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Role of the Nociceptin/Orphanin FQ-Nop Receptor System in Stress Responses

Permanent Link: http://ufdc.ufl.edu/UFE0021696/00001

Material Information

Title: Role of the Nociceptin/Orphanin FQ-Nop Receptor System in Stress Responses
Physical Description: 1 online resource (114 p.)
Language: english
Creator: Green, Megan Kay
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: anxiety, defeat, hpa, nociceptin, nofq, nop, orphanin, social, stress
Psychology -- Dissertations, Academic -- UF
Genre: Psychology thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Intracerebroventricular (ICV) microinjections of the neuropeptide nociceptin/orphanin FQ (N/OFQ) produce elevations in hypothalamic-pituitary-adrenal (HPA) axis activity and anxiety-related behaviors. Injections into the amygdala also elevate anxiety-related behaviors, although to a lesser extent than do ICV injections. Therefore, the effects of N/OFQ were examined following injections into another limbic structure, the bed nucleus of stria terminalis (BNST). Injections into the BNST produced elevated anxiety-related behaviors and circulating corticosterone. These results suggest that N/OFQ is involved in regulation of affective behaviors and the HPA axis through limbic actions. The potent effects of N/OFQ following ICV injections may involve additive or synergistic activity at multiple limbic sites. To verify diffusion patterns of N/OFQ following these injections, rats were injected with 3H-N/OFQ into either the lateral ventricle, amygdala, or BNST. Following intraparenchymal injections, diffusion of N/OFQ was localized to the target structures. Following the ICV injections, N/OFQ diffused throughout the ventricles and into periventricular structures. A number of limbic, hypothalamic, and brainstem structures were identified that could contribute to the effects observed following ICV injections. The possibility that N/OFQ and NOP receptor gene expression and receptor binding are modified by exposure to acute and repeated social stress was also explored. Rats were exposed to social stress (acute, repeated, repeated plus acute, or no stress) and then their brains were analyzed for mRNA expression and receptor binding. There were no statistically significant differences in prepro-N/OFQ mRNA expression or in receptor binding in any of the groups. However, the rats exposed to acute stress displayed greater NOP receptor mRNA expression in the septum, BNST, amygdala, and PVN, as compared to the expression in the unstressed controls. The rats exposed to repeated stress displayed greater NOP receptor mRNA expression in the BNST and PVN, and those exposed to repeated plus acute stress displayed greater expression in the amygdala and PVN. Overall, it appears that the N/OFQ-NOP receptor system is involved in the regulation of affective states through actions in limbic structures. Additionally, this may be a dynamic system that can be modified by acute and chronic social stress exposure, especially in limbic regions and the PVN.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Megan Kay Green.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Devine, Darragh P.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021696:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021696/00001

Material Information

Title: Role of the Nociceptin/Orphanin FQ-Nop Receptor System in Stress Responses
Physical Description: 1 online resource (114 p.)
Language: english
Creator: Green, Megan Kay
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: anxiety, defeat, hpa, nociceptin, nofq, nop, orphanin, social, stress
Psychology -- Dissertations, Academic -- UF
Genre: Psychology thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Intracerebroventricular (ICV) microinjections of the neuropeptide nociceptin/orphanin FQ (N/OFQ) produce elevations in hypothalamic-pituitary-adrenal (HPA) axis activity and anxiety-related behaviors. Injections into the amygdala also elevate anxiety-related behaviors, although to a lesser extent than do ICV injections. Therefore, the effects of N/OFQ were examined following injections into another limbic structure, the bed nucleus of stria terminalis (BNST). Injections into the BNST produced elevated anxiety-related behaviors and circulating corticosterone. These results suggest that N/OFQ is involved in regulation of affective behaviors and the HPA axis through limbic actions. The potent effects of N/OFQ following ICV injections may involve additive or synergistic activity at multiple limbic sites. To verify diffusion patterns of N/OFQ following these injections, rats were injected with 3H-N/OFQ into either the lateral ventricle, amygdala, or BNST. Following intraparenchymal injections, diffusion of N/OFQ was localized to the target structures. Following the ICV injections, N/OFQ diffused throughout the ventricles and into periventricular structures. A number of limbic, hypothalamic, and brainstem structures were identified that could contribute to the effects observed following ICV injections. The possibility that N/OFQ and NOP receptor gene expression and receptor binding are modified by exposure to acute and repeated social stress was also explored. Rats were exposed to social stress (acute, repeated, repeated plus acute, or no stress) and then their brains were analyzed for mRNA expression and receptor binding. There were no statistically significant differences in prepro-N/OFQ mRNA expression or in receptor binding in any of the groups. However, the rats exposed to acute stress displayed greater NOP receptor mRNA expression in the septum, BNST, amygdala, and PVN, as compared to the expression in the unstressed controls. The rats exposed to repeated stress displayed greater NOP receptor mRNA expression in the BNST and PVN, and those exposed to repeated plus acute stress displayed greater expression in the amygdala and PVN. Overall, it appears that the N/OFQ-NOP receptor system is involved in the regulation of affective states through actions in limbic structures. Additionally, this may be a dynamic system that can be modified by acute and chronic social stress exposure, especially in limbic regions and the PVN.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Megan Kay Green.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Devine, Darragh P.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021696:00001


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ROLE OF THE NOCICEPTIN/ORPHANIN FQ-NOP RECEPTOR SYSTEM INT STRESS
RESPONSES




















By

MEGAN K. GREEN


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2007


































O 2007 Megan K. Green
































To Susan L. Menet.
She left this world on November 5, 2007, but she will never leave our hearts.









ACKNOWLEDGMENTS

I would like to thank my advisor, Dr. Darragh Devine, for all of his guidance,

commitment, hard work, and long hours. I would also like to thank all of my committee

members, Drs. Margaret Bradley, Mohamed Kabbaj, Henrietta Logan, John Petitto, and Ken

Rice, for their time, effort, and input. I thank Brandon Brown and Emily Barbieri for their

contributions to the work presented herein, and Amber Muehlmann and Nate Weinstock for their

assistance with day-to-day tasks. Finally, I would like to thank Michael Menet for his

unwavering support and my mother for all that she has done to make my life successes possible.












TABLE OF CONTENTS


page

ACKNOWLEDGMENT S .............. ...............4.....


LI ST OF T ABLE S ............_...... ...............7....


LIST OF FIGURES .............. ...............8.....


AB S TRAC T ............._. .......... ..............._ 1 1..


CHAPTER


1 INTRODUCTION ................. ...............13.......... ......


Stress and Stress Response ................ ...............13................
Stress............... .. .... ...... .. .........1
HPA Axis Activation Following Stress Exposure. ............_..... ................14
Anatomy of the Limbic-HPA Axi s ................ ...............15...............
Autonomic Nervous System ................. ...............17........... ....
Chronic Stress and Disease ................. ...............17........... ...
Animal Models of Chronic Stress............... .................19
Chronic Variable Stress ................. ...............19........... ....
Other Chronic Stress Regimens............... ...............20
Social Defeat .............. ...............20....
Nociceptin/Orphanin FQ .............. ...............22....


2 ANATOMICAL ANALYSIS OF ANXIOGENIC BEHAVIORAL EFFECTS OF
EXOGENOUS NOCICEPTIN/ORPHANIN FQ .............. ...............26....


Back ground ............. ...... ._ ...............26...
M ethod s .............. ...............27....
Animal s............... ...............27

D rugs .............. ...............28....
Surgery .............. ...............28....
Equipm ent............... ..... ............2
Anxiety-Testing Procedure ............_...... ...............29....
Statistical Analyses............... ...............31
Re sults....................... ...__ ...............3 1..
Anxiety-Related Behavior ............. ...... .__ ............... 1...
Circulating Corticosterone............... .............3
Organ M asses .............. ...............32....
Discussion ............... .. ... ..__ .. .......__ .. ...........3
Anxiety-Related Behaviors and Corticosterone .............. ...............33....
Organ M asses .............. ...............36....
Summary ................. ...............36.................












3 DIFFUSION OF NOCICEPTIN/ORPHANIN FQ AFTER
INTRACEREBROVENTRICULAR AND INTRAPARENCHYMAL INJECTIONS ........46


Back ground ............. ...... ._ ...............46....
M ethod s .............. ...............46....
Animals............... ...............46

D rugs .............. ...............46....
Surgery .............. ...............47....
Inj section Procedure ........._..... .....___ ...............47...
Re sults............... .. .._ .. ... .............4
Intracerebroventricular Injections .............. ...............48....
Intra-Amy gdaloid Inj sections ........._.._.. ...._... ...............49....
Intra-BNST Inj sections ........._.._.. ...._... ...............50....
Discussion ........._..... ...._... ...............51.....


4 NOCICEPTIN/ORPHANIN FQ AND NOP RECEPTOR GENE REGULATION AND
NOCICEPTIN/ORPHANIN FQ BINDING TO THE NOP RECEPTOR AFTER
SINGLE OR REPEATED SOCIAL DEFEAT EXPOSURE............... ...............57


Back ground ................. ...............57.......... ......
M ethods .............. ...............58....
Animals............... ...............58

D rugs .............. ...............59....
Surgery .............. ..... ...............59.
Social Defeat Procedure .............. ...............59....
In Situ Hybridization .............. ...............61....
Autoradiography ................. ...............63.......... ......
Densitometry and Statistics .............. ...............64....
Re sults ................. ...............64.................
Social Defeats ................. ...............64.................
In Situ Hybridization .............. ...............65....
Prepro-N/OFQ ............ _...... ...............66....
NOP Receptor .............. ...............66....
Autoradiography ................. ...............67.................
Organ M asses .............. ...............67....
Discussion ................. .............. ...............67.......

Prepro-N/OFQ mRNA............... ...............68..
NOP Receptor mRNA ................ ...............69........... ....
NOP Receptor Binding ................. ...............70........... ....
Organ M asses .............. ...............71....

5 GENERAL DI SCUS SSION ................. ...............99.......... ....


LIST OF REFERENCES ................. ...............101................


BIOGRAPHICAL SKETCH ................. ...............114......... ......










LIST OF TABLES


Table


page


4-1 Social defeat regimen. ................. ...............73..__._. ....










LIST OF FIGURES


Figure page

2-1 Anxiety-related behaviors following intra-amygdaloid inj sections of N/OFQ. ........._......38

2-2 Anxiety-related behaviors following ICV inj sections of N/OFQ ........._.. .........._........3 9

2-3 Concentrations of circulating corticosterone following ICV and intra-amygdaloid
injections of N/OF Q............... ...............40

2-4 Photograph of the open field and a diagram of the zones used for scoring.............._.._.. ...41

2-5 Anxiety-related behaviors following intra-BNST inj sections of N/OFQ.............._._..........42

2-6 Anatomical map of BNST placements. ............. ...............43.....

2-7 Concentrations of circulating corticosterone following intra-BNST inj sections of
N/OF Q........._ ...... .___ ...............44....

2-8 Analysis of glandular masses ........._. ...... .___ ...............26...

3-1 Representative x-ray images of 3H-N/OFQ diffusion following ICV injections
overlaid on the corresponding cresyl violet stained section (A, C, and E) and x-ray
images alone (B, D, and F). ............. ...............54.....

3-2 Representative x-ray images of 3H-N/OFQ diffusion following intra-amygdaloid
inj sections overlaid on the corresponding cresyl violet stained section.............._.._.. ..........5 5

3-3 Representative x-ray images of 3H-N/OFQ diffusion following intra-BNST
inj sections overlaid on the corresponding cresyl violet stained section (A, C, and E)
and x-ray images alone (B, D, and F). ............. ...............56.....

4-1 Photograph of a social defeat interaction ................. ...............72........... ..

4-2 Photograph of stage 2 of the social defeat procedure. ................ .......... ................72

4-3 Prepro-N/OFQ sequence. .............. ...............73....

4-4 NOP receptor sequence. ........... ..... ._ ...............74..

4-5 Number of social defeats per group per day. ............. ...............75.....

4-6 Total amount of time the rats were defeated per day. ......____ .... ... ._ ...............75

4-7 Representative x-ray images of brain sections treated with sense riboprobes. ................. .76

4-8 Prepro-N/OFQ mRNA expression in the septum.. ............ ...............77.....










4-9 Prepro-N/OFQ mRNA expression in the bed nucleus of stria terminalis. ................... ......78

4-10 Representative x-ray images of brain sections showing prepro-N/OFQ mRNA
expression in the septum and BNST. .............. ...............79....

4-11 Prepro-N/OFQ mRNA expression in the amygdala. ............. ...... ............... 8

4-12 Prepro-N/OFQ mRNA expression in the zona incerta and reticular nucleus of the
thal am us .. ............... ...............8.. 1..............

4-13 Representative x-ray images of brain sections showing prepro-N/OFQ mRNA
expression in the amygdala, zona incerta, and reticular nucleus of the thalamus.. ...........8

4-14 Prepro-N/OFQ mRNA expression in the dorsal raphe.. ............ .....................8

4-15 Representative x-ray images of brain sections showing prepro-N/OFQ mRNA
expression in the dorsal raphe ................. ...............83........... ...

4-16 NOP rector mRNA expression in the septum. .............. ...............84....

4-17 Representative x-ray images of brain sections showing NOP receptor mRNA
expression in the septum. .............. ...............85....

4-18 NOP receptor mRNA expression in the bed nucleus of stria terminalis............................86

4-19 Representative x-ray images of brain sections showing NOP receptor mRNA
expression in the bed nucleus of stria terminalis. ............. ...............87.....

4-20 NOP receptor mRNA expression in the amygdala. ............. ...............88.....

4-21 Representative x-ray images of brain sections showing NOP receptor mRNA
expression in the amygdala and the paraventricular nucleus of the hypothalamus. ..........89

4-22 NOP receptor mRNA expression in the paraventricular and ventromedial nuclei of
the hypothalamus and in the zona incerta of the thalamus. ................ ........_.._.........90

4-23 Representative x-ray images of brain sections treated with 1 pLM N/OFQ plus
1251 [14Tyr] -N/OF Q.. ............... ...............91.......... ...

4-24 125 _14Tyr]-N/OFQ binding to the NOP receptor in the septum............... .................9

4-25 Representative x-ray images of brain sections showing 125 _14Tyr]-N/OFQ binding
to the NOP receptor in the septum ................. ...............93........... ..

4-26 125 _14Tyr]-N/OFQ binding to the NOP receptor in the ventromedial BNST. .................. 94

4-27 Representative x-ray images of brain sections showing 125 _14Tyr]-N/OFQ binding
to the NOP receptor in the BNST. ............. ...............95.....










4-28 125 _14Tyr]-N/OFQ binding to the NOP receptor in the amygdala............... ................9

4-29 125 _14Tyr]-N/OFQ binding to the NOP receptor in the PVN. ............. .....................9

4-30 Representative x-ray images of brain sections showing 125 _14Tyr]-N/OFQ binding
to the NOP receptor in the amygdala and the PVN. ............. ...............97.....

4-31 Gland masses after social defeat ......... ........ ......... ................ ...............98









Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

ROLE OF THE NOCICEPTIN/ORPHANIN FQ-NOP RECEPTOR SYSTEM INT STRESS
RESPONSES

By

Megan K. Green

December 2007

Chair: Darragh P. Devine
Major: Psychology

Intracerebroventricular (ICV) microinj sections of the neuropeptide nociceptin/orphanin FQ

(N/OFQ) produce elevations in hypothalamic-pituitary-adrenal (HPA) axis activity and anxiety-

related behaviors. Injections into the amygdala also elevate anxiety-related behaviors, although

to a lesser extent than do ICV inj sections. Therefore, the effects of N/OFQ were examined

following inj sections into another limbic structure, the bed nucleus of stria terminalis (BNST).

Inj sections into the BNST produced elevated anxiety-related behaviors and circulating

corticosterone. These results suggest that N/OFQ is involved in regulation of affective behaviors

and the HPA axis through limbic actions. The potent effects of N/OFQ following ICV inj sections

may involve additive or synergistic activity at multiple limbic sites.

To verify diffusion patterns of N/OFQ following these injections, rats were inj ected with

3H-N/OFQ into either the lateral ventricle, amygdala, or BNST. Following intraparenchymal

injections, diffusion of N/OFQ was localized to the target structures. Following the ICV

injections, N/OFQ diffused throughout the ventricles and into periventricular structures. A

number oflimbic, hypothalamic, and brainstem structures were identified that could contribute to

the effects observed following ICV inj sections.










The possibility that N/OFQ and NOP receptor gene expression and receptor binding are

modified by exposure to acute and repeated social stress was also explored. Rats were exposed

to social stress (acute, repeated, repeated plus acute, or no stress) and then their brains were

analyzed for mRNA expression and receptor binding. There were no statistically significant

differences in prepro-N/OFQ mRNA expression or in receptor binding in any of the groups.

However, the rats exposed to acute stress displayed greater NOP receptor mRNA expression in

the septum, BNST, amygdala, and PVN, as compared to the expression in the unstressed

controls. The rats exposed to repeated stress displayed greater NOP receptor mRNA expression

in the BNST and PVN, and those exposed to repeated plus acute stress displayed greater

expression in the amygdala and PVN.

