increased CRF expression in the amygdala of these mice, which may contribute to greater anxiety-like behavior, perhaps through stimulation of the CRF 1 receptor.

Septal administration of lower doses of the CRF 2 receptor antagonist astressin-

2B (50 and 200 ng) decreased the anxiogenic effect of urocortin 2 combined with stress in the light-dark box, open field, and novel object test, but had no effect on anxiety-like behavior in vehicle-treated mice. In contrast, the highest tested dose of astressin-2B (400 ng) decreased anxiety-like behavior in the stress condition for both vehicle- and drug-treated mice in all three behavioral assays. These results suggest the possibility that the lower doses of septal astressin-2B blocked the anxiogenic effect of septal urocortin 2, but the highest dose may also reverse the additive anxiogenic effect of the restraint stress. This data supports previous findings demonstrating that antagonism of the septal CRF 2 receptor decreases anxiety-like behavior in both rat

(Bakshi et al., 2002) and mouse (Radulovic et al., 1999).

Experiments 3 and 4 in this chapter tested the effect of the CRF 1 receptor selective antagonist antalarmin on the effects of septal urocortin 2. Septal administration of antalarmin (100 ng and 300 ng) 10 minutes prior to urocortin 2 treatment had no effect on the behavior in the light-dark box, open field, or novel object test, and did not block the anxiogenic effect of urocortin 2 in restraint-stressed mice. In contrast, when antalarmin was infused into the central amygdala, the higher dose of the antagonist (300 ng) blocked anxiety-like behavior induced by restraint stress, indicating that this dose was sufficient to antagonize anxiety-like behavior putatively mediated by the CRF 1 receptor.

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These results indicate that blockade of CRF 1 receptors in the amygdala, but not in the lateral septum, will decrease anxiety-like behavior in rodents. This finding corroborated data reported by earlier studies; Robison and colleagues (2004) observed that 250 ng of antalarmin infused bilaterally into the mouse amygdala decreased anxiety-mediated defensive behavior in C57BL/6 mice. Bakshi and colleagues (2002) also found that the CRF 1 selective antagonist NBI27914 (1 µg) blocked shock-induced freezing in rat when injected into the central amygdala, but had no effect in the lateral septum.

In summary, septal urocortin 2 had no significant effects in CRF 2 receptor KO mice, and the anxiogenic effect of urocortin 2 was blocked by pretreatment with the

CRF 2 receptor selective antagonist astressin-2B, but not the CRF 1 selective antagonist antalarmin. These data support the conclusion that the effects of urocortin 2 in the current experiments are mediated through the septal CRF2 receptor.

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Figure 6: No effect of septal urocortin 2 in CRF 2 receptor KO mice.

Effect of septal urocortin 2 (100 ng) in CRF 2 receptor wildtype (WT) and knockout (KO) mice treated with no stress or 30 minutes of restraint stress on a) latency to enter light side of light-dark box (seconds), b) total time spent in light side of light-dark box (seconds), c) locomotion in open field (total square entries), d) percent time in center of open field, e) locomotion in open field with novel object (total square entries), and f) percent time spent in center with novel object. In wildtype mice, urocortin 2 had no effect on anxiety-like behavior in the no stress condition, but increased anxiety-like behavior in the light-dark box, open field and novel object test when combined with restraint stress. Urocortin 2 had no effect in CRF 2 receptor knockout mice in the light-dark box (a,b). CRF 2 receptor knockout mice showed increased anxiety-like behavior compared to wildtype, spending less time in the center of the open field (d) and novel object test (f) compared to wildtype, regardless of drug treatment. Knockout mice treated with vehicle also exhibited greater latency to enter the light-side of the light-dark box compared to vehicle-treated wildtype mice (a). Data presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001.

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Figure 7: CRF 2 antagonist astressin-2B blocked effect of urocortin 2 and stress on anxiety .

Effect of septal CRF 2 receptor antagonist astressin-2B (50, 200, or 400 ng) administered 10 minutes before urocortin 2 (100 ng) in CRF 2 receptor wildtype mice treated with no stress or 30 minutes of restraint stress on a) latency to enter light side of light-dark box (seconds), b) total time spent in light side of light-dark box (seconds), c) locomotion in open field (total square entries), d) percent time in center of open field, e) locomotion in open field with novel object (total square entries), and f) percent time spent in center with novel object. Astressin-2B decreased anxiety-like behavior in the light-dark box, open field and novel object test in the stress condition, but had little effect in the no-stress condition. In the light-dark box, 50 and 200 ng astressin-2B decreased the anxiogenic effect of urocortin 2 in the stress condition, but had no effect in vehicle-treated mice, while the 400 ng dose reduced anxiety-like behavior in all stressed mice, decreasing the latency to enter the light side of the light-dark box (a) and increasing time spent in the light side (b). In the open field, 50 ng astressin-2B decreased the anxiogenic effect of urocortin 2, but had no effect in vehicle-treated mice in the stress condition (d). In the novel object test, 50 and 200 ng of the antagonist inhibited the anxiogenic effect of urocortin 2, but had no significant effect in vehicle- treated mice (f), while 400 ng decreased anxiety-like behavior in both groups. Data presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001.

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Figure 8: Septal administration of the CRF 1 receptor antagonist antalarmin does not block the effect of urocortin 2 .

Effect of septal administration of the CRF 1 receptor antagonist antalarmin (vehicle, 100 ng, 300 ng) on the anxiogenic-like effect of septal urocortin 2 (100 ng) on the a) latency to enter light side of light-dark box (seconds), b) total time spent in light side of light-dark box (seconds), c) locomotion in open field (total square entries), d) percent time in center of open field, e) locomotion in open field with novel object (total square entries), and f) percent time spent in center with novel object. Urocortin 2 significantly increased anxiety-like behavior in the light-dark box (a,b), open field (d), and novel object test (f) in restraint-stressed mice pretreated with septal antalarmin or vehicle. Antalarmin did not block the anxiogenic effect of urocortin 2.

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Figure 9: Administration of the CRF 1 antagonist antalarmin into the central amygdala reduced the anxiogenic effect of restraint stress .

Effect of administration of the CRF 1 receptor antagonist antalarmin into central amygdala (vehicle, 100 ng, 300 ng) on the anxiogenic effect of 30 minutes of restraint stress on a) latency to enter light side of light-dark box (seconds), b) total time spent in light side of light-dark box (seconds), c) locomotion in open field (total square entries), d) percent time in center of open field, e) locomotion in open field with novel object (total square entries), and f) percent time spent in center with novel object. 300 ng of antalarmin reduced the anxiogenic effect of immobilization stress in the light-dark box (a,b) and open field (d), with a trend towards decreased anxiety-like behavior in the novel object test (f).The lower dose of 100 ng did not have a significant effect. Data presented as mean ± SEM. ** p < 0.01, *** p < 0.001.