Overall, it appears that the N/OFQ-NOP receptor system is involved in the regulation of

affective states through actions in limbic structures. Additionally, this may be a dynamic system

that can be modified by acute and chronic social stress exposure, especially in limbic regions and

the PVN.









CHAPTER 1
INTTRODUCTION

Stress and Stress Response

Stress

Stress is defined as a physiological response to a change or threat of change, real or

perceived, in an organism's environment (Herman and Cullinan, 1997). The physiological

responses include activation of systems involved in preparation for high-energy behavioral

responses and the subsequent return to homeostatic balance, such as activation of the autonomic

nervous system and the hypothalamic-pituitary-adrenal (HPA) axis. The stimuli that initiate

these organismic responses are referred to as stressors and can be classified as systemic or

processive. Systemic stressors involve direct threats to tissues and organ systems, including

events such as cold exposure or food deprivation (Herman and Cullinan, 1997). These systemic

stressors appear to be processed primarily by brainstem inputs to the hypothalamus, and may not

require processing by higher order limbic and cortical systems. On the other hand, processive

stressors, or "emotional stressors", do seem to require higher order processing involving cortical

and limbic inputs to the hypothalamus. These types of stressors do not necessarily represent

immediate threats to tissues, but derive their qualitative value relative to the organism's previous

experience (Herman and Cullinan, 1997).

There is anatomical evidence for at least some differentiation between these two stressor

classifications. For example, ether exposure, a systemic stressor, and novel open Hield exposure,

a processive stressor, produce different patterns of c-fos activation, with open Hield exposure

producing greater c-fos activation in the lateral septum, medial amygdala, and dorsomedial

hypothalamus (Emmert and Herman, 1999). Additionally, lesions of the medial prefrontal cortex

(MPFC; Diorio et al., 1993) and of the amygdala (Feldman et al., 1994) alter stress responses to










processive stressors, such as restraint, photic, or acoustic stimuli, but not to ether. Rats with

MPFC lesions display prolonged elevations in adrenocorticotropic hormone (ACTH) and

corticosterone (CORT) following restraint, but not following ether (Diorio et al., 1993). Rats

with central and medial amygdaloid lesions display attenuated corticotrophin releasing hormone

(CRH), ACTH, and CORT responses to photic and acoustic stimuli but not to ether (Feldman et

al., 1994). These studies provide evidence that limbic structures respond strongly to processive

stressors and less so to systemic stressors.

HPA Axis Activation Following Stress Exposure

The HPA axis is one important system involved in the processing of stressor-related

information and in controlling physiological responses and emotionally-relevant behaviors.

Cortical and limbic structures, as well as brainstem structures, have converging inputs at the

paraventricular nucleus of the hypothalamus (PVN) where they are involved in regulation of the

HPA axis (Herman et al., 1989). Additionally, there are inputs from cortical, limbic, and

brainstem regions to the local surround, the peri-PVN.

The PVN is comprised of subdivisions that can be differentiated in terms of their

cytoarchitecture, neurotransmitter content, and proj sections. The dorsal parvocellular division,

the ventral extent of the medial parvocellular division, and the lateral parvocellular division

proj ect to the brainstem and the spinal cord and are involved in autonomic regulation. The

posterior magnocellular division releases arginine vasopressin (AVP) and oxytocin and proj ects

to the posterior pituitary. The dorsolateral extent of the medial parvocellular division primarily

contains and releases CRH, although approximately 50% of these cells coexpress AVP. These

factors are released into the hypophyseal portal circulation at the external median eminence and

primarily affect the anterior pituitary (for review see Whitnall, 1993; Herman et al., 2002b).









The dorsolateral extent of the medial parvocellular PVN is heavily involved in HPA axis

regulation. This region integrates the excitatory and inhibitory inputs from the cortex, limbic

structures, brainstem, and peri-PVN to produce a net response (for reviews see Herman et al.,

2002a and b). Net activation of the dorsolateral extent of the medial parvocellular PVN results

in co-release of CRH and AVP. CRH stimulates the anterior pituitary to release

adrenocorticotropic hormone (ACTH) into the bloodstream, and AVP acts as a powerful

synergist in this process (Gillies et al., 1982). This effect may be further potentiated after

repeated stress as the number of cells coexpressing CRH and AVP increase (for example de

Goeij et al., 1992; Aubry et al., 1999). Once ACTH is released into the bloodstream, it induces

the adrenal synthesis and release of glucocorticoids. The glucocorticoids, including

corticosterone (CORT) in rats and cortisol in humans, increase energy utilization and

cardiovascular tone, shift the immune response from pro-inflammatory to anti-inflammatory, and

terminate further HPA axis activity.

Anatomy of the Limbic-HPA Axis

HPA axis activation is affected by inputs from a number of cortical, limbic, and brainstem

regions. The hippocampus, MPFC, septum, amygdala, and bed nucleus of stria terminalis

(BNST) all proj ect to the PVN and /or its surround, a region that is dense with glutamatergic and

GABA-ergic projections to the PVN (Sawchenko and Swanson 1983; Whitnall, 1993; Boudaba

et al., 1996; Boudaba et al., 1997; Ziegler and Herman, 2001; Herman et al., 2002a). The

hippocampus is involved in regulation of HPA axis tone and inputs from this structure provide

inhibitory modulation of the PVN (Herman and Cullinan, 1997). The MPFC is involved in

negative feedback inhibition of the HPA axis, especially in response to processive stressors

(Diorio et al. 1993). The septum provides direct and indirect inputs to the PVN (Sawchenko and

Swanson, 1983; Risold and Swanson, 1997). The lateral septum generally provides excitatory









input to the HPA axis while the medial septal input is inhibitory (Dunn, 1987a). The amygdala

has few direct inputs to the PVN (Prewitt and Herman, 1998), although the medial amygdala

(MeA) does have some inputs to the peri-PVN (Canteras et al., 1995; Prewitt and Herman,

1998). The amygdalar involvement in HPA axis control is complex. While electrical

stimulation of the MeA and the basomedial amygdala (BMA) increase levels of circulating

CORT and stimulation of the central amygdala (CeA), basolateral amygdala (BLA), and lateral

amygdala (LA) decrease CORT (Dunn and Whitener, 1986), lesions of both the CeA and MeA

decrease the ACTH and CORT response to processive stressors while not affecting basal levels

(Feldman et al., 1994). The CeA and MeA have dense projections to the BNST and to

hypothalamic nuclei other than the PVN (LeDoux et al., 1988; Canteras et al., 1995; Prewitt and

Herman, 1998; Dong et al., 2001). Therefore, the complex actions on HPA axis activity are

likely indirect. The BNST is an important limbic structure as it integrates inputs from the

amygdala, the lateral septum, and the hippocampus and has dense inhibitory and excitatory

projections to the PVN and peri-PVN (Sawchenko and Swanson, 1983; LeDoux et al., 1988;

Cullinan et al., 1993; Whitnall, 1993; Canteras et al., 1995; Boudaba et al., 1996; Cullinan et al.,

1996; Boudaba et al., 1997; Risold and Swanson, 1997; Prewitt and Herman, 1998; Dong et al.,

2001; Dong and Swanson, 2004). The inputs to the PVN from the BNST are also complex.

Electrical stimulation of the anterior and medial regions of the BNST increase levels of

circulating CORT (Dunn, 1987a,b), stimulation of the lateral regions of the BNST decrease

levels of circulating CORT, and stimulation of the posterior regions produces mixed results

(Dunn, 1987b). Additionally, lesions of the anterior regions of the BNST decrease CRH mRNA

in the medial parvocellular PVN, while lesions of the posterior regions increase CRH mRNA

(Herman et al., 1994). However, lesions of the lateral BNST attenuate the ACTH and CORT










response to stressors (Gray et al., 1993). None of these lesions alter the basal activity of ACTH

or CORT (Gray et al., 1993; Herman et al., 1994).

Autonomic Nervous System

Several regions of the PVN, including the dorsal parvocellular, ventromedial parvocellular,

and lateral parvocellular neurons, proj ect to brainstem autonomic neurons (for review see

Herman et al., 2002b). Sympathetic activation stimulates the release of norepinephrine from

sympathetic neurons and epinephrine from the adrenal medulla (Martin and Haywood, 1992;

Kvetnansky et al., 2006). This activation further increases cardiovascular tone (Martin and

Haywood, 1992), as well as inhibits the inflammatory immune response and stimulates anti-

inflammatory responses via innervation at the lymphoid organs (for review see Elankov et al.,

2000; Eskandari and Sternberg, 2002).

There is a tight interplay between the HPA axis and the sympathetic nervous system

(SNS). Stressors activate both systems (Kvetnansky et al., 2006), and activation of one system

typically results in activation of the other (for review see Elenkov et al., 2000). Therefore, there

is a dual response from both systems, enhancing the full stress response.

Chronic Stress and Disease

Responses to acute stressors are thought to be adaptive in the short term by increasing

access to energy stores and increasing cardiovascular tone. However, the effects of chronic

cortisol elevations can be detrimental. In rats chronic stress is associated with increased basal

CORT (Blanchard et al., 1998), blunted ACTH responses to stressors (Hauger et al., 1990),

altered CRH and AVP levels in the pituitary (Hauger et al., 1990; de Goeij et al., 1991),

hippocampal atrophy (Manji et al., 2001), adrenal hypertrophy (Blanchard et al., 1998; Hauger et

al., 1990), thymus involution, and depressive-like behaviors such as decreased grooming

(Blanchard et al., 1998).









In humans, similar changes are seen in individuals with depression. For example,

individuals with depression display alterations in HPA axis activity including elevated levels of

CRH in their cerebrospinal fluid (CSF; Nemeroff et al., 1984; Nemeroff et al., 1991; Heuser et

al., 1998) and cortisol in their blood plasma (Board et al., 1956; Gold et al., 1986; Arborelius et

al., 1999). Individuals with depression also display blunted ACTH responses to CRH (Gold et

al., 1986; Holsboer et al., 1986), but normal to elevated cortisol responses (Gold et al., 1986;

Holsboer et al., 1986; Juruera et al., 2004). There are also increases in CRH mRNA and in CRH

and AVP proteins in the PVN in post-mortem tissues of individuals with depression (Herman

and Cullinan, 1997). In addition, these individuals display hippocampal atrophy (Manji et al.,

2001), adrenal hypertrophy (Rubin et al., 1995), and immune dysfunction (Weisse, 1992). These

symptoms seem to be a function of hypersecretion of CRH (Labeur et al., 1995; Linthorst et al.,

1997) as well as impaired negative feedback (Nemeroff et al., 1991; Juruera et al., 2004). This

HPA axis dysfunction appears to be a primary factor in causing depressive symptoms. For

example, individuals with depression who respond well to anti-depressant treatment often

display normalization of the HPA axis (Board et al., 1956; Gold et al., 1986; Amsterdam et al.,

1988; Nemeroff et al., 1991; Heuser et al., 1998), as well as normalization of hippocampal and

adrenal size (Manji et al., 2001; Rubin et al., 1995) and immune functioning (Weisse, 1992).

Other disorders associated with alterations in HPA axis activity include panic disorder, obsessive

compulsive disorder, post-traumatic stress disorder, Tourette's syndrome, alcoholism and alcohol

withdrawal, anorexia, fibromyalgia, and chronic fatigue syndrome (Crofford, 1998; Arborelius et

al., 1999; Juruera et al., 2004).

These physiological changes and the development of psychopathology, particularly

depression and anxiety disorders, are further associated with chronic processive stress in humans










(for review see Arborelius et al., 1999). Stress factors associated with chronic depression

include unemployment, lack of insurance, lower levels of education, low income, general

medical conditions, low quality of life, low social adjustment, and generalized anxiety (Gilmer et

al., 2005). The quality of life measures involved questions regarding work, economics, and

social and familial relationships (Wisniewski et al., 2005). In another study, depression and

generalized anxiety disorder were associated with recent stressful events including injury or

illness and relationship problems (including death of a close individual and relationship

breakdown). Additionally, both conditions were associated with lifetime stressors, such as a

history of bullying or sexual abuse (Jordanova et al., 2007). Therefore, chronic stress exposure

leading to HPA axis dysregulation may be implicated in human psychopathology.

Animal Models of Chronic Stress

Chronic Variable Stress

The convergence of the effects of chronic stress in rats and of chronic stress in humans

suggests that animal studies may provide important insights into the biochemical basis of stress-

induced psychopathology in humans. However, while a number of processive stressors are

commonly used in research with rats (e.g., restraint, exposure to a novel environment, and

exposure to soiled cages, bright lights, or loud noises), many of these are limited in their long-

term effects, because the rats habituate to repeated exposure to these stimuli (for example see

Barnum et al., 2007).

One common stress procedure used is the chronic variable stress (CVS) regimen. Many

studies have reported increased basal HPA axis activity and the absence of habitutation (for

example see Marin et al., 2007). However, as with Marin and colleagues' study, often these

CVS regimens include at least one stressor with a strong systemic component, such as cold

exposure or food/water deprivation. In our lab, when only processive stressors were used in the









CVS regimen, no increases in baseline HPA axis functioning, particularly in CORT levels, were

observed (Simpkiss and Devine, 2003). Additionally, Ostrander and colleagues (2006) found

that rats displayed habituation to novel processive stressors after CVS, but maintained

responsivity to systemic stressors. Therefore, the utility of these CVS regimens as a model of

human chronic processive stress and the development of psychopathology is limited. When only

processive stressors are used, CVS does not cause the kinds of chronic, basal changes seen in

human populations.

Other Chronic Stress Regimens

Exposure to predatory stress, social separation, and inescapable tail shock have produced

more substantial and reliable alterations in behavioral and physiological activities than the CVS

regimen has. For example, repeated visual and olfactory exposure to a cat produces increased

basal CORT, adrenal hypertrophy and thymus involution, and depressive behaviors such as

decreased grooming in rats (Blanchard et al., 1998). Young squirrel monkeys that have been

separated from their peers display elevated CORT even several days later (Lyons et al., 1999).

Additionally, even a single exposure to inescapable tail shock produces elevated CORT

responses 24 hrs later (Deak et al., 1999) and HPA hyperresponsivity as many as 10 days later

(Johnson et al., 2002). These models of stress produce changes that look more like those seen in

human chronic stress and depression.

Social Defeat

Recently, our lab has begun to work with a model of social stress, the social defeat

procedure. In this procedure a small naive male intruder rat is placed into the cage of a larger

conspecific (the resident). This resident has experience with territorial interactions and will

typically display dominance behaviors when the intruder is present. These behaviors include

pinning the intruder, biting the intruder, and kicking bedding at the intruder. In response to the










resident' s dominance behaviors, the intruder typically exhibits submissive behaviors, including

freezing and lying on its back with paws up and abdomen exposed (supine posture). These types

of social interactions produce activation in limbic-hypothalamic structures of intruder rats as

evidenced by increases in c-fos expression in the hypothalamus, septum, BNST, amygdala, and

relevant brainstem nuclei such as the locus coeruleus and the nucleus of the solitary tract

(Martinez et al., 1998; Chung et al., 1999; Nikulina et al., 2004). Additionally, several studies

have reported activation of the HPA axis in intruder rats following social defeat exposure, as

evidenced by increases in circulating ACTH (Heinrichs et al., 1992; Ebner et al., 2005) and

CORT (Heinrichs et al., 1992; Covington and Miczek, 2001; Wommack and Delville, 2003;

Ebner et al., 2005). Also, unlike CVS, social defeat does not result in habituation of the HPA

axis response, or even of the body temperature response (i.e., induction of fever; Barnum et al.,

2007). Additionally, rats exposed to repeated social defeat display increases in basal CORT (de

Goeij et al., 1992; Barnum et al., 2007; Stone et al., in preparation). Repeated social defeat also

causes other long-term changes, including decreases in thymus and seminal vesicle masses (Buwalda

et al., 2001). These results suggest that social defeat affects the functioning of the HPA axis and

HPA axis regulation, as well as other organs involved in immune response and reproduction.

In addition to physiological changes, repeated social defeat also induces long-term behavioral

changes. Rats that are exposed to social defeat display increased anxiety-related behaviors in the

elevated plus maze (Heinrichs et al., 1992; Calfa et al. 2006) and increased behavioral despair in

the Porsolt test (Rygula et al., 2005; Stone et al., in preparation). All of these results suggest that

repeated social defeat may be a valuable tool in studying potential long-term effects of chronic

stress exposure.









Nociceptin/Orphanin FQ

One neurotransmitter system that has been shown to be important in the neural response to

stress is nociceptin/orphanin FQ (N/OFQ). N/OFQ and its cognate receptor NOP constitute a

highly conserved (Danielson and Dores, 1999) peptide neurotransmitter system that affects an

interesting range of very important behavioral and physiological activities. N/OFQ is a 17 amino

acid peptide that is structurally similar to the endogenous opioids, particularly dynorphin A

(Meunier et al., 1995; Reinscheid et al., 1995). However, N/OFQ does not bind to the CI, 6, or x

opioid receptors with high affinity (Shimohigashi et al., 1996), but does bind with high affinity to

the NOP receptor (Reinscheid et al., 1995; Shimohigashi et al., 1996; Butour et al., 1997). The

NOP receptor is a 7-transmembrane, G-protein coupled receptor (Bunzow et al., 1994; Chen et

al., 1994; Wang et al., 1994; Wick et al., 1994; Lachowicz et al., 1995; Reinscheid et al., 1996)

that is negatively linked to adenylate cyclase (Mollereau et al., 1994; Lachowicz et al., 1995;

Reinscheid et al., 1995; Reinscheid et al., 1996), increases inward rectifying K+ channel

conductance (Connor et al., 1996a; Vaughan and Christie, 1996; Vaughan et al., 1997), and

inhibits Ca2+ COnductance (Connor et al., 1996b). The NOP receptor shows high structural

homology with the opioid receptors (Bunzow et al., 1994; Chen et al., 1994; Mollereau et al.,

1994; Wang et al., 1994; Wick et al., 1994; Lachowicz et al., 1995), although it does not bind

any of the opioids with high affinity (Bunzow et al., 1994; Wang et al., 1994; Lachowicz et al.,

1995; Butour et al., 1997). This low affinity between N/OFQ and opioid receptors and between

NOP receptors and opioid peptides suggests that the N/OFQ-NOP receptor system is functionally

distinct from the opioid system.

N/OFQ, the NOP receptor, and their mRNAs are widely distributed throughout the brain,

spinal cord, and periphery (Bunzow et al., 1994; Chen et al., 1994; Mollereau et al., 1994; Wang

et al., 1994; Wick et al., 1994; Lachowicz et al., 1995; Nothacker et al., 1996; Neal et al., 1999a









and b; Devine et al., 2003), consistent with a wide range of functions including pain modulation

(Meunier et al., 1995; Reinscheid et al., 1995; Tian et al., 1997), motor performance (Reinscheid

et al., 1995; Devine et al., 1996), spatial learning (Sandin et al., 1997; Sandin et al., 2004), and

feeding (Pomonis et al., 1996; Nicholson et al., 2002). However, N/OFQ and NOP receptor

expression are relatively high in some limbic regions including the hypothalamus, septum,

BNST, and amygdala (Bunzow et al., 1994; Wang et al., 1994; Lachowicz et al., 1995;

Nothacker et al., 1996; Neal et al., 1999a and b; Devine et al., 2003). This limbic localization is

consistent with an additional role of N/OFQ in emotional regulation, particularly stress and

anxiety responses. For example, Devine and colleagues (2003) found that N/OFQ is released

from forebrain neurons following exposure to acute restraint stress.

Exogenous N/OFQ administration has also been found to increase anxiety-related

behaviors and HPA-axis activation in response to stressful/anxiety-provoking stimuli. To

examine anxiety-related behaviors, standard neophobic tests are generally used, such as the open

field test, elevated plus maze, and light-dark test. These tests take advantage of rats' motivation

to explore during foraging activities, while, on the other hand, displaying a natural aversion to

brightly lit and open spaces. For example, rats show thigmotaxis in the open field (for example

see Simon et al., 1994), and they show a preference for the enclosed arms of the elevated plus

maze (Handley and Mithani, 1984; Pellow et al., 1985) and for the dark box of the light-dark

test (Crawley and Goodwin, 1980; Costall et al., 1989; Onaivi and Martin, 1989; Chaouloff et

al., 1997). The balance between exploration and avoidance can be manipulated in a highly

reproducible manner by anxiolytic drugs (i.e., drugs that humans report to be anxiety-reducing,

such as diazepam) and by anxiogenic drugs (i.e., drugs that humans report to be

anxiety-inducing, such as FG 7142). Rats increase their exploration of open or lit spaces









following administration of anxiolytic compounds and decrease their exploration following

administration of anxiogenic compounds (Hughes, 1972; Crawley and Goodwin, 1980; Crawley,

1981; Handley and Mithani, 1984; Pellow et al., 1985; Pellow and File, 1986; Costall et al.,

1989; Onaivi and Martin, 1989; Stefanski et al., 1992; Simon et al., 1994; Chaouloff et al., 1997;

Fernandez et al., 2004).

In our lab, a modified version of the open field test is used. A start box was attached to

one wall of the open field, which allows the use of latency to enter the open field and time spent

in the open field as measures of anxiety-related behavior (in addition to thigmotactic behavior

that has been reported in previous versions of the open field). The test has been calibrated

(lighting, handling, etc.) so that vehicle-treated rats spend approximately 25% of the test time in

the open field, allowing observation of both increases and decreases in anxiety-related behaviors.

Under these conditions, rats that have been treated with diazepam generally show shorter

latencies to enter the open field, more total time spent in the open field, and more exploration

away from the start box and into the middle of the open field, as compared to vehicle-treated

rats. On the other hand, rats treated with FG 7142 generally show longer latencies to enter the

open field, less total time spent in the open field, and less exploration away from the start box

and into the center of the open field (Fernandez et al., 2004). These data provide evidence that

the modified open field is a valid and sensitive tool for measuring changes in the expression of

anxiety-related behaviors.

Intracerebroventricular (ICV) inj sections of N/OFQ have been shown to increase

anxiety-related behaviors in this open field test, as well as the elevated plus maze and the

light-dark test (Fernandez et al. 2004; Green et al., in press). N/OFQ-treated rats, as compared to

vehicle-treated rats, display longer latencies to enter and spend less total time in the open area of









the open field test, the open arms of the elevated plus maze, and the lit box in the light-dark test.

These behaviors resemble the effects following inj sections of other anxiogenic drugs, such as FG

7142 and are opposite from the effects following injections of anxiolytic drugs, such as

diazepam. These results suggest that N/OFQ has an anxiogenic action after ICV administration.

In addition to these behavioral effects, ICV inj sections of N/OFQ increase HPA axis

activity in rats. When rats are inj ected into the lateral ventricle under unstressed conditions (i.e.,

the rats are allowed to recover from the stress of handling and cannula implantation prior to the

delivery of the drug), and mildly stressed conditions (N/OFQ inj sections are administered without

allowing rats to recover from the stress of handling) they exhibit substantial elevations in

circulating ACTH and CORT (Devine et al., 2001; Nicholson et al., 2002; Legget et al., 2006).

Elevations of ACTH and CORT are also observed when rats are inj ected and then exposed to the

open field test (Fernandez et al., 2004). Additionally, injections ofN/OFQ affect gene regulation

in this system, producing increases in CRH mRNA in the PVN and increases in

proopiomelanocortin (POMC; the precursor to ACTH) mRNA in the pituitary (Legget et al.,

2006). This suggests that N/OFQ is important in the regulation of the HPA axis.

In the following experiments, the role of N/OFQ in stress and anxiety is further explored.

The potential role of limbic structures in producing increased anxiety-related behaviors and HPA

axis activation is examined, and the diffusion characteristics ofN/OFQ following ICV and

intraparenchymal injections are verified. Finally, the endogenous regulation ofN/OFQ mRNA

and NOP receptor mRNA following acute and repeated social defeat exposure is examined.









CHAPTER 2
ANATOMICAL ANALYSIS OF ANXIOGENIC BEHAVIORAL EFFECTS OF
EXOGENOUS NOCICEPTIN/ORPHANIN FQ

Background

The N/OFQ-induced elevations in circulating hormone concentrations described in Chapter

1 are mediated by limbic inputs, including the septum, BNST, and amygdala (Misilmeri and

Devine, in preparation). Specifically, unstressed injections into these limbic structures produce

elevations in circulating ACTH and CORT. Accordingly, limbic structures are implicated in the

HPA-axis modulating effects of N/OFQ under unstressed conditions.

In light of the observations that N/OFQ produces anxiogenic behavioral effects and

activation of the HPA axis, and that the hormonal alterations produced by N/OFQ are at least

partially mediated by limbic structures, we previously explored the role of one limbic structure,

the amygdala, in affecting anxiety-related behaviors and HPA axis activation in rats (Green et

al., in press). The rats that were inj ected with N/OFQ into the amygdala displayed longer

latencies to enter the open field (Figure 2-1). However, the behavioral effects of these injections

into the amygdala were not as potent as the effects after inj sections into the lateral ventricle

(Figure 2-2). Additionally, there were no significant between-groups differences in circulating

CORT concentrations when rats were given intra-amygdaloid inj sections of N/OFQ or vehicle

prior to the open field exposure (Figure 2-3). This lack of a hormonal effect might seem

surprising at first, since there are elevations in circulating CORT concentrations after intra-

amygdaloid N/OFQ when rats are tested under unstressed conditions (Misilmeri and Devine, in

preparation). However, in that unstressed experiment, the controls exhibited low basal CORT

levels, and the effects of the N/OFQ injections were small. In our behavioral experiment, the

rats were inj ected under stressed conditions (handling) and were exposed to the additional

stressor of the novel open field. In this instance the circulating CORT concentrations in the









vehicle-treated rats were much higher. Thus, the stress-induced elevations in CORT could have

obscured any HPA axis-activating effect of the amygdaloid inj sections of N/OFQ. This finding

concurs with a previous report in which the mild stress of a novel environment partially obscured

the hormonal effects of N/OFQ after an ICV inj section (Devine et al., 2001).

Because of these less potent effects, the BNST was examined as a potential site for the

anxiogenic effects of N/OFQ. The BNST is another limbic structure known to participate in the

regulation of behavioral and hormonal responses to anxiety-inducing stimuli (for examples see

Henke, 1984; Rogan et al., 1997; Walker and Davis, 1997). The amygdala and BNST can be

differentiated in terms of their roles in fear and anxiety (Lee and Davis 1997; Walker and Davis,

1997; for review see Walker et al., 2003). Although the distinctions are not entirely clear, the

amygdala appears to play a larger role in fear-related behaviors (such as startle responses to a

specific, usually conditioned, stimulus), and the BNST appears to be more important in

responding to anxiety-provoking stimuli (primarily those stimuli that are long in duration, non-

specific, and non-conditioned). For example, lesions of the BNST, but not the amygdala, block

light-enhanced startle, while amygdaloid lesions, but not BNST lesions, block fear-potentiated

startle (Walker and Davis, 1997). The modified open field test used in the present experiment

resembles tests of generalized anxiety more than it resembles tests of fear, as there is no specific

or conditioned fear stimulus. In this respect, the BNST may be more involved in the behavioral

responses during neophobic tests of anxiety such as the open field test and may be an important

potential mediator of the anxiogenic effects that were seen after ICV inj sections of N/OFQ.

Methods

Animals

Male Long Evans rats (n = 34, Harlan, Indianapolis, IN) were housed in polycarbonate

cages (43 x 21.5 x 25.5 cm) on a 12hr-12hr light-dark cycle (lights on at 7:00 am). The rats were










pair-housed in a climate-controlled vivarium (temperature 21-23 Co humidity 55-60%) until

surgery. After surgery, the rats were singly-housed in the same environment. Standard

laboratory chow and tap water were available ad libitum throughout the experiment. All

procedures in this and subsequent experiments were pre-approved by the University of Florida' s

Institutional Animal Care and Use Committee, and the experiments were conducted in

compliance with the National Research Council's Guide for the Care and Use of Laboratory

Animals.

Drugs

Ketamine and xylazine were both obtained from Henry Schein (Melville, NY) at

concentrations of 100 mg/ml. Ketamine-xylazine was mixed by adding 2 ml ofxylazine to 10

ml of ketamine yielding a 12 ml solution of 83.3 mg/ml ketamine and 16.7 mg/ml xylazine.

Ketorolac tromethamine (30 mg/ml) and AErrane (99.9 % isoflurane) were also purchased from

Henry Schein.

N/OFQ was obtained from Sigma-Aldrich (St. Louis, MO) and was dissolved in artificial

extracellular fluid (aECF) composed of 2.0 mM Sorenson's phosphate buffer (pH 7.4) containing

145 mM Na 2.7 mM K 1.0 mM Mg2 1.2 mM Ca2+, 150 mM C1F, and 0.2 mM ascorbate.

These ion concentrations replicate the concentrations found in extracellular fluid in the brain

(Moghaddam and Bunney, 1989). N/OFQ was prepared at concentrations of 0.01, 0.1i, and 1.0

nmole per 0.5 Cll aECF.

Surgery

Each rat (265-407g) was implanted with a guide cannula under ketamine-xylazine

anesthesia (62.5 mg/kg ketamine + 12.5 mg/kg xylazine, i.p. in a volume of 0.75 ml/kg).

Ketorolac tromethamine (2 mg/kg, s.c.) was inj ected for analgesia at the time of surgery.

AErrane was administered as supplemental surgical anesthesia as necessary. Once the rat was









anesthetized, a stainless steel guide cannula (11mm, 22 gauge) was implanted into the right

BNST (0.3 mm posterior to bregma, 3.0 mm lateral from the midline, and 5.4 mm ventral from

dura; n = 28). Cannulae were implanted at a 14o angle to minimize intrusion of the cannula into

the ventricle. Six additional rats were each implanted with a cannula aimed at extra-BNST sites

(anatomical controls). Each cannula was secured with dental cement anchored to the skull with

stainless steel screws (0.80 x 3/32"). An obturator that extended 1.2 mm beyond the guide

cannula tip was inserted at the time of surgery and removed on the day of the experiment at the

time that an intracranial injection was administered. Following surgery, each rat was given at

least 7 days to recover from surgery.

Equipment

Anxiety-related behavior was measured in an open Hield test. The open Hield was

composed of a 90 x 90 x 60 cm Hield with a 20 x 30 x 60 cm start box attached to the outside of

the open Hield, at the midpoint of one side (see Figure 2-4). The bottom and sides were

constructed of black acrylic. A black acrylic guillotine door separated the start box and the open

Hield. This door was attached to a rope and pulley system, which allowed the door to be opened

from outside the testing room. The tops of the start box and open Hield were open and a camera

was mounted on the ceiling above the testing apparatus to record the rats' behaviors.

Illumination of the start box and open Hield were approximately equal (14-30 lux).

Anxiety-Testing Procedure

After the surgical recovery period, each rat was handled for 5 minutes on each of 3

consecutive days, and then given one day with no disturbance. On the 5th day, each rat was

fitted with a 28-gauge stainless steel inj ector connected with polyethylene (PE20) tubing to a 1

Cll Hamilton syringe. Each rat then received a 0.5 Cll inj section of aECF vehicle or aECF

containing 0.01, 0.1, or 1.0 nmole N/OFQ by an experimenter blind to the dose. The injections









were administered over a 2-minute period using a syringe pump, and the inj ector was left in

place for 3 additional minutes to allow diffusion of the inj section volume. Each rat was freely

moving in its home cage during the inj section procedure.

These inj sections and the subsequent behavioral tests were completed 90-210 minutes after

the vivarium lights were turned on, the time during which the HPA axis is at its daily nadir (Ixart

et al., 1977; Kwak et al., 1993). Five minutes after the injection, each rat was individually

placed in the start box of the open field test, and the door to the testing room was closed,

isolating the rat from the experimenter and any other disruptive influences. The rat was then

given 1 minute to acclimate to the novel environment of the start box. After 1 minute the

guillotine door was opened remotely, and it remained open throughout the test period. The rat

was given 5 minutes to explore the start box and open field. Each rat was then returned to its

home cage until 30 minutes elapsed after the start of the inj section. At this time, the rat was

rapidly decapitated.

Immediately after decapitation, 6 ml of trunk blood was collected into polypropylene tubes

containing 600 Cll of Na2EDTA (20 Clg/CIl) on ice. The tubes were centrifuged at 1000x gravity.

The plasma fraction was collected, aliquotted, and frozen at -80oC. Later, RIA was performed

for quantification of plasma concentrations of CORT using a kit from Diagnostic Products Corp.

(Los Angeles, CA). The interassay variability for this kit increased with sample CORT

concentrations, ranging from less than 5% for lower plasma CORT concentrations to less than

15% for higher plasma CORT concentrations.

Additionally, each brain was removed, frozen in 2-methylbutane at -400C, and stored at

-800C. Later, each brain was sectioned at 30 Clm and stained with cresyl violet for cannula










placement verification. Adrenal glands, thymus glands, and spleens were dissected out and

weighed for verification of the health status of the rats.

An observer who was blind to the treatment conditions scored the exploratory behavior of

the rats from the videotapes, using a grid that was superimposed on the video monitor. This grid

divided the open field into 25 equal squares. The outer 16 squares defined an outer zone and the

inner 9 squares defined an inner zone (Fig. 2-4). Between-groups differences in the latency to

enter the open field, total time spent in the open field, latency to enter and time spent in the inner

zone, and the number of entries into the open field and the inner zone were used as measures of

anxiety. An entry into the open field or movement from the periphery to the inner zone was

counted when all 4 paws of the rat left one zone and entered a new zone.

Statistical Analyses

In order to analyze differences between the N/OFQ-treated groups and the vehicle-treated

group, one-way ANOVAs were calculated for latency to enter the open field and the inner zone,

total time spent in the open field and the inner zone, number of entries into the open field and

inner zone, plasma CORT concentrations, and organ masses. All significant effects (p< 0.05)

were further analyzed using Fisher' s LSD post-tests.