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Hammack, S.E., Schmid, M. J., LoPresti, M.L., Der-Avakian, A., Pellymounter, M.A., Foster, A.C., Watkins, L.R., Maier, S.F. (2003). "Corticotropin releasing hormone type 2 receptors in the dorsal raphe nucleus mediate the behavioral consequences of uncontrollable stress." J Neurosci 23(3): 1019-25.

Heinrichs, S.C., Lapsansky, J., Lovenberg T.W., De Souza E.B., Chalmers D.T. (1997). "Corticotropin-releasing factor CRF1, but not CRF2, receptors mediate anxiogenic-like behavior." Regul Pept 71(1): 15-21.

Hikichi, T., Akiyoshi, J., Yamamoto, Y., Tsutsumi, T., Isogawa, K., Nagayama, H. (2000). “Suppression of conditioned fear by administration of CRF receptor antagonist CP-154,526.” Pharmacopsychiatry. 33(5):189-93.

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Isogawa, K., Akiyoshi, J., Tsutsumi, T., Kodama, K., Horinouti, Y., Nagayama, H. (2003). “Anxiogenic-like effect of corticotropin-releasing factor receptor 2 antisense oligonucleotides infused into rat brain.” J Psychopharmacol. 17(4): 409-13.

Kehne, J.H., Coverdale, S., McCloskey, T.C., Hoffman, D.C., Cassella, J.V. (2000). “Effects of the CRF(1) receptor antagonist, CP 154,526, in the separation-induced vocalization anxiolytic test in rat pups.” Neuropharmacology. 39(8):1357-67.

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Robison, C.L., Meyerhoff, J.L., Saviolakis, G..A, Chen, W.K., Rice, K.C., Lumley, L.A. (2004) A CRH 1 antagonist into the amygdala of mice prevents defeat-induced defensive behavior. Ann NY Acad Sci 1032: 324-328.

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Chapter 5:

The Role of the Edinger-Westphal Nucleus

ABSTRACT

The Edinger-Westphal (EW) nucleus is the primary site of urocortin 1 expression in the brain and appears to play an important role in the response to stress.

Rodents exposed to various stressors, including restraint stress and ether treatment, show significant increases in EW c-fos expression and upregulation of urocortin 1 mRNA in this nucleus. There is a dense urocortin 1 projection from the EW to the rodent lateral septum, suggesting that the release of urocortin 1 in the septum could have a significant effect on septal CRF 2 receptor function. While administration of urocortin into the brain is consistently reported to increase anxiety-like behavior, the effect of removing the peptide on measures of anxiety is not yet clearly understood.

Such data would provide converging evidence for the role of urocortin in the lateral septum on anxiety-like behaviors. The present study examined the effect of lesioning the Edinger-Westphal nucleus on anxiety-like behavior following the administration of septal urocortin 2 into the mouse lateral septum. Mice were infused with quinolinic acid (1 µg) or vehicle (sham treatment) into the EW and subsequently prepared with bilateral cannulae in the lateral septum. Animals were administered vehicle or 100 ng of urocortin 2 into the septum in combination with 30 minutes of restraint stress or no stress, and anxiety-like behavior was assessed in the light-dark box, open field, and

95 96 novel object test. In confirmation of our previous findings, the results indicated that treatment with urocortin 2 increased anxiety-like behavior in sham-operated animals in the stress condition. Interestingly, however, this well established effect of urocortin

2 injection into the lateral septum was absent in EW-lesioned mice in the stress condition in both the light-dark box and novel object test, but the lesion of the EW nucleus did not affect baseline anxiety-like behaviors. These data suggest that the anxiogenic-like effect of urocortin 2 in the lateral septum during stress is critically dependent on urocortin 1 input from the EW.

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INTRODUCTION

The Edinger-Westphal (EW) nucleus is the primary site of urocortin expression in the brain. The EW has long been established as a critical component of the pathway that regulates oculomotor function (Warwick et al., 1954), including pupil control and lens accommodation. When exposed to bright light, the retina activates light sensitive neurons in the olivary pretectal nucleus (OPN). The OPN stimulates the EW to send a signal through the oculomotor nerve to constrict the ciliary muscles, causing the eye pupil to constrict, a reaction known as the pupillary light reflex (Klooster et al., 1998). There has been some controversy over the exact function of neurons in the EW; some neurons but not others are activated by stimulation of the optic nerve (Gamlin et al., 1994) and a retrograde tracer in the oculomotor nerve of the cat labeled only a small percentage of the EW nucleus neurons (Loewy et., al, 1978).

More recent studies have shown that the EW nucleus neurons project to other sites outside the ciliary ganglion and target regions throughout the brain, including the lateral septum, parabrachial nucleus, trigeminal nucleus, facial nucleus, dorsal column nuclei, inferior olive, and reticular formation, spinal cord and the cerebellum

(Bittencourt et al., 1999). Relevant to the present project is the fact that urocortin 1 positive neurons have been found in the human EW nucleus, but these neurons do not express choline-acetyltransferase. This finding suggests that these urocortin 1 positive neurons in the EW differ from the parasympathetic cholinergic neurons projecting to

98 the ciliary ganglion (Ryabinin et al., 2005), supporting the idea that the function of the

EW nucleus is not limited to controlling visual acuity.

Most Urocortin 1 projections in the brain originate from the Edinger-Westphal nucleus and predominately project to the hindbrain (Bittencourt et al., 1999), the exception being a strong forebrain projection to the lateral septum. As described above, infusion of urocortin 1 into the brain elicits anxiety-like behavior in rats (Spina et al., 2002; Jones et al., 1998) and mice (Moreau et al., 1997) and strongly inhibits food intake (Benoit et al., 2000). A number of studies have suggested that the EW nucleus may play a role in responding to stress. Weninger and colleagues (2000) reported that restraint stress significantly upregulated urocortin 1 mRNA in the mouse

Edinger-Wesphal nucleus and Korosi and colleagues (2005) also reported that ether stress in mice increased urocortin 1 message and induced c-fos expression in the EW.

Gaszner and colleagues (2004) reported that rats exposed to restraint, ether stress or lipopolysaccaride stress showed significant c-fos induction in the EW, while Turek and Ryabinin (2005) showed that ethanol exposure in mice also increased EW c-fos expression.

To examine the effect of removing urocortin from the brain on rodent physiology and behavior, two separate strains of urocortin 1 KO mice have been developed and tested, but the effects of urocortin deficiency on anxiety-like behavior have not been consistent. Wang and colleagues (2002) reported that their urocortin KO mouse showed normal anxiety-like responses in the elevated plus maze, light-dark box test, and open field test, but showed decreased response to a startling stimulus, potentially indicative of a decreased response to stressors in the environment. In

99 contrast, Vetter and colleagues (2002) report that a second strain of urocortin KO mice showed increased anxiety-like behavior in the elevated-plus maze and open field test, but no difference in the light-dark box test. These mice also exhibited marked hearing deficits at 3 and 6 months of age, which could explain the decreased response to acoustic startle reported by Wang and colleagues (2002).