Differences between anatomical controls and intra-BNST vehicle controls for each of the

listed measures were analyzed by individual t-tests.

Results

Anxiety-Related Behavior

The intra-BNST N/OFQ-treated rats showed greater expression of anxiety-related

behaviors than the aECF vehicle-treated rats did. The N/OFQ-treated rats displayed significantly

longer latencies to enter the open field (Fig. 2-5a; F(3,24) = 4.009, p<0.05) than did the

vehicle-treated rats, but the groups did not differ significantly in latency to enter the inner zone










(Fig. 2-5b; F(3,24) = 2.593, p>0.05). There were also no significant differences between

N/OFQ-treated and vehicle-treated rats in time spent in the open field (Fig. 2-5c; F(3.24) =

2.075, p>0.05) or the inner zone (Fig. 2-5d; F(3,24) = 2.669, p>0.05). However, the

N/OFQ-treated rats displayed significantly fewer entries into the open field (Fig. 2-5e; F(3,24)

3.055, p<0.05) and significantly fewer entries into the inner zone (Fig. 2-5f; F(3,24) = 3.321,

p<0.05) than did the vehicle-treated rats.

The BNST placements were fairly evenly distributed throughout the medial and lateral

divisions (Fig.2-6). The effects of vehicle injections into these BNST sites were compared

against the effects of N/OFQ inj sections into extra-BNST sites (anatomical controls).

Comparisons between the vehicle-treated rats and the N/OFQ-treated anatomical controls

showed no significant differences on any measure of latency (Fig. 2-5a and b), time (Fig. 2-5c

and d), or entries (Fig. 2-5e and f).

Circulating Corticosterone

Inj sections of N/OFQ into the BNST produced significant elevations in circulating CORT

(Fig. 2-7; F(3,22) = 3.171, p<0.05) as compared to the CORT concentrations in the

vehicle-treated rats. Concentrations of circulating CORT were not significantly different

between N/OFQ-treated anatomical controls and vehicle-treated rats.

Organ Masses

There were no significant differences in adrenal weights (Fig. 2-8A), thymus gland

weights (Fig. 2-8B), or spleen weights (Fig. 2-8C) between the vehicle-treated and

N/OFQ-treated rats and between the vehicle-treated rats and anatomical controls.









Discussion

Anxiety-Related Behaviors and Corticosterone

We previously found that inj sections of N/OFQ into the right lateral ventricle produce dose

orderly elevations in all measured anxiety-related behaviors in the modified open field and

elevations in circulating CORT (Fernandez et al., 2004; Green et al., in press). Injections into

the right amygdala also elevated anxiety-related behaviors, although the behavioral and

hormonal effects were less potent than those seen following ICV inj sections. Similarly, injections

into the right BNST produced elevations in anxiety-related behaviors and elevations in

circulating CORT, and once again, these effects were less potent than those observed after ICV

injections. The behavioral results are somewhat surprising as the amygdala, and especially the

BNST appeared to be ideal candidates for mediating the behavioral actions ofN/OFQ. Both

structures are involved in responses to emotionally-salient stressors, and both structures have

relatively high levels of NOP receptor mRNA expression and binding (Neal et al., 1999b). It is

possible, however, that ICV inj sections are having effects at multiple sites, accounting for the

greater potency of this route of administration. Because inj sections into the amygdala and into the

BNST produce partial effects on anxiety-related behaviors, there may be additive or synergistic

effects at these and other limbic, cortical, and brainstem structures following ICV injections. For

example, the lateral septum may be another structure involved. Like the amygdala and the

BNST, the lateral septum is involved in responses to stressors and has at least moderate levels of

NOP receptor binding.

The hormonal effects following intraparenchymal inj sections are less surprising. The

modest effects following amygdaloid inj sections can be expected considering there are only

sparse connections between the amygdaloid complex and the PVN (Prewitt and Herman, 1998).

Unlike the amygdala, though, the BNST does have many connections to the PVN (for example









see Prewitt and Herman, 1998; Dong et al., 2001; Dong and Swanson, 2004). This anatomical

difference is consistent with the greater elevations in circulating CORT observed in stressed (the

present experiment) and unstressed rats (Misilmeri and Devine, in preparation) following intra-

BNST inj sections versus intra-amygdaloid inj sections.

One concern with the smaller effects of our intraparenchymal inj sections versus ICV

inj sections is the fact that they were done unilaterally (inj sections were into the right hemisphere in

all groups). Previous analysis of the diffusion of N/OFQ after unilateral ICV injections

suggested that there was at least some bilateral distribution, primarily at midline periventricular

structures (D. P. Devine, personal communication). It is not clear if bilateral injections into the

BNST or the amygdala would have increased the effects. To our knowledge, the effects of

bilateral BNST injections of anxiolytic or anxiogenic compounds have not been reported with

any standard tests of fear or anxiety. Thus it is possible that inj sections of N/OFQ into the right

and left BNST might produce more potent effects. However, in the case of the amygdala, the

right hemispheric structure is generally more dominantly involved in emotionally-relevant

behavioral responses (Coleman-Mesches and McGaugh, 1995a and b; Andersen and Teicher,

1999; Adamec et al., 2001; Peper et al., 2001; Scicli et al., 2004), or is at least no less involved

than the left amygdala (Good and Westbrook, 1995; LaBar and LeDoux, 1996; Izquierdo and

Murray, 2004). In fact, bilateral inj sections of drugs in to the amygdalae may add little in terms

of changes in emotionally-relevant behaviors when compared to the effects of unilateral

inj sections into the right amygdala (for example see Coleman-Mesches and McGaugh, 1995b).

Therefore, in our previous analysis of the effects of unilateral amygdaloid inj sections it does not

seem likely that bilateral injections would have produced more potent effects.









There are reports that N/OFQ and its synthetic analogue Ro64-6198 each exert anxiolytic

actions in rats and mice (Jenck et al., 1997; Jenck et al., 2000; Gavioli et al., 2002; Varty et al.

2005). Additionally, some researchers find opposing effects depending on the dose of drug used

(Kamei et al., 2004), the number of times the drug is administered (Vitale et al., 2006), or the

anxiety tests used (in this case with NOP receptor knockouts; Gavioli et al., 2007). The reasons

for these discrepancies are currently unclear; although, there are methodological differences that

may account for the contrasting results (see Fernandez et al 2004 for a full discussion of the

potential explanations). It is possible that N/OFQ may produce differing anxiogenic and

anxiolytic actions depending upon the prior history and/or the current stress status of the animals,

but we, in the Devine lab, have so far been unable to detect anxiolytic effects in our studies.

It has been argued that the increases in anxiety-related behaviors may be due to locomotor

impairment produced by N/OFQ. At higher doses of N/OFQ there are profound motoric effects

that result in postural changes and reduced locomotion (Devine et al., 1996). Recently, Vitale

and colleagues (2006) reported reduced exploration of the open arms of the elevated plus maze

after a single dose of N/OFQ administered ICV. However, a second dose given 2 hours later

resulted in a reversal of this effect. They interpreted the reduced exploration as a locomotor

effect, and concluded that the lack of this effect following the second administration was due to

tolerance (rats develop tolerance to the locomotor-inhibiting effects of N/OFQ; see Devine et al.,

1996). However, anxiogenic actions have been observed at doses that do not produce locomotor

effects (Devine et al., 1996; Fernandez et al., 2004; Green et al., in press). Additionally,

locomotor controls show that motoric effects are not the likely source of the reduced exploration

following N/OFQ injections (Fernandez et al., 2004; Green et al., in press). For example, when

the threatening stimulus of a brightly lit environment in the dark-light box is removed, thus










creating a dark-dark test, N/OFQ-treated rats and vehicle-treated rats explored the boxes in a

comparable manner (Fernandez et al., 2004). Likewise, when the threatening stimulus of a large

open field is removed and replaced with an undersized open field equal in size to the start box,

N/OFQ-treated rats and vehicle-treated rats again behaved in a comparable manner (Green et al.,

in press). The only difference in these conditions is the presence or absence of a threatening

stimulus. Thus, it seems likely that behavioral differences observed in the open field test and

light-dark box are due to differences in the anxiety states of the rats and not due to locomotor

impairment.

The results of the present experiment, as well as the results following inj sections into the

amygdala, provide additional evidence of an anxiety-related effect as opposed to a locomotor

effect. The intraparenchymal injections in these experiments were highly specific, with no

significant effects observed in the anatomical controls. Given the known roles of the BNST and

amygdala, inj sections into these structures would not be expected to affect locomotor behaviors,

but would be expected to affect anxiety-related behaviors.

Organ Masses

In the present study, the adrenal, thymus, and spleen masses were measured to establish

that there were no systematic differences in health status or stress exposure between the various

groups of rats. Since there were no significant differences in adrenal gland masses, thymus

gland masses, or spleen masses between any of the groups tested, it can be concluded that there

were no apparent differences in the health or stress history of the rats that can account for the

behavioral or hormonal differences.

Summary

The results of the present study show that N/OFQ inj sections affect anxiety and HPA axis

activity through limbic structures including the BNST. In addition, previous work has shown a










role for the amygdala. This suggests the possibility that limbic N/OFQ neurotransmission may

be involved in regulation of affect. However, because ICV injections of N/OFQ produced

greater, more potent effects than did inj sections into either the BNST or the amygdala, there may

be additive or synergistic actions between the BNST and the amygdala. Additionally, the

contralteral structures as well as other relevant limbic, cortical, and brainstem structures may be

involved.













300 e





0.0 0.01 0.1 1.0


B








0.0 0.01 0.1 1.0 AC

Dose NIOFQ (nmoles)

D
10
0

S4
* 3

0.0 0.01 0.1 1.0 AC


Dose NIOFQ (nmoles)


C
S3(

0 21
Q
q) 11


Dose NIOFQ (nmoles)


Dose NIOFQ (nmoles)


0 0.01 0.1 1.0 A(

Dose NIOFQ (nmoles)


Dose NIOFQ (nmoles)


Figure 2-1. Anxiety-related behaviors following intra-amygdaloid inj sections of N/OFQ. (A)
N/OFQ-treated rats exhibited longer latencies to enter the open Hield. However,
inj sections of N/OFQ into the right amygdala did not significantly alter (B) the latency
to enter the inner zone, (C) the time spent in the open Hield, (D) the time spent in the
inner zone, (E) the number of entries into the open field, (F) or the number of entries
into the inner zone. Values expressed are group means + SEM (n = 9-10 rats per
group). Significant differences between the N/OFQ-treated rats and the aECF-treated
controls are expressed as p < 0.05 and ** p < 0.01. AC =anatomical controls.









































U Ill
0.0 0.01 0.1 1.0

Dose NIOFQ (nmoles)


0 0.01 0.1 1.0

Dose NIOFQ (nmoles)


0 0.01 0.1 1.0

Dose NIOFQ (nmoles)


JcJe JcJe
T T


JcJe Je
T


U. I I.U


U.U 1 U. I I.U


Dose NIOFQ (nmoles)


Dose NIOFQ (nmoles)


C
300-
-

O 200-
-

O, 10
Q .


0.0 0.01 0.1 1.0

Dose NIOFQ (nmoles)


JcJe JcJe


Je


JcJe JrJr


Figure 2-2. Anxiety-related behaviors following ICV inj sections of N/OFQ. N/OFQ-treated rats
exhibited: (A) longer latencies to enter the open field and (B) the inner zone, (C) less
total time in the open field and (D) the inner zone, and (E) fewer entries into the open
field and (F) the inner zone. Values expressed are group means + SEM (n = 7-8 rats
per group). Significant differences between the N/OFQ-treated rats and the aECF-
treated controls are expressed as p < 0.05 and ** p < 0.01.


10
-

O 7
o 6
Q) 5
4










A







0 00 .

Dose NIOFQ (nmoles)

B

6400-1


o~200*


0.0 0.01 0.1 1.0 AC

Dose NIOFQ (nmoles)

Figure 2-3. Concentrations of circulating corticosterone following ICV and intra-amygdaloid
inj sections of N/OFQ. (A) Inj sections of N/OFQ into the right lateral ventricle
produced elevations in circulating CORT. (B) N/OFQ administration into the right
amygdala did not significantly alter the levels of circulating corticosterone.Values
expressed are group means + SEM (n = 7-10 rats per group). Significant differences
between the N/OFQ-treated rats and the aECF-treated controls are expressed as ** p
< 0.01. AC = anatomical controls




























Figure 2-4. Photograph of the open Hield and a diagram of the zones used for scoring. The rat is
placed in the start box for the first minute of the test. After the door opens, the rat
may move between the start box and the open Hield freely. The outer zone represents
the periphery of the open Hield. The inner zone represents the central region of the
open field.




















s O I I I
0 0.01 0.1 1.0 AC

Dose NIOFQ (nmoles)


B
300

O 0
I




0 0.01 0.1 1.0 AC


0 0.01 0.1 1.0 AC

Dose NIOFQ (nmoles)

E

10






0 0.01 0.1 1.0 AC


10

4C
3



0 0.01 0.1 1.0 AC


0 0.01 0.1 1.0 AC

Dose NIOFQ (nmoles)


Dose NIOFQ (nmoles)


Dose NIOFQ (nmoles)


Dose NIOFQ (nmoles)


Figure 2-5. Anxiety-related behaviors following intra-BNST inj sections of N/OFQ.
N/OFQ-treated rats exhibited (A) longer latencies to enter the open field and (E)
fewer entries into the open field and (F) the inner zone. However, there were no
significant differences in (B) latency to enter the inner zone or in (C) time spent in the
open field or (D) the inner zone. Values expressed are group means + SEM (n = 6-8
rats per group). Significant differences between the N/OFQ-treated rats and the
aECF-treated controls are expressed as p < 0.05 and ** p < 0.01. AC = anatomical
controls.


JcJe


h


si
E 81
7-1
W
61
51
L 41
d 31
E 21
1I
Z o~















0.~ 48 me


-0.26 mm~t









Figureg 2-.Aaoiclmpo NS lcmns.Teijctrtppaemnsaeilutae o









Figre -6.Anatomical capontol areT id aentfed s by e a n bl ck d t plae withinthe marke ilsr. td











BNST
Circulating Corticosterone
600-e







O 0.01 0.1 1.0 AC

Dose NIOFQ (nmoles)

Figure 2-7. Concentrations of circulating corticosterone following intra-BNST inj sections of
N/OFQ. Injections of N/OFQ into the BNST produced elevations in CORT at the
highest dose administered. Values expressed are group means + SEM (n = 6-8 rats
per group). Significant differences between the N/OFQ-treated rats and the
aECF-treated controls are expressed as ** p < 0.01. AC = anatomical controls

























Dose NIOFQ (nmoles)


Dose NIOFQ (nmoles)


Figure 2-8. Analysis of glandular masses. (A) Adrenal gland masses, (B) thymus gland masses,
and (C) spleen masses showed no significant differences between groups. Values
expressed are group means + SEM (n = 6-8 rats per group).


500
450


300
250
200
150
100
50
1 I
0.0 0.01 0.1 1.0 AC
Dose NIOFQ molese)









CHAPTER 3
DIFFU SION OF NO CICEP TIN/ORPHANIN F Q AF TER INTRACEREB ROVENTRICULAR
AND INTRAPARENCHYMAL INJECTIONS

Background

The behavioral and hormonal effects following inj sections of N/OFQ into the right lateral

ventricle, the amygdala, the BNST, and other limbic structures have been described (Fernandez

et al., 2004; Green et al., in press; Missilmeri and Devine, in preparation). However, the extent

of the diffusion of N/OFQ following these injections is not clear. Data from the anatomical

controls in Chapter 2 suggest that diffusion following inj sections into the BNST and the amygdala

was limited to the target structure. To confirm this and compare it with the penetration of

N/OFQ into the tissue following ICV inj sections, 3H-N/OFQ was inj ected into the lateral

ventricle, the BNST, and the amygdala.

Methods

Animals

Male Long Evans rats (n = 15, Harlan, Indianapolis, IN) were housed in polycarbonate

cages (43 x 21.5 x 25.5 cm) on a 12hr-12hr light-dark cycle (lights on at 7:00 am). The rats were

pair-housed in a climate-controlled vivarium (temperature 21-23 Co humidity 55-60%) until

surgery. After surgery, the rats were singly-housed in the same environment. Standard

laboratory chow and tap water were available ad libitum throughout the experiment.

Drugs

Ketamine and xylazine were both obtained from Henry Schein (Melville, NY) and were

mixed as described in Chapter 2. Ketorolac tromethamine (30 mg/ml) and AErrane (99.9 %

isoflurane) were also purchased from Henry Schein.

N/OFQ was obtained from Sigma-Aldrich (St. Louis, MO) and was dissolved in artificial

extracellular fluid (aECF), as described in Chapter 2. N/OFQ was prepared at concentrations of









1.0 nmole per 1.0 Cll aECF for ICV inj sections or per 0.5 Cll aECF for intra-amygdaloid and

intra-BNST injections. 3H-N/OFQ was prepared by adding 0.01 nmoleS 3H-N/OFQ (Phoenix

Pharmaceuticals, Belmont, CA) to the 1.0 nmole N/OFQ doses.

Surgery

Each rat (268-319g) was implanted with a guide cannula under ketamine-xylazine

anesthesia (62.5 mg/kg ketamine + 12.5 mg/kg xylazine, i.p. in a volume of 0.75 ml/kg).

Ketorolac tromethamine (2 mg/kg, s.c.) was inj ected for analgesia at the time of surgery.

AErrane was administered as supplemental surgical anesthesia as necessary. Once the rat was

anesthetized, a stainless steel guide cannula (6 or 11Imm, 22 gauge) was implanted into the right

lateral ventricle (ICV; 0.8 mm posterior to bregma, 1.4 mm lateral from the midline, and 2.7 mm

ventral from dura; n = 5), the right amygdala (1.8 mm posterior to bregma, 3.9 mm lateral, and

6.2 mm ventral; n = 5) or the right BNST (0.3 mm posterior to bregma, 3.0 mm lateral from the

midline, and 5.4 mm ventral from dura; n = 5). The ICV and amygdaloid cannulae were each

implanted vertically, and the BNST cannulae were implanted at a 14o angle. Each cannula was

secured with dental cement anchored to the skull with stainless steel screws (0.80 x 3/32"). An

obturator that extended 1.2 mm beyond the guide cannula tip was inserted at the time of surgery

and removed on the day of the experiment at the time that an intracranial inj section was

administered. Following surgery, each rat was given at least 7 days to recover from surgery.

Injection Procedure

Following recovery from surgery, each of the rats was handled for 5 minutes on one day.

Two days later the rats were given 3H-N/OFQ injections. Each rat received a 1.0 Cll (ICV, n= 5)

or 0.5 Cll (intra-amygdala, n= 5, and intra-BNST, n= 5) injection of aECF containing 1.0 nmole

N/OFQ plus 0.01 nmole 3H-N/OFQ. These inj sections were administered over a 2-minute period,

and the inj ector was left in place for 3 additional minutes to allow for diffusion. Each rat was









rapidly decapitated 12 minutes following the start of the 3H-N/OFQ injection. This time point

corresponded with the time at which the rats in Experiment 1 were removed from the open Hield.

Immediately after decapitation (approximately 15 min following the start of the 3H-N/OFQ

inj section each brain was removed, frozen in 2-methylbutane at -400C, and stored at -800C.

Later, each brain was sectioned at 20 Clm and placed on x-ray film (Kodak X-Omat) at -800C for

12 weeks. Following fi1m exposure, the brain sections were stained with cresyl violet for further

cannula placement verification and anatomical evaluation of the fi1m images.

Results

Intracerebroventricular Injections

Inj sections of 3H-N/OFQ into the right lateral ventricle produced differing diffusion

patterns depending on whether there was a successful ventricular placement or not. Of the 5 ICV

placements, cresyl violet staining revealed 1 clear hit, 3 marginal hits, and 1 miss. In the rat with

the clear ventricular placement (Fig. 3-1), 3H signal was seen in all aspects of the right lateral

ventricle (Fig. 3-la and b), continuing in more posterior regions into the dorsal and ventral 3rd

ventricle (Fig. 3-la-d), through the cerebral aqueduct, and into the 4th ventricle (Fig. 3-le and f).

From these locations there was visible diffusion approximately 0.5-1mm into the tissues,

reaching the lateral septum; medial and lateral subnuclei of the BNST, striatum; subfornical

organ; numerous thalamic nuclei; paraventricular, suprachiasmatic, posterior, and arcuate nuclei

of the hypothalamus; medial preoptic area; Eimbria; periaqueductal gray; some cerebellar regions

(particularly those that emerge near the 4th ventricle); tegmental nuclei; and locus coeruleus. In

general, signal was visible around the ventricular spaces throughout the anterior to posterior

extent.

In the rats with marginal ventricular hits (e.g., the cannula tip was located at the edge of

the corpus callosum near the ventricular wall) there was some visible 3H signal in the right lateral









ventricle, but only faint signal in the dorsal and ventral 3rd ventricle. No signal was visible in

the more posterior cerebral aqueduct or 4th ventricle. In these cases the diffusion from the

ventricular regions was much lighter, primarily reaching the lateral septum and striatum.

In the rat with a clear ventricular miss (the cannula tip was located in the corpus callosum

and did not pierce the ventricle) 3H signal is very faint in the right lateral ventricle and only

located in a very limited anterior to posterior span (less than 1mm). In this rat, as well as those

with marginal hits, the 3H-N/OFQ was visible primarily as an inj section bolus dispersed over the

corpus callosum.

In all cases, there was substantial signal along the edges of the cannula track, suggesting

that the drug diffused up along the sides of the cannula implant. This generally led to signal

affecting the cingulate, Ml, and M2 cortices, lateral septum, and striatum.

Intra-Amygdaloid Injections

All 5 of the intra-amygdaloid placements displayed visible signal over the amygdala. In

general, substantial signal was visible along the edges of the cannula track with an inj section

bolus of approximately 1-1.5mm radius ventral to the cannula tip. This bolus was typically

visible over the central amygdala, medial amygdala, basolateral amygdala, basomedial

amygdala, and internal capsule (Fig. 3-2a). Considering some variability in placement, other

structures that occasionally displayed signal included the BNST-intraamygdaloid nucleus,

anterior amygdala, reticular thalamus, lateral hypothalamus, and substantial innominata.

As with the ICV placements, all amygdaloid placements displayed signal along the edges

of the cannula track, affecting structures such as the S1 cortex and the striatum. To verify that

this was occurring following the injections (and not as an effect of the removal of the cannula

following decapitation), one intra-amygdaloid implanted brain was rapidly frozen prior to

removing the cannula. In this instance there was still substantial signal visible around the









cannula track, suggesting that this was, in fact, occurring during and following the inj sections

(Fig. 3-2b).

Intra-BNST Injections

All 5 of the intra-BNST placements displayed visible signal over the BNST. However,

they also all displayed some ventricular involvement, even when the cannula did not apparently

pierce the ventricular wall. Observation following cresyl violet staining revealed 1 placement

that did not appear to pierce the ventricle, 1 with marginal entry into the right lateral ventricle,

and 3 that fully passed through the right lateral ventricle.

With the one placement that did not pierce the ventricle, there was visible signal over the

medial BNST (Fig. 3-3a). As was the case with the ICV and intra-amygdaloid placements, there

was visible signal along the cannula track edges, reaching structures such as the S1 cortex, the

striatum, and some thalamic nuclei. As mentioned above, despite no obvious penetration into the

ventricles, there was still visible signal in the lateral and 3rd ventricles (Fig. 3-3b). From the

ventricular spaces there was faint diffusion visible into the subfornical organ; septum; and the

paraventricular, suprachiasmatic, and arcuate nuclei of the hypothalamus; and the median

emmnence.

In the case of the more marginal ventricular penetration, there was visible signal over the

medial and lateral BNST, anterior commissure, diagonal band, ventral pallidum, substantial

innominata, and preoptic areas (Fig. 3-3c). The visible signal along the cannula track was

evident over the Ml cortex, the lateral and medial septum, and the striatum. There was signal

visible in the right lateral ventricle; however, in more posterior regions the signal was very faint

(Fig. 3-3d). Here there was slight visible signal in the cerebral aqueduct with some diffusion into

the periaqueductal gray.









In the cases where the cannula passed through the right lateral ventricle, there was visible

signal over the medial and lateral BNST and anterior commissure (Fig. 3-3e). In some cases

there was signal over the preoptic areas, hypothalamic nuclei (PVN, arcuate, lateral), and ventral

pallidum. The signal along the edges of the cannula track was generally visible over the Ml

cortex and the striatum. Additional signal greatly resembled ICV injections, with signal visible

in the right lateral ventricle, dorsal and ventral 3rd ventricle, cerebral aqueduct, and 4th ventricle.

Diffusion from these ventricular regions affected structures such as the septum; subfornical

organ; thalamic nuclei; periventricular, paraventricular, arcuate, ventromedial, dorsomedial, and

anterior nuclei of the hypothalamus; and the medial preoptic area. In more posterior regions,

there was greater variability in the signal displayed. Two of the 3 placements displayed visible

signal in the cerebral aqueduct with diffusion into the periaqueductal gray (Fig. 3-3f). One of

these continued to display clear signal into the 4th ventricle with diffusion over the emerging

cerebellum, the tegmental nuclei, and the locus coeruleus.

Discussion

Inj sections of 3H-N/OFQ into the right lateral ventricle revealed differing diffusion patterns

depending on the accuracy of the placement. In our behavioral experiments, only rats that had a

cannula placement that was clearly within the lateral ventricle were used. This was verified by

cresyl violet staining and visualization of the cannula tip. Therefore, the diffusion pattern that is

depicted in Figure 3-1 is the best representation of the diffusion that occurred in the rats in the

behavioral studies. Accordingly, the potent hormonal and behavioral effects seen following

ICV inj sections were likely due to ipsilateral effects in rostral brain regions, with potential

bilateral involvement of periventricular, midline structures at more caudal sites. The sites that

were labeled with 3H-N/OFQ and are most likely to be involved in anxiety-related responses

include the lateral septum, the BNST, the medial preoptic area, the periaqueductal gray, the










tegmental nuclei, and the locus coeruleus. Interestingly, substantial diffusion into the amygdala

from the ventricles was not observed, although our behavioral study suggests that the amygdala

is involved in anxiogenic responses to N/OFQ. It is important to note that 3H produces a faint

signal, requiring months of film exposure. Thus the possibility cannot be ruled out that a small

amount of the ICV-inj ected N/OFQ might diffuse as far as the amygdala.

The size and shape of the diffusion patterns after lateral ventricle, BNST, and amygdala

inj sections are in agreement with previous analyses using other compounds (puromycin, CORT,

insulin-like growth factor) that were inj ected into the hypothalamus, frontal cortex, or lateral

ventricle (see Renner et al., 1984; Diorio et al., 1993; Nagaraja et al., 2005). The localization

observed following intraparenchymal inj sections suggests that the behavioral and hormonal

effects are primarily due to actions within the target structures. However, with all of the

inj sections (ICV, intra-BNST, and intra-amygdaloid), there was significant diffusion up the

cannula track. Again, this is in agreement with previous diffusion studies (for example see

Renner et al., 1984). This does not appear, however, to contribute to the behavioral or hormonal

effects observed in chapter 2. In those experiments, anatomical controls were included. In all

the anatomical controls, there would be diffusion back up the cannula track affecting the same

general extra-amygdaloid and extra-BNST sites as the targeted injections did. The lack of

behavioral and hormonal effects in the anatomical controls confirms that the effects of the BNST

and amygdaloid inj sections were indeed mediated at the target structures.

On the other hand, the possibility cannot be ruled out that the BNST inj sections of N/OFQ

described in chapter 2 produced some effects via diffusion into the ventricles, since some

ventricular diffusion was apparent even when the cannula did not pierce the lateral ventricle.

While our analyses of anxiety-related behaviors or circulating CORT did not include data from










rats in which clear penetration of the ventricle was observed, this may not have eliminated all

ventricular involvement.







































Figure 3-1. Representative x-ray images of 3H-N/OFQ diffusion following ICV inj sections
overlaid on the corresponding cresyl violet stained section (A, C, and E) and x-ray
images alone (B, D, and F). (A, B) Inj sections into the right lateral ventricle resulted in
strong 3H signal in the ipsilateral ventricle with some diffusion into the contralateral
ventricle and the 3rd ventricle. Further diffusion from these ventricles is most
prominent in the periventricular structures within approximately 0.5-1mm from the
ventricular wall. Diffusion continues posteriorly through (C, D) the 3rd ventricle and
(E,F) into the 4th ventricle.





























Figure 3-2. Representative x-ray images of 3H-N/OFQ diffusion following intra-amygdaloid
inj sections overlaid on the corresponding cresyl violet stained section. (A) Diffusion of
3H-N/OFQ from these placements is generally localized to the inj section site with
diffusion throughout the amygdaloid complex. (B) In addition there is substantial
diffusion along the cannula track that occurs during the inj section procedure, as
evidenced by freezing the tissue prior to cannula removal.















I


B


A


L;


Figure 3-3. Representative x-ray images of 3H-N/OFQ diffusion following intra-BNST inj sections
overlaid on the corresponding cresyl violet stained section (A, C, and E) and x-ray
images alone (B, D, and F). (A,B) Diffusion of 3H-N/OFQ from successful BNST
placements, with no ventricular piercing, results in 3H signal that is generally
localized to the inj section site with diffusion across numerous BNST sub-nuclei and
along the cannula track. With these placements there is slight diffusion into the
ventricles. (C, D) Cannulae that pierce the lateral ventricle or (E, F) fully pass
through it result in inj sections with diffusion into the posterior 4th ventricle and
surrounding structures. In our anxiety experiments, rats with placements piercing the
ventricle were removed from the experiments.









CHAPTER 4
NOCICEPTIN/ORPHANIN FQ AND NOP RECEPTOR GENE REGULATION AND
NOCICEPTIN/ORPHANIN FQ BINDING TO THE NOP RECEPTOR AFTER SINGLE OR
REPEATED SOCIAL DEFEAT EXPOSURE

Background

Administration of the NOP receptor antagonists J-1 13397, [Nphe ]-nociceptin (1-13)-NH2

and UFP-101 have antidepressant effects in animal models of depression (Redrobe et al. 2002;

Gavioli et al. 2003; 2004). This raises the possibility that the N/OFQ-NOP system may play an

important role in stress-associated psychopathology. If N/OFQ neurotransmission is truly

implicated in these disorders, it seems reasonable to expect that severe or repeated stress might

cause dysregulation of the expression of genes and/or proteins in the N/OFQ-NOP receptor

system. In fact, Devine and colleagues (2003) found that the system is tightly regulated. Acute

restraint stress caused a decrease in the N/OFQ content in the forebrain of rats. This decrease

occurred rapidly and was replenished within a 24 hour period. Thus, it appears that there is a

release of N/OFQ from basal forebrain neurons following acute stress exposure and the content

is restored rapidly in those neurons. Following from this, the primary aim of this study was to

examine the gene regulation of N/OFQ and the NOP receptor in response to acute and repeated

social stressors.

N/OFQ is cleaved from a precursor protein, prepro-N/OFQ. In rats, prepro-N/OFQ is 181

amino acids, containing one copy of N/OFQ at the 135-151 amino acid positions (Nothacker et

al., 1996). Therefore, the gene regulation of prepro-N/OFQ was examined using in situ

hybridization with a radiolabelled riboprobe complimentary to the prepro-N/OFQ mRNA. In

addition, repeated N/OFQ release might affect gene regulation of the NOP receptor, a 367 amino

acid protein (Wang et al., 1994). Therefore, in situ hybridization was conducted using a










radiolabelled riboprobe aimed at the NOP receptor mRNA in order to explore changes in the

receptor' s gene expression.

Changes in receptor mRNA expression, however, may not accurately represent changes in

protein synthesis. Post-transcriptional processes could destroy the mRNA, blocking protein

synthesis. Additionally, post-translational mechanisms could result in the breaking down of the

protein or in the packaging of receptors in vesicles for later use, but not resulting in insertion of

the receptors into the membrane. To determine if changes in receptor mRNA expression results

in changes in receptor insertion and functionality, a preliminary examination of the binding of

N/OFQ to the NOP receptor was conducted using autoradiography with 125I-N/OFQ.

In order to expose rats to social stressors, the social defeat procedure described in chapter 1

was used. This allowed the examination of changes in gene regulation in response to a single,

acute social defeat, repeated social defeat with no acute exposure, and repeated social defeat with

an acute exposure.

Methods

Animals

Forty-eight male Long Evans rats and 24 female Long Evans rats (Harlan, Indianapolis)

were used in this experiment. Twenty-four of the male rats and the 24 female rats were used as

resident pairs in the social defeat procedure. These male rats, weighing 400-500g at arrival (600-

700g at the time of the experiment), were pair-housed prior to vasectomy surgery, and then

singly housed for 1 week during recovery. The females (200-225g at arrival) were pair-housed

until the males recovered from surgery, at which time the males and females were housed

together. Four of the male rats were used as intruders for the purposes of training the resident

males. The remaining 20 males, weighing approximately 300g, were used as experimental

intruder rats. The intruder rats were pair-housed throughout the course of the experiment. All of









the rats were housed in 43 x 21.5 x 25.5 cm polycarbonate cages on a 12hr-12hr light-dark cycle

(lights on at 7:00 am) in a climate-controlled vivarium (temperature 21-23oC, humidity 55-60%).

Standard laboratory chow and tap water were available a~d libitum.

Drugs

Ketamine and xylazine were both obtained from Henry Schein (Melville, NY) and mixed

as described in Chapter 2. Ketorolac tromethamine (30 mg/ml) and AErrane (99.9 % isoflurane)

were also purchased from Henry Schein.