There is indirect evidence to suggest that urocortin may mediate anxiety-like behavior based on observations of CRF KO mice, at least in the absence of CRF. Dunn and Swiergiel (1999) tested the behavior of mice lacking CRF in several behavioral tests. They observed that both CRF wildtype and KO mice showed a normal response to restraint-stress induced anxiety-like behavior in the elevated-plus maze and a novel object task, but almost no corticosterone response to stress. These results suggest that while HPA axis function is impaired in the absence of CRF, another factor or factors continue to mediate centrally mediated anxiety-like behaviors. Weninger and colleagues (1999) also reported that both CRF wildtype and KO mice showed the same anxiety-like response to stress. The non-selective CRF antagonist αhCRF 9-41 and the CRF 1 receptor selective antagonist CP-154, 526 decreased shock-induced freezing in both genotypes, indicating that a CRF-like peptide (potentially urocortin, with high affinity for both receptors) is acting at one or both of the CRF receptors to induce anxiety-like behavior in mice lacking CRF. In support of the later hypothesis,

Weininger and colleagues (2000) reported that urocortin mRNA in the EW nucleus is upregulated 2-3 fold in CRF receptor KO mice compared to wildtype animals. Thus, there is indirect evidence to suggest that urocortin in the EW may mediate the anxiety- like behavior observed in these mice.

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A few recent studies have examined the effect of lesioning the EW nucleus on rodent behavior. Bachtell and colleagues (2003) demonstrated that consumption increases c-fos expression in urocortin-positive EW cells and a higher degree of EW urocortin immunoreactivity is correlated with increased alcohol self- administration. In a subsequent study from the same lab, Bachtell and colleagues

(2004) performed electrolytic lesions of the EW nucleus in C57BL/6 mice and observed their response to alcohol treatment. Mice with EW lesions showed significantly less alcohol-induced hypothermia, decreased ethanol preference and consumed less total alcohol and less sucrose in a two-bottle choice test when compared to controls. Weitemier and Ryabinin (2005) reported that C57BL/6 mice with EW lesions consumed significantly less food and water following the surgery and ate less food after a 24 hour food deprivation, but did not exhibit differences in anxiety-like behavior in the elevated-plus maze when compared to sham-lesioned mice.

The purpose of the study described in the current chapter was to examine the effect of a lesion of the Edinger-Westphal nucleus on septal urocortin 2 administration into the mouse lateral septum. This experiment tested the hypothesis that the anxiogenic effect of urocortin 2 during stress is dependent on urocortin 1 input from the EW.

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METHODS

Animals

Male ICR mice were purchased from Harlan (Indianapolis, IN) and treated as described in the general method section in Chapter 2.

Drugs

Urocortin 2 was synthesized under the direction of Dr. Jean Rivier of the

Clayton Foundation Laboratories for Peptide Biology, Salk Institute. The peptide was dissolved in sterile physiological saline containing 0.1M bovine serum albumin, pH

7.4. Quinolinic acid was purchased from Sigma Aldrich (St.. Louis, MO) and prepared in 0.1 M phosphate buffered saline, pH 7.4.

Surgery and drug administration

Mice under anesthesia were placed into the stereotaxic instrument with the nose elevated and the tooth bar set at +5. This angled placement was adopted to avoid damaging the midsaggital sinus, following the EW lesion procedures used by Bachtell and colleagues (2004). A 33 gauge cannula was targeted at the EW nucleus (-3.2 mm posterior to bregma, 0.0 mm lateral from the midline, -3.8 ventral from skull surface

(Franklin and Paxinos, 1997). Quinolinic acid (1 µg/ 6 nmol) or vehicle (sham treatment) was infused into the region in a volume of 0.25 µl over a period of five minutes. After the infusion into the EW, the mice were prepared with bilateral cannulae in the lateral septum as described in Chapter 2. One week after the surgery,

102 septal urocortin 2 was administered as previously described and each animal tested for anxiety-like behavior in the light-dark box, open-field test, and novel object test as explained in the behavioral testing section in the Chapter 2.

Histology and Immunocytochemistry

Mice were euthanized with CO 2 asphyxiation and the animals were immediately transcardially perfused with 0.1 M phosphate buffered saline (PBS; pH

7.4) before fixation with 4% paraformaldehyde chilled to 4 °C. Brains were postfixed overnight before transfer to 30% sucrose for 48 hrs. Horizontal brain sections 35 microns thick were cut on a microtome and stored in 0.1% sodium azide in PBS. The location of cannulae placement was verified in sections stained with cresyl violet acetate. Sections of the Edinger-Westphal nucleus (4 matched sections per animal) in lesioned and sham treated animals were tested for the presence of urocortin 1-positive neurons with an immunocytochemistry assay using a Urocortin 1 antibody raised in guinea pig and developed by Joan Vaughn in the Vale Laboratory (Figure 1). Animals that did not show correct cannula or lesion placement were excluded from analysis.

EW sections were washed three times (10 minutes per wash) in 0.1 M PBS at room temperature, then incubated with the urocortin 1 antibody (1:10,000) with 0.2%

Triton X and 5% normal donkey serum for 48 hours at 4 οC. After this period, the sections were washed 6 times (5 minutes per wash) in 0.1 M PBS, then incubated with biotinylated anti-guinea pig IgG (1:1000) for 1 hour at room temperature. The tissue was again washed in PBS three times (10 minutes per wash), then incubated with an avidin-biotin-horseradish peroxidase complex (ABC; Vector) for one hour, and stained

103 with 10% 3,3’- diaminobenzidine (3 min, DAB, Pierce) in stable peroxide substrate buffer. Stained sections were mounted on gelatin-coated slides, dehydrated, and coverslipped.

RESULTS

In confirmation of our previous findings, the data showed that treatment with urocortin 2 increased anxiety-like behavior in sham-operated effects in the stress condition. Most importantly, this effect was absent in EW-lesioned mice in the stress condition in both the light-dark box and the novel object test, but the lesion of the

Edinger-Westphal nucleus did not affect anxiety-like behavior in the no stress condition. Statistical analysis (mixed 3-way ANOVA with stress, lesion, and drug treatment as the factors) revealed a two-way interaction between lesion and peptide treatment on the latency to enter the light side of the light-dark box (F (1,56) = 4.971, p <

0.05), and the percent time spent in the center with the novel object (F (1,56) = 9.201, p <

0.01). There were no main effects of the EW lesion in any of the three tests.

Two-way ANOVA in sham mice (conducted according to our a priori hypothesis that urocortin 2 will affect behavior in this group of mice, as seen previously) showed a significant interaction between stress and peptide on the latency to enter the light side of the light–dark box (F (1,29) = 13.496, p < 0.001), on time spent in the light side of the light-dark box (F (1,29) = 6.820, p < 0.05), the percent time in the center of the open field (F (1,29) = 15.680, p < 0.001), and percent time spent in the center with the novel object (F (1, 29) = 7.372, p < 0.05). In sham-treated mice in the stress condition, post-hoc tests revealed that urocortin 2 significantly increased the

104 latency to enter the light side of the light-dark box (p < 0.001) (Fig. 11a), decreased time spent in the light side of the light-dark box (p < 0.01) (Fig. 11b) and decreased percent time in the center of the open field (p < 0.01) (Fig. 11f) and with the novel object (p < 0.001) (Fig. 11f).

In contrast, septal urocortin 2 had did not have a significant effect on anxiety- like behavior in the light-dark box (Fig 11a,b) or the novel object test (Fig 11f) during stress in mice treated with a quinolinic acid lesion of the EW nucleus. There was a significant interaction between stress and peptide in EW-lesioned mice in the open field test (F (1,27) = 4.758, p < 0.05); a post-hoc test showed that urocortin 2 decreased time in the center region in the stress, but not the no stress condition, in these mice

(Fig 11d).

DISCUSSION

The data from the current study indicated that a quinolinic acid lesion of the

Edinger-Westphal nucleus decreased the anxiogenic-like effect of septal urocortin 2 with restraint stress, but the lesion did not have a main effect on anxiety-like behavior.

In the no stress condition, there was no difference between sham or lesioned mice on any measure of anxiety-like behavior. However, in the stress condition, treatment with urocortin 2 increased anxiety-like behavior in sham, but not lesioned, mice in the light-dark box and novel object task, although the peptide treatment decreased time in the center of the open field in both sham and lesioned animals. There was no effect of

105 the lesion on total square entries in the open field or novel object task, indicating that damage to the EW nucleus had no effect on overall locomotion.

Administration of urocortin 1 has been reported to increase anxiety-like behavior in several studies (Spina et al., 2002; Declan et al., 1998, Moreau et al.,

1997), but the effects of removing the peptide on anxiety are not yet well understood.

As discussed above, while Wang and colleagues (2002) reported no change in anxiety- like behavioral responses in a urocortin KO mouse, Vetter and colleagues (2002) observed increased anxiety-like behavior in a separately developed KO strain. Only one study to date has examined the effect of a selective lesion of the EW nucleus on anxiety-like behavior. Weitemier and Ryabinin (2005) reported that a lesion of EW in

C57BL/6 mice did not significantly affect anxiety-like behavior in the elevated-plus maze compared to sham lesioned mice and the lesion did not alter locomotor activity.

However, this study did not examine the effect of EW ablation on stress-induced behavior.

In replication of these previous findings, the results reported in this chapter also show that a lesion of the EW nucleus did not have an effect on baseline anxiety- like behavior or locomotion. Most importantly, however, lesions of the EW nucleus, decreased the anxiogenic-like effect of septal urocortin 2 when the peptide treatment was combined with restraint stress. As previously described, a number of studies have suggested that urocortin 1 may play a role in the stress response. Weninger and colleagues (2000) subjected mice to 3 hours of restraint stress and reported a significant increase in urocortin 1 mRNA in the EW. These authors also reported an increase in EW urocortin mRNA in CRF KO mice, a mouse reported to respond

106 normally to restraint stress-induced anxiety-like behavior despite lack of CRF (Dunn and Swiergiel, 1999). Korosi and colleagues (2005) also reported that acute ether stress in mice also significantly increased urocortin mRNA in the EW nucleus.

The immediate early gene c-fos has consistently been shown to be an effective marker of neuronal activation in stress-sensitive brain regions (Bullitt, 1990; Chan et al., 1993; Cullinan et al., 1995). Acute stressors such as restraint induce Fos expression in CRF-expressing neurons in regions such as the hypothalamic paraventricular nucleus (Stamp et al., 1999; Viau et al., 2002), and recent studies have observed a similar effect with urocortin. Gaszner and colleagues (2004) observed that several different stressors induced Fos expression in a considerable number of urocortin positive neurons in the EW nucleus (52% for ether stress, 45% for restraint stress, and 60% for lipopolysaccharide stress). Korosi and colleagues (2005) also reported that most urocortin positive neurons in the EW expressed Fos following acute ether stress in mice.

The data from the current study suggest that the anxiogenic effect of septal urocortin 2 could be dependent upon urocortin 1 input from the Edinger-Westphal nucleus. A dense urocortin 1 projection from the EW to the rodent lateral septum has been identified in both rat (Bittencourt et al., 1999) and mouse (Bachtell et al., 2004), and stressors such as restraint stress appear to activate urocortin in this region

(Weninger et al., 2000; Korosi et al., 2005; Gaszner et al., 2004). In the no stress condition, infusion of a low dose of septal urocortin 2 may have no effect on anxiety.

However, in the presence of a stressor such as restraint stress, increased urocortin

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release in the lateral septum combined with the administered CRF 2 receptor agonist produces a significant increase in anxiety-like behaviors.

It is relevant to note that the lesion of the EW nucleus blocked the anxiogenic effect of septal urocortin 2 in the stress condition in the light-dark box and the novel object test, but the peptide treatment still decreased time in the center of the open field in both sham and lesioned mice. While the light-dark box, open field and novel object tests all measure approach-avoidance behavior, there are differences in these three behavioral tests. The open field test is considered to be the most anxiogenic (Dulawa et al., 1999) and a better measure of avoidance behavior, as the rodent is focused on avoiding the open space in the center region. The light-dark box and the novel object test induce more approach/exploration behavior, as rodents are motivated to explore a second novel compartment in the light-dark box or a novel stimulus in the novel object test. It is conceivable that the effect of the EW lesion may have been diminished in the more anxiogenic open field test, or this brain region may be more effective at specifically modulating approach versus avoidance behaviors.

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SHAM (Vehicle Treatment) LESION (Quinolinic acid)

Figure 10: Effect of a quinolinic acid lesion on Urocortin 1 positive neurons in the

Edinger-Westphal nucleus in ICR mice

Mice were infused with vehicle or 6 nmol of quinolinic acid targeted at the Edinger-Westphal nucleus, and matched tissue sections were tested for the presence of urocortin 1 positive neurons using an immunocytochemistry assay with a urocortin 1 antibody raised in guinea pig. The section on the left is an example of a vehicle-treated mouse where urocortin 1 neurons are clearly labeled in the EW; the section on the right is an example of a lesioned mouse that does not show urocortin 1 expression.

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Figure 11: A quinolinic acid lesion of the Edinger-Westphal nucleus reduces the anxiogenic-like effects of septal urocortin 2, but does not alter anxiety-like behavior under baseline conditions in the absence of stress.

Effect of a quinolinic acid lesion of the Edinger-Westphal nucleus on the anxiogenic-like effect of septal urocortin 2 (100 ng) and restraint stress on the a) latency to enter the light side of light-dark box (seconds), b) total time spent in light side of light-dark box (seconds), c) locomotion in open field (total square entries), d) percent time in center of open field, e) locomotion in open field with novel object (total square entries), and f) percent time spent in center with novel object. Septal urocortin 2 increased anxiety-like behavior in the light-dark box (Fig 11 a,b) and novel object test (Fig 11f) in the stress condition in sham but not lesioned mice. The peptide did reduce the time spent in the center of the open field in both sham and lesioned mice subjected to restraint stress (Fig 11d). There was no significant effect of the lesion itself on anxiety-like behavior.