Surgery

Each of the male resident rats was vasectomized under ketamine-xylazine anesthesia (62.5

mg/kg ketamine + 12.5 mg/kg xylazine, i.p. in a volume of 0.75 ml/kg). Ketorolac tromethamine

(2 mg/kg, s.c.) was inj ected for analgesia at the time of surgery. AErrane was administered as

supplemental surgical anesthesia as necessary. Vasectomy was completed by making a 1 cm

ventral midline incision just rostral to the penis. Each vas deferens was isolated using forceps

and a 0.5 cm section of each duct was removed using a mini cautery tool. The abdominal wall

was sutured with absorbable 4-0 "Ethilon" monofilament nylon non-wicking suture (Ethicon

Inc.), and the external incision was closed with stainless steel wound clips (9mm, World

Precision Instruments Inc.). Following surgery, each rat was returned to its home cage and

allowed 7-10 days for recovery, at which time the wound clips were removed.

Social Defeat Procedure

The resident male and female rats were pair-housed for at least 10 days and each of the

male resident rats was trained and tested for territorial behavior prior to introducing the

experimental intruder rats. In each training session, resident females were removed from the

home cage 10 minutes before resident-intruder encounters. After 10 minutes, the intruder was

placed into the cage with the resident. The encounter continued for 5 minutes or until the









intruder displayed submissive behavior defined as entering into a supine posture (Fig. 4-1) 3

times for at least 2 seconds each or freezing for a total of 90 seconds. Encounters were also

terminated if either rat was severely bitten, which rarely occurred. The resident rat was

considered dominant if the intruder submitted as defined above. The 14 residents that most

consistently displayed dominance behaviors at the end of the training period were used for the

experiment.

The social defeat procedure with the experimental intruder rats occurred in 2 stages. The

first stage was identical to the training session and was terminated by the same criteria (3

submissions, 90 seconds of freezing, 5 minutes maximum, or severe biting). Immediately

following termination of stage 1, each intruder rat was placed, individually, into a 10cm x 10cm

x 15cm (inner dimensions) double-walled wire mesh cage and placed back into the resident's

cage to allow further stress exposure while protecting the rat from potential injury (Fig. 4-2).

The intruder remained in the cage until 10 minutes had passed from the start of stage 1. This

allowed for equalization of the duration of stress exposure of the intruder rats regardless of how

quickly stage I was terminated. Following the 10-minute session, the intruder rat was returned to

its home cage and the female was returned to the resident' s cage. The social defeat procedure

was conducted over 6 days wherein specific treatment groups were exposed to differing amounts

of repeated and/or acute exposure (Table 1). Group 1 experienced no social defeats on any of the

6 days (No Stress Controls). Group 2 experienced a social defeat encounter on the 6th day only

(No Repeated, Acute). Group 3 experienced a social defeat encounter on each of the first 5 days,

but did not experience an encounter on the 6th day (Repeated, No Acute), and group 4

experienced social defeat encounters on all 6 days (Repeated, Acute). On the 6th day, all of the









intruder rats were rapidly decapitated at 3 hours following the start of the defeat session, or at the

equivalent time in the groups that were not acutely defeated before termination.

Immediately after termination of the rats, each brain was removed, frozen in

2-methylbutane at -40oC, and stored at -80oC until use. The thymus glands, adrenal glands, and

spleens were dissected out and weighed for examination of the health/stress status of each rat.

The social defeat interactions were recorded and subsequently scored for the number of

defeats and total time spent in submissive postures (total time defeated) for each interaction.

There was an uneven number of rats per group, with 4 rats included in groups 1 and 4 and 6 rats

included in groups 2 and 3. The in situ hybridization procedure has limitations in the number of

slides that can be processed per experiment; therefore, the number of rats was reduced to 4 per

group in all 4 groups. In group 4, there was variability in the number of defeats that each rat

received; therefore, the groups were balanced such that each group included some rats that

received a high number of defeats and some rats that had a low number of defeats.

In Situ Hybridization

Each brain was sectioned into 15 Clm slices and mounted onto polylysine-subbed slides.

Every third section was processed for prepro-N/OFQ, NOP receptor mRNA, or NOP receptor

autoradiography. The 504 base cDNA fragment corresponding to the 5' end of the coding region

of the rat prepro-N/OFQ (Fig. 4-3) was cloned into plasmid pAMP and was provided by Dr.

Olivier Civelli. The 529 base cDNA fragment corresponding to 102 bases in the 3' untranslated

region through 427 bases of the open reading frame of the NOP receptor (Fig. 4-4) was cloned

into BSSK and was provided by Dr. Huda Akil. Antisense and sense riboprobes were generated

and labeled with 35S-UTP (>1000 Ci/nmole; GE Healthcare). The pAMP plasmid was linearized

with EcoR1 and transcribed with SP6 polymerase to generate the antisense prepro-N/OFQ

riboprobe. This plasmid was also linearized with HindIII and transcribed with T7 to yield the









sense probe. The BSSK plasmid was linearized with EcoR1 and transcribed with T7 to generate

the antisense riboprobe for NOP receptor mRNA, and this plasmid was also linearized with

Xhol and transcribed with T3 to yield the sense probe for the NOP receptor. The brain sections

were prepared for hybridization by soaking in 4% formaldehyde for 1 hour, then rinsing in 3

washes of 2x SSC and one wash of ddH20 all at room temperature (RT). The sections were

soaked in 0.1M Triethanolamine plus 0.25% vol/vol acetic anhydride for 10 min., then rinsed in

ddH20 and dried using graded ethanols (50, 70, 80, 95, 95, 100, and 100%). The radiolabelled

riboprobes were prepared in 50% hybridization buffer and were hybridized to the mounted

sections. The slides were coverslipped and incubated overnight in humidified boxes at 55oC.

Coverslips were then removed and the sections were treated with RNAse A (to remove single-

stranded, nonspecific label), then washed in 2x SSC (RT), lx SSC (RT), and 0.1x SSC (67oC) to

further remove excess label. The sections were dried in graded ethanols from 50% to 100%.

Once the sections and slides were completely dried they were placed on Kodak X-OMAT fi1m in

x-ray cassettes and exposed at room temperature for 1 week (NOP receptor) or 2 weeks

(N/OFQ). The fi1ms were developed then photographed and digitized with an MCID camera and

software system (Imaging Research Inc., Canada).

Due to the limitations in the total number of slides that can be processed in any single in

situ hybridization experiment, separate experiments were conducted for the anterior and posterior

halves of the brain for each mRNA probe. This division was between -3.6 (last anterior section)

and -4.8 (first posterior section) mm posterior to bregma, according to the Paxinos and Watson

(1998) rat brain atlas. Optical densities were measured in selected brain regions that are known

to participate in physiological stress responses and/or emotional regulation. Those regions were

the MPFC; the dorsolateral, interomediolateral, and ventrolateral septum (DLS, ILS, andVLS,










respectively); the anteromedial BNST, the posteromedial BNST, the lateral BNST, and the

ventromedial BNST; the CeA, MeA, BMA, BLA, and LA; the PVN, arcuate, and ventromedial

(VMH) hypothalamus; the MPOA; the reticular nucleus and zona incerta of the thalamus; CA1,

CA2, CA3, and dentate gyrus regions of the hippocampus; the substantial nigra pars reticularis,

pars lateralis, and pars compact; the periaqueductal gray; the dorsal raphe; and the locus

coeruleus.

Autoradiography

All sections from the 4 groups were processed together for autoradiographic analysis of

N/OFQ binding to the NOP receptor so that identical radioligand binding conditions were used

across groups. Slides were incubated with 125 _14Tyr]-N/OFQ such that there were

approximately 6000 counts per minute per section. Controls for nonspecifie binding were also

incubated with 1 CLM N/OFQ. The iodinated probe was applied in an incubation chamber (RT,

60-80% humidity) in a buffer of 50 mM Tris HCI (pH 7.0), 1.0 mM EDTA, 0.1% BSA with

proteinase inhibitor consisting of 0. 1 mM phenylmethylsulfonyl fluoride, 1 Cpg/ml aprotinin, 1

Cpg/ml leupeptin, 1.0 Cpg/ml pepstatin, and 1.0 mM iodoacetamide. The incubation was

terminated after 1 hour with four 4-minute washes in 50 mM Tris HCI on ice (pH 7.0). The

sections were then rinsed in ddH20 to remove excess salt. Dry sections were placed on Kodak

X-OMAT film in x-ray cassettes and exposed at room temperature for 5 days. The films were

developed then photographed and digitized with the MCID camera and software system.

The total number of slides did not exceed the number that could be processed at one time,

so all sections were processed together. Optical densities were measured in the same regions as

in the in situ hybridization experiments.










Densitometry and Statistics

Potential differences between the repeated and the repeated plus acute groups in the

number of defeats and the total time defeated over the first 5 days were analyzed by a 2x5

repeated measures two-way ANOVA. All significant effects (p< 0.05) were further analyzed

using Bonferroni post-tests. Additionally, potential differences between the acute and repeated

plus acute groups in the number of defeats and the total time defeated on the 6th day were

analyzed using T-tests.

Densitometric analysis of radioactive signal was analyzed using the MCID Basic software.

A standard outline, using the Paxinos and Watson Rat Brain Atlas (1998), was determined for

each region analyzed. Bilateral optical density measures were sampled from each region. If

multiple sections were used per region, the optical densities for these sections were integrated to

create one data point per rat per region per probe. Background measures were taken from the

striatum and cerebellum since these are regions with very low levels of expression of N/OFQ

mRNA, NOP receptor mRNA, and NOP receptor protein. Background values were subtracted

from values obtained in the analyzed regions to control for potential differences in non-specific

binding. The group means were compared by a one-way ANOVA for each region analyzed. All

significant effects (p< 0.05) were further analyzed using Dunnet' s post-tests.

In order to analyze differences in organ masses between the stressed and non-stressed

groups, group means were compared by one-way ANOVAs for adrenal gland weights, thymus

gland weights, and spleen weights.

Results

Social Defeats

Whereas I attempted to balance the stress-exposed groups by including the data from some

rats that were defeated multiple times and some rats that were defeated fewer times, there was









still variability in the number of defeats between groups (Fig. 4-5). In general, over the first 5

days the rats experiencing repeated defeats and those experiencing repeated plus acute defeats

were exposed to, on average, 11.25 defeats and 9.75 defeats, respectively. However, these

differences were not statistically significant (F(1,24)= 1.385, p>0.05). Additionally, there were no

significant differences in the number of defeats between days 1 through 5 (F(4,24)= 1.068, p>0.05)

or in the interaction between stress group and day (F(4, 24)= 1.748, p>0.05). On the 6th day the

acutely defeated group and the repeatedly plus acutely defeated group received, on average, 3

defeats and 1.25 defeats, respectively. Again, though, these differences were not statistically

significant (t(6)= 1.849, p>0.05).

There was also some observed variability in the total time defeated (Fig. 4-6). The

repeatedly stressed group experienced an average total of 104.75 sec of defeat over the first 5

days, and the repeatedly plus acutely stressed group experienced an average total of 136.75 sec

of defeat over the first 5 days. However, these differences were not statistically significant

(F(1,24)= 2.796, p>0.05). There were also no significant differences in the time defeated between

days 1 through 5 (F(4,24)= 1.286, p>0.05) or in the interaction between stress group and day

(F(4,24)= 1.016, p>0.05). On day 6, the acutely stressed group was defeated for an average 20 sec

while the repeatedly plus acutely stressed group was defeated for an average of 12.5 sec. Again,

these differences were not statistically significant (t(5)= 0.756, p>0.05).

In2 Situ Hybridization

Sense controls for prepro-N/OFQ and NOP receptor mRNAs displayed no specific signal.

The background levels were approximately equal to those treated with antisense probes, and

none of the sampled regions expressed signal substantially higher than background after

hybridization with the sense probes (Fig. 4-7). Therefore, signal measured from antisense-

treated sections can be considered specific for prepro-N/OFQ or NOP receptor mRNA.










Prepro-N/OFQ. There were no significant differences across groups in prepro-N/OFQ mRNA

expression in any of the regions examined. However, there were consistent trends toward

elevated expression in several limbic regions following acute social defeat exposure. These

elevations occurred in the dorsolateral and ventrolateral septal nuclei (Fig. 4-8, 4-10), all the

nuclei of the BNST (Fig. 4-9, 4-10), the CeA and MeA (Fig. 4-1 1, 4-13), the zona incerta and

reticular nucleus (Fig. 4-12, 4-13), and the dorsal raphe (Fig. 4-14, 4-15). Signal was low and

there were no visible trends in the MPFC, BMA, BLA, LA, PVN, arcuate, VMH, MPOA CA1,

CA2, CA3, DG, SNpr, SNpl, SNpc, PAG, or LC (data not shown).

NOP Receptor. There were significant stress-induced increases in NOP receptor mRNA

expression in several important limbic regions. In the septum (Fig. 4-16, 4-17), there were

significant elevations in NOP receptor mRNA expression following acute stress exposure in both

the DLS (Fig. 4-16a; F(3, 15)= 7.134, p<0.01), and the VLS (Fig. 4-16c; F(3, 15)= 3.656, p<0.05).

The ANOVA for the ILS revealed a significant overall effect that appears to result from a trend

toward increases in NOP receptor mRNA expression in the acutely defeated groups, (Fig. 4-16b;

F(3, 15)= 3.52, p<0.05). However, the post-tests revealed no significant differences between any

of the stressed groups and the control group for NOP receptor mRNA expression in the ILS. In

the BNST, there was a significant elevation in NOP receptor mRNA expression in the

ventromedial region following repeated social defeat exposure (Fig. 4-18, 4-19; F(3, 14)= 6.132,

p 0.05). In the amygdala, NOP receptor mRNA was elevated in several regions, particularly

following acute social defeat exposure (Fig. 4-20). In the CeA, there were elevations in NOP

receptor mRNA following exposure to acute and repeated plus acute social defeat exposure (Fig.

4-20a, 4-21; F(3, 15)= 6.449, p<0.01). In the MeA, these elevations occurred following acute social

defeat (Fig. 4-20b; F(3, 15)= 5.174, p<0.05). There were also significant elevations in NOP










receptor mRNA following acute social defeat in the BMA (Fig. 4-20c, 4-19; F(3, 15)= 6.02,

p<0.01), the BLA (Fig. 4-20d, 4-21; F(3, 15)= 8.66, p<0.01), and the LA (Fig. 4-20e; F(3, 15)=

9.802, p<0.01). In the PVN there were significant elevations in NOP receptor mRNA in all

stress-exposed groups (Fig. 4-21, 4-22a; F(3, 15)= 10.47, p<0.01). Differences in NOP receptor

mRNA expression approached significance in the VMH (Fig. 22b, F(3, 15)= 3.398, p=0.0536) and

in the zona incerta (Fig. 22c, F(3, 15)= 3.25, p=0.0599). These differences appear to be due to

elevations following exposure to acute and to repeated plus acute social defeat. There were no

significant differences in NOP receptor mRNA expression in the reticular nucleus of the

thalamus, in any of the hippocampal regions, or in any of the midbrain or brainstem regions.

Autoradiography

Controls for non-specifie binding displayed little to no signal and background levels were

approximately equivalent to those treated with 125 _14Tyr]-N/OFQ (Fig. 4-21). Therefore, signal

measured from 125 _14Tyr]-N/OFQ -treated sections can be considered specific for binding to the

NOP receptor. However, there were no significant differences in N/OFQ binding between any of

the stress-exposed groups for any of the regions analyzed (Figs. 4-24 to 4-30).

Organ Masses

There were no significant differences in adrenal weights (Fig. 4-31a), thymus gland

weights (Fig. 4-31Ib), or spleen weights (Fig. 4-31c) between the non-stressed controls and the

socially defeated rats.

Discussion

In general, basal expression of prepro-N/OFQ and NOP receptor mRNAs, as well as

NOP receptor binding agrees with previous anatomical examinations (Neal et al., 1999a and b).

Stress exposure appears to alter these basal levels, particularly in the expression of NOP receptor

mRNA. In the BNST, it appears that the N/OFQ-NOP receptor system plays a role in limbic










plasticity following repeated, severe stress exposure. Additionally, in the PVN, NOP receptor

regulation appears to be sensitive to acute and repeated social stressors. This suggests that

stressor exposure might produce significant changes in HPA responses to N/OFQ.

Prepro-N/OFQ mRNA

Devine and colleagues (2003) previously found release ofN/OFQ following acute stress

exposure, and the N/OFQ content was replenished within 24 hours. In order for the peptide

content to be replenished, transcription and protein synthesis need to occur. Therefore, it was

expected that acute and repeated plus acute social defeat would produce increases in

prepro-N/OFQ mRNA. While there were not statistically significant changes, there were

consistent trends toward elevated mRNA expression following acute social defeat exposure.

These trends were repeated over multiple limbic structures, including the septum, the BNST, and

the amygdala. All of these regions are involved in responses to fear- and anxiety-provoking

stimuli (for example see Goldstein, 1965; Van de Kar et al., 1991; Gray et al., 1993; Calfa et al.,

2006; Menard and Treit, 1996; Lee and Davis, 1997; Walker and Davis, 1997; Vyas et al., 2003).