110

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Benoit, S.C., Thiele, T.E., Heinrichs, S.C., Rushing, P.A., Blake, K.A., Steeley, R.J. (2000). “Comparison of central administration of corticotropin-releasing hormone and urocortin on food intake, conditioned taste aversion, and c-Fos expression Peptides.” 21(3):345-51.

Bittencourt, J.C., Vaughan, J., Arias, C., Rissman, R.A., Vale, W.W., Sawchenko, P.E. (1999). “Urocortin expression in rat brain: evidence against a pervasive relationship of urocortin-containing projections with targets bearing type 2 CRF receptors.” J Comp Neurol. 415(3), 285-312.

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Dulawa, S.C., Grandy, D.K., Low, M.J., Paulus, M.P., Geyer, M.A. (1999). “Dopamine D4 receptor-knock-out mice exhibit reduced exploration of novel stimuli.” J. Neurosci. 19, 9550-9556 (1999).

Dunn, A.J, Swiergiel, A.H. (1999). “Behavioral responses to stress are intact in CRF- deficient mice.” Brain Res. 845(1):14-20.

Gamlin, P.D., Zhang, Y., Clendaniel, R.A., Mays, L.E. (1994). “Behavior of identified Edinger-Westphal neurons during ocular accommodation.” J Neurophysiol. 72(5):2368-82.

Gaszner, B., Csernus, V., Kozicz, T. (2004). “Urocortinergic neurons respond in a differentiated manner to various acute stressors in the Edinger-Westphal nucleus in the rat.” J Comp Neurol. 480(2):170-9.

Jones, D.N., R. Kortekaas, Slade, P.D., Middlemiss, D.N., Hagan, J.J. (1998). "The behavioural effects of corticotropin-releasing factor-related peptides in rats." Psychopharmacology (Berl) 138(2): 124-32.

Klooster, J., Vrensen, G.F. (1998). “New indirect pathways subserving the pupillary light reflex: projections of the accessory oculomotor nuclei and the periaqueductal gray to the Edinger-Westphal nucleus and the thoracic spinal cord in rats.” Anat Embryol (Berl). 198(2):123-32.

Korosi, A., Schotanus, S., Olivier, B., Roubos, E.W., Kozicz, T. (2005). “Chronic ether stress-induced response of urocortin 1 neurons in the Edinger-Westphal nucleus in the mouse.” Brain Res. 1046(1-2):172-9.

Loewy, A.D., Saper, C.B., Yamodis, N.D. (1978). “Re-evaluation of the efferent projections of the Edinger-Westphal nucleus in the cat.” Brain Res.141(1):153-9.

Moreau, J.L., Kilpatrick, G., Jenck, F. (1997). Urocortin, a novel neuropeptide with anxiogenic-like properties. Neuroreport. 8(7):1697-701.

Ryabinin, A.E., Tsivkovskaia, N.O., Ryabinin, S.A. (2005).”Urocortin 1-containing neurons in the human Edinger-Westphal nucleus.” Neuroscience.134(4):1317-23.

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Spina, M.G., E. Merlo-Pich, Akwa, Y., Balducci, C., Basso, A.M., Zorrilla, E.P., Britton, K.T., Rivier, J., Vale, W.W., Koob, G.F. (2002). "Time-dependent induction of anxiogenic-like effects after central infusion of urocortin or corticotropin-releasing factor in the rat." Psychopharmacology (Berl) 160(2): 113-21.

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Turek, V.F., Ryabinin, A.E. (2005). “Expression of c-Fos in the mouse Edinger- Westphal nucleus following ethanol administration is not secondary to hypothermia or stress.” Brain Res. 1063(2):132-9.

Vetter, D.E., Li, C., Zhao, L., Contarino, A., Liberman, M.C., Smith, G.W., Marchuk, Y., Koob, G.F., Heinemann, S.F., Vale, W., Lee, K.F. (2002). “Urocortin-deficient mice show hearing impairment and increased anxiety-like behavior.” Nat Genet.31(4):363-9.

Viau, V., Sawchenko, P.E. (2002). “Hypophysiotropic neurons of the paraventricular nucleus respond in spatially, temporally, and phenotypically differentiated manners to acute vs. repeated restraint stress: rapid publication.” J Comp Neurol. 445(4):293-307.

Wang, X., Su, H., Copenhagen, L.D., Vaishnav, S., Pieri, F., Shope, C.D., Brownell, W.E., De Biasi, M., Paylor, R., Bradley, A.(2002). “Urocortin-deficient mice display normal stress-induced anxiety behavior and autonomic control but an impaired acoustic startle response.” Mol Cell Biol. 22(18):6605-10.

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114 corticotropin-releasing hormone (CRH) receptor, but not CRH.” Proc Natl Acad Sci U S A. 96(14):8283-8.

Weninger, S.C., Peters, L.L., Majzoub, J.A.Endocrinology. (2000). “Urocortin expression in the Edinger-Westphal nucleus is up-regulated by stress and corticotropin-releasing hormone deficiency.” 141(1):256-63.

Chapter 6:

Summary and Conclusions

The role of the CRF 2 receptor in anxiety-like behavior has been a subject of controversy for the past few years. CRF 2 receptor knockout mice exhibit greater anxiety-like behavior in the elevated plus maze and the light-dark box compared to wildtype mice (Bale et al., 2000; Kishimoto et al., 2000), or show no difference in anxiety-like measures (Coste et al., 2000). Selective CRF 2 receptor agonists (urocortin

2 and 3) decreased anxiety-like behavior in the elevated plus maze in rats (Valdez et al., 2002, 2003), and reduced anxiety-like behavior in the open field and light-dark box in C57BL/6 mice (Venihaki et al., 2004). However, these CRF 2 receptor agonists increased anxiety-like behavior in Balb/c mice (Pelleymounter et al., 2002, 2004) and acoustic startle in C57BL/6 mice (Risbrough et al., 2003, 2004). Further, antagonism of septal CRF 2 receptors has also been reported to decrease anxiety-like behavior

(Bakshi et al., 2002; Radulovic, 1999). Thus, the current literature suggests that CRF 2 receptor activation may mediate both anxiolytic and anxiogenic effects depending on the stress level of the organism, the degree of receptor activation, and brain location.

The purpose of this dissertation project was to examine the hypothesis that the effect of CRF 2 receptor activation on anxiety-like behavior depends on the stress level of the

115 116 animal. In the initial experiments, as described in Part A of Chapter 3, mice were administered the CRF 2 receptor selective agonist urocortin 2 in the lateral septum and tested for anxiety-like behavior in the light dark box, open field, and novel object test under baseline conditions, without manipulating the level of stress in the environment.

These data from these studies showed that 500 ng of urocortin 2 infused into the mouse septum increased anxiety-like behavior in the light-dark box and novel object test, with a strong trend towards anxiety-like behavior in the open field test.

There was no consistent effect on anxiety-like behavior with the lower doses of the peptide. 100 ng of urocortin 2 decreased transitions in the light-dark box, but did not alter the latency to enter the light or the time spent in the light side and had no effect on anxiety-like behavior in the open field or novel object test. 10 ng of the peptide had no effect on anxiety-like behavior in the light-dark box or open field, but did reduce time in the center with the novel object. The lowest dose tested, 1 ng, had no effect in any test. Urocortin 2 in this dose range (1 – 500 ng) did not alter locomotion in either the open field or novel object test, indicating that effects on anxiety-like behavior were not confounded by changes in motor activity.

To determine if the anxiogenic effect of urocortin 2 infusion in the septum could be attributed to spread of the peptide into the medial septum or into the lateral ventricle, the peptide was administered directly into both areas. Urocortin 2 infusion into the medial septum had no effect on behavior in the light-dark box, open field, or novel object test. Urocortin 2 injection into the lateral ventricle significantly decreased overall locomotion in the open field and the novel object test at the highest dose tested

(5 µg), but did not have any effect on measures of anxiety-like behavior or locomotion

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at lower doses.These results support the hypothesis that CRF 2 receptors within the septum mediated the increased anxiety-like behaviors observed in the current experiment.

In part B of Chapter 3, I examined the consequence of environmental stress on the behavioral effect of septal CRF 2 receptor activation. To determine if the function of the CRF 2 receptor may depend on the stress level of the animal during behavioral evaluation, mice were subsequently tested under low and high stress conditions. In the low stress condition, mice were handled each day for one week before the test and habituated to the test room before drug administration and behavioral testing. In the high stress condition, mice were restrained for 30 minutes after infusion of the peptide, a stressor that increases anxiety-like behavior.

In the first experiment in this section, 100 ng of urocortin 2 was infused into the lateral septum of ICR mice alone or in combination with 30 minutes of restraint stress. This dose was chosen in the current and subsequent experiments to determine if stress would interact with a dose of the CRF 2 receptor agonist that was inactive in the low stress condition. The results showed that 100 ng of septal urocortin 2 did not have a significant effect on anxiety-like behavior or locomotion by itself, but significantly increased anxiety-like behavior in the light-dark box, open field and novel object test compared to vehicle when combined with restraint stress. In this experiment, restraint stress decreased total locomotion in open field and novel object test, but there was no difference in locomotor activity between peptide- and vehicle-treated mice in the high stress condition. These results indicated that the effect of septal urocortin 2 on anxiety- like behavior is influenced by the stress level of the mouse.

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This effect was subsequently replicated in mice on a mixed C57BL/6 x 129 background. These mice were treated with 1, 10 and 100 ng of urocortin 2 infused into the septum with and without restraint stress. During the high stress condition, 100 ng of septal urocortin 2 significantly increased anxiety-like behavior compared to vehicle in the light-dark box and novel object test, but had no effect in the low stress condition. The 10 ng dose significantly decreased time in the center with the novel object in the high stress condition, but did not differ from vehicle during in the low stress condition, and there was no effect of the 1 ng dose under any condition.

The final experiment in this chapter examined the effect of septal urocortin 2 on HPA axis activation. Glucocorticoids have been demonstrated to induce anxiety- like behavior associated with stressors in the environment, increasing freezing in response to footshock (Thompson et. al, 2004), increasing acoustic startle (Lee et al.,

1994), and increasing anxiety-like behavior in the light-dark box in mice (Ardayfio et al., 2006). To determine if septal urocortin 2 could be increasing anxiety-like behavior by activating the HPA axis, we assessed the levels of blood plasma corticosterone after administration of the peptide. The results showed that septal urocortin 2 (100 ng and 500 ng) did not have any effect on plasma corticosterone in the mouse. These data would suggest that the anxiogenic effects of urocortin 2 in the septum were not mediated by peripheral release of glucocortiocoids, but were induced by the action of the CRF 2 receptor agonist within the brain.

While the above studies demonstrated anxiogenic effects of urocortin 2 infused into the septum, the receptor that mediates these changes in anxiety-like behavior had not been clearly defined. The purpose of the experiments run in Chapter 4 was to

119 determine if the anxiogenic effect of septal urocortin 2 is dependent on activation of the CRF 2 receptor and/or the activation of the CRF 1 receptor by testing the peptide in

CRF 2 receptor KO mice and assessing the effect of selective CRF receptor antagonists.

Septal administration of urocortin 2 had no effect in CRF 2 receptor KO mice in the light-dark box, but increased anxiety-like behavior in the light-dark box, open field and novel object test compared to vehicle in wildtype mice in the high stress condition. The data also revealed that vehicle-treated CRF 2 receptor knockout mice exhibited more anxiety-like behavior compared to wildtype, spending less time in the light side of the light-dark box and the center of the open field, with a strong trend towards less time spent in the center region with the novel object. The KO mice in the stress condition spent very little time in the center of both the open field and novel object test (< 6% of the time) regardless of drug treatment, so it is difficult to determine if urocortin 2 had any effect in these behavioral assays. It is possible that the anxiety-like response reached a floor effect under these conditions. The results from this experiment provide support for the argument that the effect of urocortin 2 on anxiety-like behavior is mediated through the CRF 2 receptor, but also report an apparent contradiction in that mice lacking the CRF2 receptor showed higher levels of anxiety-like behavior overall. It is relevant to note that the CRF 2 receptor KO mice are reported to show increased CRF mRNA in the central amygdala as well as an increased number of urocortin 1 mRNA-positive cells in the Edinger-Westphal nucleus (Bale et al., 2000), suggesting that increased anxiety-like behavior in these mice could be due to the developmental overexpression of these peptides.

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Septal administration of the selective CRF 2 antagonist astressin-2B did not have any effect on anxiety-like behavior in the no stress condition, but lower doses (50 and 200 ng) decreased the anxiogenic-like effect of urocortin 2 in the high stress condition and at the highest dose (400 ng) appeared to also block the anxiogenic-like effect of restraint stress prior to infusion of urocortin 2 into the septum. In contrast, infusion of the CRF 1 selective antagonist antalarmin had no effect on septal urocortin

2-induced anxiety-like behavior, but decreased restraint-induced anxiety-like behavior when infused into the central amygdala. These findings provide additional support for the argument that the anxiogenic-like effect of septal urocortin 2 is mediated by activation of the CRF 2 receptor.

Several experiments in this dissertation have demonstrated that a low dose of septal urocortin 2 has little effect on anxiety-like behavior when administered alone, but increases anxiety-like behavior in combination with restraint stress; however, the mechanism mediating the synergistic or additive effects of the peptide treatment and the environmental stressor had not been identified. The Edinger-Westphal nucleus sends a strong urocortin 1 projection to the lateral septum and is activated during exposure to several types of stressors, suggesting that the EW nucleus could modulate the effect of stress on CRF 2 receptor activity in the septum.

The study in Chapter 5 tested the hypothesis that the anxiogenic effect of urocortin 2 during stress is dependent on urocortin 1 input from the EW. In this experiment, mice were infused with quinolinic acid (1 µg) or vehicle (sham treatment) into the EW and subsequently prepared with bilateral cannula in the lateral septum.