Several of the regions where trends were present are in the basal forebrain, the same region

where Devine and colleagues (2003) found release of N/OFQ. This concordance and the

consistency of the trends over multiple limbic structures suggest that the effect is a real one. One

potential reason for the lack of statistical significance is the variability in the amount of stress

exposure, with some rats experiencing fewer social defeats and less total time in submissive

postures. While between groups differences in the number and total duration of defeats were not

statistically significant, we do not know if these variables are critical in determining the affective

and physiological responses of the intruders (or if the mere presence of the resident is the

important factor). It is possible that the variability in residents' behaviors could contribute to

variability in the mRNA expression of intruders, reducing statistical power. Another









consideration is that Devine and colleagues (2003) found that N/OFQ content was replenished 24

hours later. It is possible that in the current study the timepoint selected was not during the peak

of the mRNA upregulation. It is also possible that there is a relatively small upregulation of

mRNA (as we observed in the consistent trend across many forebrain regions), and that N/OFQ

is slowly replenished over an extended portion of that 24 hour period.

NOP Receptor mRNA

There were significant changes in NOP receptor mRNA expression in several brain regions

following social defeat exposure. NOP receptor mRNA expression was significantly increased

in several limbic-hypothalamic regions, including the septum, the BNST, the amygdala, and the

PVN. These changes primarily occurred following acute social defeat exposure, although in the

amygdala there were also increases in NOP receptor mRNA in the repeated plus acute group and

in the BNST there were increases in mRNA in the repeated (but no acute) group. This increase

in the BNST following repeated stress is interesting given the most recent hypothesis about the

roles of the amygdala and the BNST. It has been shown that the amygdala and the BNST can be

differentiated in terms of their roles in fear- and anxiety-like responses, with the amygdala

primarily involved in fear-like behaviors and the BNST involved in anxiety-like behaviors

(Walker and Davis, 1997; for review see Walker et al., 2003). It has been proposed that the

involvement of these structures in the response to stressor exposure is related to the length of the

exposure, with the amygdala involved in the early, immediate response and the BNST involved

in the later, long-term responses (Walker et al., 2003). Our findings are consistent with this

interpretation, with the amygdala showing changes in response to an acute social defeat and the

BNST showing changes in response to repeated defeat sessions. These results suggest that the

N/OFQ-NOP receptor system may respond differently, depending on the type and amount of

stress exposure and the region of the brain.









NOP receptor mRNA was also expressed in greater amounts in the PVN following all

social defeat regimens (acute, repeated, and repeated plus acute), as compared to the expression

in the no stress control group. It is known that administration of N/OFQ into the ventricles

produces activation of the HPA axis in unstressed rats and prolongs the activation in mildly

stressed rats (Devine et al., 2001). One likely site for this activation is the PVN, since this

structure contains the neurons that synthesize and release CRH, activating the HPA axis.

Therefore, NOP receptor upregulation in the PVN could suggest an increased sensitivity of the

HPA axis.

The upregulation of NOP receptor mRNA in response to stress exposure is somewhat

surprising given the increased release of N/OFQ following stressful events (Devine et al., 2003).

In some receptor systems, an increase in neurotransmitter release (or the overconsumption of a

drug that acts at the receptor) results in long-term desensitization of the receptor--a mechanism

that accounts for many tolerance effects. It has already been observed that rats display rapid

tolerance to the locomotor-inhibiting effects of N/OFQ (Devine et al., 1996); thus, the system is

capable of tolerance and the receptors may desensitize in response to N/OFQ release. There is

evidence that the NOP receptor does, in fact, undergo rapid desensitization (Dautzenberg et al.,

2001; Spampinato and Baiula, 2006). However, Spampinato and Baiula (2006) demonstrated

that at higher levels of N/OFQ exposure, the NOP receptor undergoes internalization and

recycling resulting in resensitization and, even, supersentization. These experiments, however,

are in artificial systems and it is not clear how these results compare to stress-induced release of

N/OFQ in vivo.

NOP Receptor Binding

There were no significant increases in N/OFQ binding in any of the regions analyzed,

despite the upregulation of NOP receptor mRNA in several regions. There may be some post-










transcriptional or post-translational mechanism interfering with the synthesis of the receptors or

with the insertion of the receptors into the cell membrane. However, it may be more likely that

the time point selected to examine binding was not the ideal time point. In the acute groups,

binding was examined 3 hours after stressor exposure. At this time point, protein synthesis and

receptor insertion may not have yet occurred. In the repeated but no acute groups, binding was

examined 27 hours after the last stressor exposure. At this point, receptor levels may have

returned to baseline. Devine and colleagues (2003) have already shown the peptide content is

replenished within 24 hours. Therefore, the N/OFQ-NOP receptor systems appears to be a

tightly regulated system in which changes in response to stressors are transient.

To fully examine the potential changes in receptor synthesis and insertion and potential

changes in binding, a more thorough analysis would be required. This would involve a full

Scatchard analysis to examine binding, protein analysis by immunohistochemistry under non-

permeabilizing and permeabilizing conditions (to assay membrane insertion), and protein

quantification by Western blot.

Organ Masses

There were no statistically significant changes in adrenal, thymus, or spleen masses in any

of the stress-treated groups. Chronic stress exposure tends to produce thymus and spleen

involution and adrenal hypertrophy (for examples see Selye, 1936; Bryant et al., 1991; Watzl et

al., 1993; Blanchard et al., 1998; Dominguez-Gerpe and Rey-Mendez, 2001; Hasegawa and

Saiki, 2002). The thymus glands appear to be most sensitive to stress exposure. Selye (1936)

found that even single exposures to potent systemic stressors caused rapid thymus involution.

There was a trend toward decreased thymus weights in all of the groups that received social

defeat, including the acute group. Therefore, there is some evidence in this experiment that the

social defeat procedure was potent enough to produce changes in thymus glands.






























Figure 4-1. Photograph of a social defeat interaction. The intruder is engaging in submissive
behaviors by displaying a supine posture while the resident is displaying dominant
behaviors by standing over the intruder. One defeat was counted when the rats
engaged in this interaction for at least 2 seconds prior to disengaging. This first stage
of the social interaction continued until 3 defeats occurred, the intruder froze for 90
total seconds, or until 5 minutes had elapsed.


Figure 4-2. Photograph of stage 2 of the social defeat procedure. Following 3 defeats or 5
minutes, the intruder was placed into a double-walled wire mesh cage placed into the
resident' s cage. The residents continued to display dominant behaviors by climbing
atop the cage and kicking bedding at the intruder. The intruder remained in this cage
until 10 minutes had elapsed from the start of the procedure.














Table 1: Groups of intruder rats

Groups Repeated stress Acute
stress
Day 1 Day 2 Day 3 Day 4 Day 5 Day 6
Group 1






Table 4-1. Social defeat regimen. Four groups of rats were exposed to differing amounts of
social defeat. Group 1 was not exposed to any social defeat but was left undisturbed
in their cages (No stress controls). Group 2 was exposed to a single social defeat
session on day 6 (Acute). Group 3 was exposed to one social defeat session on each
of 5 days, but no social defeat on day 6 (Repeated, No Acute). Group 4 was exposed
to one social defeat session on each of the 6 days (Repeated, Acute).


cctcttgctt
ctgctctcca
cacccggctc
ttcccccgcc
agccctgctg
cagcacctga
gaggcagatg
aaaaggtttg
cggttcagtg
cgcactatgc
cactgcaacc
gcaggccggg
acagcatgtc
ctaacatttt
gaggaacatg
gagctgttct
aaatggttaa


cccacggctg
gcgtgttcag
cgggcagctt
ctatctggac
atccagagct
agagaatgcc
cagagcctgt
ggggcttcac
agtttatgag
accagaatgg
catgagcate
agtcaggatt
tcaccacaat
aatggcccca
aaatcagacc
ttttgactga
taaa


caccatgaaa
cagctgtecc
caacctgaag
totttgcacc
cacgtecgct
gcgtgtcagg
cgcagatgag
tggggcccgg
gcagtacctg
taatgtgtag
caggtgagcc
cctccttacc
cctgttgcta
tottcttgct
tggggttttg
ttgtttgaaa


atcctgtttt
gaggactgcc
ctgtgcatcc
aaagccatgg
gctatttacc
agtgtggtgc
gccgatgagg
aagtcagccc
gtectgagca
ccagaaggag
cccgtacage
tgaggcactg
catcagagtg
catcctatgc
cctcaccact
caactttctc


gtgatgtect
tcacctgcca
tecagtgtga
ccagtgactc
agtcgaaage
aagcccgaga
tggagcagaa
ggaagttggc
tgcagtcaag
cccctcccag
atgtgtecac
aacacccgcg
tatttttgta
cctategtag
gccataactg
cattaaactt


gctgctcage
ggagaggctc
agagaaggtc
tgagcagctc
etcggagatg
cgcagagcct
gcagctgcag
caaccagaag
ccaacgccgg
ctgcaccggc
accaagacct
gcacctcccc
attactccag
ggccaggtga
gtttgtaaag
ctactgagca


translation=MKILFCDVLLLSLL SSVFSSCPEDCLTCQELPPSNKCLCEVPPWLTA
ASDSEQLSPADPELTSAALYQSKASEMQHLKRMPRVRSVVADEEDEPAEDVQQLKFGTA
KSARKLANQKRFSEFMRQYLVLSMQSSQRRRTLHQNGNV


Figure 4-3. Prepro-N/OFQ sequence. The 972 bases for the prepro-N/OFQ gene are displayed
(Accession # NM_0 13 007). The highlighted region represents the portion of the
sequence that was included in the prepro-NOFQ plasmid insert used for the in situ
hybridization. Sequencing was performed by the University of Florida DNA
Sequencing Core, Interdisciplinary Center for Biotechnology Research.












1 gcggccgcct
61 tgtgccgttg
121 tcctgctcca
181 aaatgagacc
241 tggactcaag
301 gaactgcctc
361 ttacatattt
421 cacagacatc
481 tatcgactac
541 ctatgtggct
601 ggctgttaat
661 gggttcagca
721 ggactattgg
781 gctgatcatc
841 ttcaggctcc
901 ggtggctgtg
961 gggtgttcag
1021 ctatgtcaac
1081 ctgctttaga
1141 tgtgcggagc
1201 gccagcatga
1261 acccaacacg
1321 tggagccttg
1381 agcaccatgg
1441 ccctccctgg
1501 gtggacatgc
1561 ctccctgctg
1621 actcttgtga
1681 gctgaacata
1741 cttaagcttg
1801 tacccgaggg
1861 cagtacatgg
1921 ataatcctgg
1981 ctaaggctct
2041 actgcttcta
2101 cacaacatgg
2161 atatgctctc
2221 tgctatcaga
2281 agtcaggatg
2341 acatatctga
2401 ctaaagtggc
2461 gaagagtcta
2521 tttctatggg
2581 gacctcggtc
2641 gacctgggct
2701 tctaga


ttctgctaag cattggggtc tattttggcc cagcttctga agaggctgtg


gaggaactgt
tactgggagg
gtaccccacc
gtcaccatcg
gtcatgtatg
aatctggcac
ctactgggct
tacaacatgt
atctgccacc
gtggccatat
caagtggaag
ggccctgtat
tctgtctgct
cgggagaagg
tttgtgggct
ccaggtagtg
agttgtctca
aagttctgct
attgccaagg
ctaggcgtgg
gagctcacac
aatggctttt
gacaggtcaa
tataggacca
ctggtgagcc
ccctggctct
ctacatgttg
cctggtattg
gcgttgcctt
tgagcatcag
agtctatgaa
gctatcttct
ttccctccaa
ggtgtgtggg
aggcaccaca
tctcgattct
ggtcaggagt
tgttctactc
gtaaggcctg
tcatttgcaa
taccttggag
tcagatatca
agatggtttc
tctggggagg


actgagtggc
tcttgtatgg
acctgctcct
tggggctcta
tcatcctcag
tggctgatac
tctggccatt
ttaccagcac
ctatccgtgc
gggccctggc
atgaagagat
tcgccatctg
acagcctcat
accgaaacct
gctggacgcc
agactgcagt
atcccattct
gtgcttcatc
atgttggcct
acctgcccat
aggtcactgc
cttttggatc
agcatcaagg
gagaggacca
catgtaggta
agctgggctc
tgtgctgttg
cagtggggag
ggagcgtctt
tggtttcttg
ggggagtcac
tggcaagatg
aaccactgtg
aggtaatcag
tgctggtctt
ctacaaactc
tgtactgcta
tatatccaca
agtgtgctgc
ggactattat
atctatttga
aaataccagc
atgtcatgca
ccagggttct


tttgcagggt
cagccacttt
caatgctagt
cttggctgtg
gcacaccaag
cctggtcttg
tgggaatgca
ttttactctg
ccttgatgtt
ttcagtggtt
cgagtgcctg
catcttcctt
gattcgacga
gcggcgtatc
tgtgcaggtg
tgccatcctg
ctatgctttc
cctgcaccgg
tggttgcaag
ggtgcctgtc
tctctaggtt
aggatgctca
tggtctccat
aaggaactga
ttcatgcttc
aacctgaggt
ctctcggcct
cattaatttt
ctacttctga
gatggctgtt
aattcatctg
acagtggggg
aactcttatc
gagaaagctt
gcctgcttag
cctcagttct
gaagcatact
gtgaccacct
caaattggag
ggtttggaat
tggttcacag
aacgttggat
gagaacctag
tcctttgaca


gacagcatgg
caagggaacc
cacagcgcct
tgcatcgggg
atgaagacag
ctaacactgc
ctctgcaaga
accgccatga
cggacatcca
ggtgttcctg
gtggagatcc
ttttccttca
cttcgtggtg
actcgactgg
tttgtcctgg
cgcttctgca
ctggatgaga
gagatgcagg
acttctgaga
agcccacaga
gaccctgaac
gtcctagagg
ggcctctgtc
atagaaacat
acttgactct
attgtagtgg
ttcagtattt
cttttaaagt
cttcactgat
ttctgaagat
gtactgccac
agacaagaca
ctacagactg
tgtggcctct
tacaggcagg
ccagcagagt
tgtagcttgg
gettcatata
gttggtatga
agcaatgggg
aagaggtttt
agattctgac
gctggttcct
cttgtgcggg


agtccctctt
tgtccctcct
tcctgcccct
ggctcctggg
ctaccaacat
ccttccaggg
ctgtcattgc
gcgtagaccg
gcaaagccca
ttgccatcat
ctgcccctca
tcatccctgt
tecgtctgct
tgctggtagt
ttcaaggact
cagccctggg
acttcaaggc
tttctgatcg
cagtaccacg
gcccatctac
cttgagcatc
aagacctttt
agattaagtt
ccacaacaca
tctctggctt
tcatgtagtc
ccacaggact
gagactggcc
gcagtcagat
tcttcccatc
tacctgctct
cagagcttcc
ttcggcaage
gtaggctgct
acagagcaga
ctcttttact
gaagagtggc
tagggttagg
gagctgatgc
ggcatgggaa
gtaaacgccc
cttttactga
gtgtcagaga
agccgttagc


translation=MESLFPAPYWEVLYGSHFQGNLSLLNETVHLLASFPGLTILYACGLNCMYL
RHTKMKTATNIYI FNLALADTLVLLTLPFQGTDI LLGFWPFGNALCKTVIAI DYYNMFTSTFTLTAMSVDRYVAICHP IRALDVRT
SSKAQ!AVNVAIWALASVVGVPVAIMGSAQ!VEDEEIECLVEIAQYGVACFLFSFIIPVLII SVCYSLMIRRLRGVRLLS
GSREKDRNLRRI TREVLVVRAVFVGCW\TPVQ!VFVLVQGLGVQPGSETAVAILFTLYVSLPLYAFLDENFKACFRKFCC
ASSLHREMG!VSDRVRSIAKDVGLGCKTSETVPRPA




Figure 4-4. NOP receptor sequence. The 2706 bases for NOP receptor gene are displayed
(Accession # NM_03 1569.2). The highlighted region represents the portion of the
sequence that was included in the NOP receptor plasmid insert used for the in situ
hybridization. Sequencing was performed by the University of Florida DNA
Sequencing Core, Interdisciplinary Center for Biotechnology Research.

























II _ _


50'
o 40








One Two Three Four Five Six
Day


Mean Number of Defeats per Day


j41
3


O 1


mA


SOne TwGo Thr~ee Four
Day


Five Slix


Figure 4-5. Number of social defeats per group per day. Values shown are group means & SEM
(n = 4 rats per group). A = acute stress group, R = repeated stress group, R+A =
repeated plus acute stress group.


Total Time Defeated per Day


mA


Figure 4-6. Total amount of time the rats were defeated per day. Values shown are group means
SSEM (n = 4 rats per group). A= acute stress, R= repeated stress, R+A= repeated
plus acute.









































Figure 4-7. Representative x-ray images of brain sections treated with sense riboprobes. Sections
treated with (A,B) N/OFQ sense strands and (C,D) NOP receptor sense strands did
not display any specific signal, and background levels were comparable to those of
antisense-treated sections.