The results indicated that treatment with septal urocortin 2 (100 ng) increased anxiety-

121 like behavior in sham but not EW-lesioned mice in the stress condition in both the light-dark box and novel object test, but the lesion of the EW nucleus did not affect baseline anxiety-like behavior in the no stress condition. These results indicate that the

EW may have a limited effect on anxiety-like behavior during low stress conditions

(as reported by Ryabinin et al., 2005), but supported the hypothesis that anxiogenic- like effect of septal urocortin 2 during stress could be dependent upon urocortin 1 input from the EW nucleus. In the no stress condition, infusion of a low dose of septal urocortin 2 may have no effect on anxiety-like behavior. However, in the presence of a stressor such as restraint stress, increased urocortin release in the lateral septum may combine with the administered CRF 2 receptor agonist to produce a significant increase in anxiety-like behavior.

There is some indication that the mice showed increased sensitivity to the anxiogenic effects of septal urocortin 2 during the novel object test. The results showed that a low dose of urocortin 2 (10 ng) increased anxiety-like behavior in the novel object test by itself (Experiment 1, Chapter 3, Part A) and also in combination with restraint stress (Experiment 2, Chapter 3, Part B), but this dose had no effect on anxiety-like behavior in the light-dark box or open field tests. In all experiments, mice were tested first in the light-dark box, second in the open field, and last in the novel object test. Exposure to a novel environment by itself has been shown to induce a stress response in mice (Hebda-Bauer et al., 2004). It is possible that the mice were more sensitive to the anxiogenic effects of urocortin 2 after 30 minutes of stress induced by successive exposure to three novel situations, resulting in increased anxiety-like behavior in the novel object test. Another possibility is that urocortin 2

122 may have time-dependent effects on behavior. Previous studies have reported delayed effects of CRF 2 receptor agonists on anxiety-like behavior (Valdez et al., 2002) and feeding (Zorrilla et al., 2004), and it is conceivable that the effect of septal urocortin 2 on anxiety-like behavior may differ 30 to 60 minutes after administration. Finally, while the light-dark box, open field and novel object tests all measure approach- avoidance behavior, the novel object test elicits the greatest approach or exploration behavior (Dulawa et al., 1999) and may also be the most sensitive measure of the behavioral effects of urocortin 2 during stress.

The current literature suggests that stress may play a role in determining the function of the CRF 2 receptor in anxiety-like behaviors. Valdez and colleagues (2002,

2003) reported that intraventricular treatment with urocortin 2 or 3 decreased anxiety- like behavior in rats previously handled for a week and habituated to the testing environment for two hours before peptide delivery and behavioral testing. Venihaki and colleagues (2004) reported anxiolytic effects of intraventricular urocortin 3 in

C57BL/6 mice habituated to human handling and cannula manipulation. In contrast,

Risbrough and colleagues (2003, 2004) reported that urocortin 2 increased acoustic startle in mice, interpreted as an increase in anxiety-like defensive behavior. This latter effect is measured during a stressful procedure where animals are restricted in movement inside a small chamber and subjected to loud noises. It is also conceivable that “trait” as well as “state” anxiety may affect receptor function; Pelleymounter and colleagues (2002, 2004) report anxiogenic-like effects of urocortin 2 in Balb/c mice, a genotype characterized by high levels of endogenous anxiety-like behavior in behavioral tests that include the open field and light-dark box (Belzung et al., 2001).

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Some data suggest that stress level could influence the function of CRF 2 receptors located specifically in the lateral septum. Ho and colleagues (2001) reported that septal administration of CRF 2 receptor antisense oligonucleotides decreased shock- induced freezing in rats. Bakshi and colleagues (2002) also reported that antagonism of septal CRF 2 receptors decreased freezing behavior in response to shock. Both studies reported an anxiolytic-like effect of blocking septal CRF 2 receptors in animals tested in high stress conditions. However, Radulovic and colleagues (1999) reported that septal administration of the CRF 2 receptor antagonist antisauvagine-30 had no effect on anxiety-like behavior under baseline (low stress) conditions, but decreased anxiety-like behavior induced by administration of CRF into the lateral septum. It is relevant to note that in the experiments presented in this dissertation, septal adminstration of the selective CRF 2 receptor antagonist astressin-2B decreased anxiety-like behavior induced by restraint stress, but had no effect in the low stress condition.

The key to understanding how the CRF 2 receptor affects anxiety may depend on defining the role of the lateral septum in anxiety and stress. The lateral septum receives both urocortin 1 from the Edinger-Westphal nucleus and putative urocortin 3 input from the perifornical region adjacent to the hypothalamus (Bachtell et al., 2004;

Li et al., 2002). It sends afferent projections to many regions of the brain critical for regulating affect, including much of the hypothalamus and the extended amygdala.

The major neurotransmitter expressed throughout the lateral septum is γ – aminobutyric acid (GABA) (Risold et al., 1997), and there is considerable evidence that the septum acts to inhibit areas of the brain that mediate stress and anxiety.

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Lesions of the septum are reported to result in a “septal rage” phenomena in rats that become hyperdefensive and aggressive (Risold et al., 1997). This behavior can be ameliorated by lesioning the anterior hypothalamus/perifornical region, an area that receives dense innervation from the lateral septum. Septal lesions also produce anxiety-like behavior in the open field, an increase in reactivity to footshock, and increased startle, signs of anxiety-like behavior that are decreased when the amygdala is also lesioned (Decker et al., 1995); this pattern of results indicates that the lateral septum may act to inhibit anxiety-like behavior mediated by the amygdala. There are also preliminary findings reporting that electrical stimulation of the rat lateral septum inhibits neurons in the central nucleus of the amygdala (Thomas et al., 2001, 2004).

A detailed analysis of lateral septum anatomy (Risold et al., 1997) reveals considerable intra-septal connections throughout the caudal-rostral, dorsal-ventral, and medial-lateral axes. Many septal neurons extend collaterals to other neurons in the septum as they descend to target other regions of the brain, while the presence of classical interneurons contained within the septum has yet to be confirmed (Phelan et al. (1989), Sheehan et al, 2004). Several papers have reported that electrical stimulation of the fimbria activates an excitatory hippocampal glutamergic projection to the lateral septum, inducing an initial depolarization of septal neurons, followed by a hyperpolarization effect attributed to intraseptal GABAergic release (Stevens et al,

1987; DeFrance et al., 1975; Gallagher et al., 1989). These groups conclude that neurons within the septum can inhibit other septal neurons via the release of GABA from recurrent axon collaterals. Eberly and colleagues (1983) reported that in vitro application of CRF on slices of rat lateral septum inhibited spontaneous firing in about

125 half the neurons tested. A more recent study (Liu et al., 2004), found that the application of urocortin 1 decreased and astressin –2B increased glutamatergic excitatory postsynaptic currents in an in vitro preparation of rat lateral septum, suggesting that activation of the CRF 2 receptor in the septum can inhibit neuronal firing.

There is also evidence that CRF 2 receptor activation may exert different effects in the septum depending on the stress level of the animal. Liu and colleagues (2005) reported that urocortin 1 depressed the activity of septal neurons by activating CRF 2 receptors in the mediolateral nucleus of the lateral septum (intermediate lateral septum), but had the opposite effect following the stress of cocaine withdrawal, facilitating excitatory glutamatergic post-synaptic currents. These results suggest that the stress could actually reverse the effect of CRF2 receptor activation, at least in this region of the brain. Pernar and colleagues (2004) reported a similar phenomenon in the dorsal raphe, observing that low doses of urocortin 2 inhibit serotonergic neurons in the rat dorsal raphe, but higher doses excite these neurons, hypothesizing that such a reversal of the effect of urocortin 2 may be mediated by activation of CRF 2 receptors on GABAergic interneurons.

Some studies suggest that the septal CRF 2 receptor might reduce anxiety-like behavior by inhibiting CRF 1 receptor function. Bale and colleagues (2000) reported increased CRF mRNA in the amygdala of CRF 2 receptor KO mice, a region innervated by inhibitory septal neurons. These results suggest that increased anxiety- like behavior in CRF 2 receptor KO mice may be mediated by overactivation of the

CRF 1 receptor. Bale and Vale (2003) also reported that the CRF 1 receptor antagonist

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antalarmin decreased depression-like behavior in CRF 2 receptor KO mice tested in the

Porsolt swim test. In contrast, both Radulovic and colleagues (1999) and Bakshi and colleagues (2002) reported that antagonism of septal CRF 2 receptors reduces anxiety- like under high stress conditions (restraint stress or shock-induced freezing). Taken as a whole, these findings suggest it is likely that the activation of the septal CRF 2 receptor may have a complex effect on anxiety.

The model suggested here (Figure 12) postulates that CRF 2 receptors in the septum will have variable effects on anxiety-like behavior depending on the level of receptor activation. According to this model, CRF 2 receptors are located on lateral septum neurons that inhibit each other via GABAergic collaterals and/or send inhibitory GABAergic projections towards brain regions that mediate anxiety-like behavior, such as the amygdala. Low CRF 2 receptor activation during mild stress may have little overall effect on septal output, but high activation, such as the one caused by increased urocortin input induced by restraint stress (intense stress) when combined with urocortin 2 administration (or a mild stress), may increase the activity of inhibitory intraseptal GABAergic neurons, decreasing overall activity of GABAergic projections from the septum to other brain areas, and thus disinhibiting the amygdala and increasing anxiety-like behavior. In contrast, the complete absence of any CRF 2 receptor activity on septal neurons that directly inhibit the amygdala (in the CRF 2 receptor knockout mice) may also increase anxiety-like behavior. In other words, both the hyperactivation and the complete removal of the septal CRF 2 receptor could increase anxiety-like behavior compared to a low activation state.

127

In conclusion, the present set of studies demonstrated that the effect of septal

CRF 2 receptor activation on anxiety-like behavior appears dependent on stress level.

In the low stress condition, a high dose of 500 ng of urocortin 2 was required to increase anxiety-like behavior, but lower doses did not have a consistent effect.

However, in combination with restraint stress, 100 ng of septal urocortin 2 increased anxiety-like behavior in the behavioral paradigms used. The anxiogenic effect of the

CRF 2 receptor agonist was blocked by pretreatment with the selective CRF 2 receptor antagonist astressin-2B and by a lesion of the Edinger-Westphal nucleus, indicating that urocortin 1 input to the lateral septum during stress may mediate the anxiogenic effect of septal urocortin 2. The current findings do not resolve the controversy over the reported anxiolytic effects of CRF 2 receptor activation, but suggest that environmental factors influence the effect of receptor activation on behavior. Our data provide strong and converging evidence that septal CRF 2 receptor activation is more likely to increase anxiety-like behavior in a high stress environment, but has less effect on anxiety-like behavior under low stress conditions.

128

Figure 12: A model for the role of the septal CRF 2 receptor in anxiety-related behavior

This model postulates that CRF 2 receptors in the septum will have variable effects on anxiety-like behavior depending on the level of receptor activation. We hypothesize that CRF 2 receptors are located on lateral septum neurons that inhibit each other via GABAergic collaterals and/or send inhibitory GABAergic projections towards brain regions that mediate anxiogenic behavior, such as the amygdala. Low CRF 2 receptor activation in a low stress environment may have little overall effect on septal output; in this condition, the septum effectively inhibits regions that mediate anxiety, such as the amygdala, bed nucleus of the stria terminalis (BNST) and the hypothalmus. In a high stress situation, increased urocortin 1 input from the Edinger-Westphal nucleus (elicited by an intense stress such as restraint) combines with the administered urocortin 2 agonist (or a mild stress) to increase the activity of inhibitory intraseptal GABAergic neurons, decreasing overall GABA release from the septum, and thus disinhibiting regions such as the amygdala, increasing anxiety-like behavior.

129

Low Stress : Low septal CRF 2 receptor activation during mild stress may have little effect on anxiety-like behavior, as the lateral septum continues to inhibit regions that mediate anxiety, such as the amygdala.

Urocortin 2 infusion (or mild stress)

+ Lateral Septum +

R2 R2 GABA

GABA - Amygdala/ BNST/ Hypothalamus

130

Figure 12: A model for the role of the septal CRF 2 receptor in anxiety-related behavior, Continued

This model postulates that CRF 2 receptors in the septum will have variable effects on anxiety-like behavior depending on the level of receptor activation. We hypothesize that CRF 2 receptors are located on lateral septum neurons that inhibit each other via GABAergic collaterals and/or send inhibitory GABAergic projections towards brain regions that mediate anxiogenic behavior, such as the amygdala. Low CRF 2 receptor activation in a low stress environment may have little overall effect on septal output; in this condition, the septum effectively inhibits regions that mediate anxiety, such as the amygdala, bed nucleus of the stria terminalis (BNST) and the hypothalmus. In a high stress situation, increased urocortin 1 input from the Edinger-Westphal nucleus (elicited by an intense stress such as restraint) combines with the administered urocortin 2 agonist (or a mild stress) to increase the activity of inhibitory intraseptal GABAergic neurons, decreasing overall GABA release from the septum, and thus disinhibiting regions such as the amygdala, increasing anxiety-like behavior.

131

High Stress : Intense stress (restraint) increases urocortin 1 input to the lateral septum; the combination of two stressors induces high septal CRF 2 receptor activation that stimulates inhibitory intraseptal GABAergic neurons, thus reducing GABAergic output from the septum and disinhibiting regions that mediate anxiety, such as the amygdala.

Urocortin 2 infusion Edinger -Westphal (or mild stress) releases Urocortin 1 (Intense stress)

+ +

+ Lateral Septum R2 R2 GABA

Septal GABA output decreases

Amygdala/ BNST/ Hypothalamus

132

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