"l"


,NS


Stress Exposure


0.11



0.0!


Stress Exposure


Figure 4-8. Prepro-N/OFQ mRNA expression in the septum. There were trends toward greater
mRNA expression in (A) the dorsolateral septum and (B) the ventrolateral septum
following exposure to acute social defeat. Values expressed are group means in
optical density & SEM (n = 4 rats per group). NS= no stress, A= acute stress, R=
repeated stress, R+A= repeated plus acute.



























NS A R R+A


?n ~ -- --~I I I


0.10-




0.05-


A R


Stress Exposure


Stress Exposure


0.10-


..


0.(


NS A R R+A


A R R+A


Stress Exposure


Stress Exposure


Figure 4-9. Prepro-N/OFQ mRNA expression in the bed nucleus of stria terminalis. There were
trends toward greater mRNA expression in the (A) anteromedial BNST, (B)

posteromedial BNST, (C) ventromedial BNST, and (D) lateral BNST following
exposure to acute social defeat. Values expressed are group means in optical density
SSEM (n = 4 rats per group). NS= no stress, A= acute stress, R= repeated stress,
R+A= repeated plus acute.


0.10.




0.05-





NS


0.10.









0.00
NS














































Figure 4-10. Representative x-ray images of brain sections showing prepro-N/OFQ mRNA
expression in the septum and BNST. Sections were exposed to 125I-labelled
riboprobes complimentary to segments of prepro-N/OFQ mRNA. (A) No stress
control rats display low to moderate signal in the dorsolateral septum,
intermediolateral septum, ventromedial septum, and lateral BNST. (B) Rats exposed
to acute social defeat displayed trends toward increased expression in these regions.











79













0.10-







NS A R R+A

Stress Exposure


B
0.10.


0.05



NS A R R+A

Stress Exposure



Figure 4-11. Prepro-N/OFQ mRNA expression in the amygdala. There were trends toward
greater mRNA expression in (A) the central amygdala and (B) the medial amygdala
following exposure to acute social defeat. Values expressed are group means in
optical density & SEM (n = 4 rats per group). NS= no stress, A= acute stress, R=
repeated stress, R+A= repeated plus acute.












A


Stress Exposure


Stress Exposure



Figure 4-12. Prepro-N/OFQ mRNA expression in the zona incerta and reticular nucleus of the
thalamus. There were trends toward greater mRNA expression in (A) the zona incerta
and (B) the reticular nucleus following exposure to acute social defeat. Values
expressed are group means in optical density & SEM (n = 4 rats per group). NS= no
stress, A= acute stress, R= repeated stress, R+A= repeated plus acute.


0.10



0.05



NS


A R R+A


0.10



0.05



0.00
NS


A R R+A











































Figure 4-13. Representative x-ray images of brain sections showing prepro-N/OFQ mRNA
expression in the amygdala, zona incerta, and reticular nucleus of the thalamus.
Sections were exposed to 125I-labelled riboprobes complimentary to segments of
prepro-N/OFQ mRNA. (A) No stress control rats display moderate signal in the
amygdala and low signal in the zona incerta and reticular nucleus. (B) Rats exposed
to acute social defeat displayed trends toward increased expression in these regions.
NS= no stress, A= acute stress, R= repeated stress, R+A= repeated plus acute.











P repro-NIO FQ mnRNA Express ion
in the Dorsal Raphe
0.10.







NS A R R+A
Stress Exposure

Figure 4-14. Prepro-N/OFQ mRNA expression in the dorsal raphe. There were trends toward
greater mRNA expression in the dorsal raphe following exposure to acute social
defeat. Values expressed are group means in optical density & SEM (n = 4 rats per
group). NS= no stress, A= acute stress, R= repeated stress, R+A= repeated plus acute.


Figure 4-15. Representative x-ray images of brain sections showing prepro-N/OFQ mRNA
expression in the dorsal raphe. Sections were exposed to 125I-labelled riboprobes
complimentary to segments of prepro-N/OFQ mRNA. (A) No stress control rats
display moderate signal dorsal raphe. (B) Rats exposed to acute social defeat
displayed trends toward increased expression in these regions. NS= no stress, A=
acute stress, R= repeated stress, R+A= repeated plus acute.










A
0.10.


0.05.


NS A R R+A
Stress Exposure

B
0.10.





NS A R R+A
St ress Exposure

C
0.10*





NS A R R+A
Stress Exposure

Figure 4-16. NOP rector mRNA expression in the septum. mRNA expression was significantly
greater in the (A) dorsolateral septum, (B) intermediolateral septum, and (C)
ventrolateral septum following exposure to acute social defeat. Values expressed are
group means in optical density & SEM (n = 4 rats per group). Significant differences
between the stress-exposed rats and the no stress controls are expressed as p < 0.05,
Y**p < 0.01. NS= no stress, A= acute stress, R= repeated stress, R+A= repeated plus
acute.










































Figure 4-17. Representative x-ray images of brain sections showing NOP receptor mRNA
expression in the septum. Sections were exposed to 125I-labelled riboprobes
complimentary to segments of NOP receptor mRNA. (A) No stress control rats
display low signal in the dorsolateral, intermediolateral, and ventrolateral septum. (B)
Rats exposed to acute social defeat displayed significantly greater mRNA expression
in these regions regions.










NOP mRNA Expression
in Ventromedial BNST









NS A R R+A

Stress Exposure

Figure 4-18. NOP receptor mRNA expression in the bed nucleus of stria terminalis. mRNA
expression was significantly greater in the ventromedial BNST following exposure to
repeated social defeat. Values expressed are group means in optical density & SEM (n
= 4 rats per group). Significant differences between the stress-exposed rats and the no
stress controls are expressed as ** p < 0.01. NS= no stress, A= acute stress, R=
repeated stress, R+A= repeated plus acute.










































Figure 4-19. Representative x-ray images of brain sections showing NOP receptor mRNA
expression in the bed nucleus of stria terminalis. Sections were exposed to 1251
labelled riboprobes complimentary to segments of NOP receptor mRNA. (A) No
stress control rats display low signal in the ventromedial BNST. (B) Rats exposed to
repeated social defeat displayed significantly greater mRNA expression in the
ventromedial BNST.











A B
0.10~ 0.10





CJ D



So o **a

NS A R R+A NS A R R+A
Stress Exposure Stress Exposure



0.10~ .

0.05



NS A R R+A N +
Stress Exposure te Epsr

Fiue4-0NOreetrmN exrsinithamgaamRAepesows







Figrgreat NP ecer mRNA expression in theceta amygdala. folowng repeated plusact


social defeat. Values expressed are group means in optical density & SEM (n = 4 rats
per group). Significant differences between the stress-exposed rats and the no stress
controls are expressed as p < 0.05 and ** p < 0.01. NS= no stress, A= acute stress,
R= repeated stress, R+A= repeated plus acute.



































Figure 4-21. Representative x-ray images of brain sections showing NOP receptor mRNA
expression in the amygdala and the paraventricular nucleus of the hypothalamus.
Sections were exposed to 125I-labelled riboprobes complimentary to segments of NOP
receptor mRNA. (A) No stress control rats display low to moderate signal in the
amygdala and PVN. Rats exposed to (B) acute and (D) repeated plus acute social
defeat displayed greater mRNA expression in the amygdala. Rats exposed to (B)
acute, (C) repeated, and (D) repeated plus acute social defeat displayed greater
mRNA expression in the PVN.











A
0.10.





NS A R R+A
Stress Exposure


B
0.10-





NS A R R+A
Stress Exposure


C
0.10.


0.05.


NS A R R+A
Stress Exposure

Figure 4-22. NOP receptor mRNA expression in the paraventricular and ventromedial nuclei of
the hypothalamus and in the zona incerta of the thalamus. (A) mRNA expression was
significantly greater in the PVN following any exposure to social defeat. In (B) the
ventromedial hypothalamus and (C) the zona incerta elevations in mRNA expression
following social defeat approached significance. Values expressed are group means in
optical density & SEM (n = 4 rats per group). Significant differences between the
stress-exposed rats and the no stress controls are expressed as p < 0.05 and ** p <
0.01. NS= no stress, A= acute stress, R= repeated stress, R+A= repeated plus acute.


































Figure 4-23. Representative x-ray images of brain sections treated with 1 CLM N/OFQ plus
125 _14Tyr]-N/OFQ Sections treated with 1 CLM N/OFQ plus 125 _14Tyr]-N/OFQ did
not display any specific signal, and background levels were comparable to those
sections treated with only 125 _14Tyr]-N/OFQ.







































u.00 a a g
NS A R R+A


u.UU g
NS A R R+A


0.10-



0.05-



0.00-


NS A R R+A

Stress Exposure











I III a m


0.10-



0.05-


Stress Exposure


0.10-



0.05-


I Ilill a


Stress Exposure


Figure 4-24. 125I_ 14Tyr]-N/OFQ binding to the NOP receptor in the septum. There were no
significant changes in binding following any stress exposure in (A) the dorsolateral
septum, (B) the intermediolateral septum, or (C) the ventrolateral septum. Values
expressed are group means in optical density & SEM (n = 4 rats per group). NS= no
stress, A= acute stress, R= repeated stress, R+A= repeated plus acute.











































Figure 4-25. Representative x-ray images of brain sections showing 125 _14Tyr]-N/OFQ binding
to the NOP receptor in the septum. Despite an increase in NOP receptor mRNA, (B)
the acutely stressed group did not display a significant increase in binding as
compared to (A) the no stress controls.











NIOFQ Binding to the NOP Receptor
in the Ventromedial BNST




ol




NS A R R+A

Stress Exposure


Figure 4-26. 125 _14Tyr]-N/OFQ binding to the NOP receptor in the ventromedial BNST. There
were no significant changes in binding following any stress exposure in the
ventromedial BNST. Values expressed are group means in optical density & SEM (n
= 4 rats per group). NS= no stress, A= acute stress, R= repeated stress, R+A=
repeated plus acute.







































Figure 4-27. Representative x-ray images of brain sections showing 125 _14Tyr]-N/OFQ binding
to the NOP receptor in the BNST. Despite an increase in NOP receptor mRNA, (B)
the repeatedly stressed group did not display a significant increase in binding as
compared to (A) the no stress controls.













0.10-



0.05-



0.00-


NS A R R+A


Stress Exposure


Stress Exposure


0.05-


0.05-


NS


Stress Exposure


Stress Exposure


0.05


NS


Stress Exposure



Figure 4-28. 125I_ 14Tyr]-N/OFQ binding to the NOP receptor in the amygdala. There were no
significant changes in binding following any stress exposure in (A) the central
amygdala, (B) the medial amydala, (C) the basomedial amygdala, (D) the basolateral
amygdala, or (E) the lateral amygdala. Values expressed are group means in optical
density & SEM (n = 4 rats per group). NS= no stress, A= acute stress, R= repeated
stress, R+A= repeated plus acute.


A R R+A


NS A R R+A


A R R+A


A R R+A










NIOFQ Binding to the NOP Receptor
in the PVN









NS A R R+A

Stress Exposure


Figure 4-29. 125 _14Tyr]-N/OFQ binding to the NOP receptor in the PVN. There were no
significant changes in binding in the PVN following any stress exposure. Values
expressed are group means in optical density & SEM (n = 4 rats per group). NS= no
stress, A= acute stress, R= repeated stress, R+A= repeated plus acute.


Figure 4-30. Representative x-ray images of brain sections showing 125I-[14Tyr]-N/OFQ
binding to the NOP receptor in the amygdala and the PVN. Despite an increase in
NOP receptor mRNA in these regions, none of the stressed groups displayed
increases in binding. Shown are representative images from (A) the no stress groups,
(B) the acutely stressed group, (C) the repeatedly stressed group, and (D) the
repeatedly plus acutely stressed group.










A













NS A R R+A
Stress Exposure

B




600 R R+
500s xosr

asoe43.Gadmse fersca eet A dea gadmse,()tyu ln
soo s n C penmssssoe osgiiatdifrne ewe rus
150 sepesd r ru ens~SM( rt e ru)









CHAPTER 5
GENERAL DISCUSSION

There is mounting evidence that N/OFQ is involved in the expression of anxiety-related

behaviors and in the activation of the HPA axis. Injections of N/OFQ into the lateral ventricles

and into limbic structures increase circulating ACTH and CORT (Devine et al., 2001; Nicholson

et al., 2002; Fernandez et al., 2004; Legget et al., 2006; Green et al., in press; Misilmeri and

Devine, in preparation) and alter anxiety-related behaviors (Jenck et al., 1997; Jenck et al., 2000;

Gavioli et al., 2002; Fernandez et al., 2004; Kamei et al., 2004; Varty et al. 2005; Vitale et al.,

2006; Gavioli et al., 2007; Green et al., in press). Additionally, exposure to acute stressors

results in release of N/OFQ from forebrain neurons (Devine et al., 2003) and in increases in NOP

receptor mRNA expression (present experiments). However, most of these effects are modest

and are generally evident in mildly to moderately stressful environments. For example, Devine

and colleagues (2001) found that when rats are more profoundly stressed (e.g., restraint), N/OFQ

does not alter the HPA axis activation. This has also been observed recently with restraint and

with unhandled rats (unpublished).

However, there is some evidence of changes in the regulation of the N/OFQ-NOP receptor

system in response to severe chronic stress, particularly in the BNST and the PVN, two regions

that are important in affective responses. The upregulation of NOP receptor mRNA in the BNST

following chronic stress is consistent with the role of the BNST in long-term responses to

ongoing stressors. Additionally, HPA axis regulation in the PVN by the N/OFQ-NOP system

may be altered by chronic social stressor exposure.

It remains evident that N/OFQ is an important neuromodulator in a number of functions,

including stress response, pain modulation (Meunier et al., 1995; Reinscheid et al., 1995; Tian et

al., 1997), motor performance (Reinscheid et al., 1995; Devine et al., 1996), spatial learning










(Sandin et al., 1997; Sandin et al., 2004), and feeding (Pomonis et al., 1996; Nicholson et al.,

2002). In general, it appears to be a fairly tightly regulated system that is generally resistant to

dysregulation. Thus it seems important in the functioning of normal, nonpathological behavior.

Its potential involvement in stress-induced psychopathology is suggested by stress-induced

changes in NOP receptor mRNA (especially in the BNST and PVN of chronically-stressed rats).

However, the lack of changes in receptor autoradiography indicates that this possibility will need

to be studied in more detail.












LIST OF REFERENCES


Adamec RE, Blundell J, Collins A (2001) Neural plasticity and stress induced changes in defense
in the rat. Neurosci Biobehav Rev 25:721-744.

Amsterdam JD, Maislin G, Winokur A, Berwish N, Kling M, Gold P (1988) The oCRH
stimulation test before and after clinical recovery from depression. J Affect Disord 14:
213-222.

Andersen SL, Teicher MH (1999) Serotonin laterality in amygdala predicts performance in the
elevated plus maze in rats. NeuroReport 10:3497-3 500.

Arborelius L, Owens MJ, Plotsky PM, Nemeroff CB (1999) The role of corticotrophin-releasing
factor in depression and anxiety disorders. J Endocrinol 160:1-12.

Aubry JM, Bartanusz V, Jezova D, Belin D, Kiss JZ (1999) Single stress induces long-lasting
elevations in vasopressin mRNA levels in CRF hypophysiotrophic neurons, but repeated
stress is required to modify AVP immunoreactivity. J Neuroendocinol 1 1:377-3 84.

Barnum CJ, Blandino P, Deak T (2007) Adaptation in the corticosterone and hyperthermic
responses to stress following repeated stressor exposure. J Neuroendocinol 19:632-642.

Blanchard RJ, Nikulina JN, Sakai RR, McKittrick C, McEwan B, Blanchard DC (1998)
Behavioral and endocrine change following chronic predatory stress. Physiol Behav 63:
561-569.

Board F, Persky H, Hambur DA (1956) Psychological stress and endocrine functions; blood
levels of adrenocortical and thyroid hormones in acutely disturbed patients. Psychosom
Med 18: 324-333.

Boudaba C, Szabo K, Tasker JG (1996) Physiological mapping of local inhibitory inputs to the
hypothalamic paraventricular nucleus. J Neurosci 16: 7151-7160.

Boudaba C, Schrader LA, Tasker JG (1997) Physiological evidence for local excitatory synaptic
circuits in the rat hypothalamus. J Neurophysiol 77: 3396-3400.

Bryant HU, Bernton EW, Kenner JR, Holaday JW (1991) Role of adrenal cortical activation in
the immunosuppressive effects of chronic morphine treatment. Endocrinology 128:3253-
3258.

Bunzow JR, Saez C, Mortrud M, Bouvier C, Williams JT, Low M, Grandy DK (1994) Molecular
cloning and tissue distribution of a putative member of the rat opioid receptor gene family
that is not a mu, delta or kappa opioid receptor type. FEB S Lett 347: 284-288.










Butour JL, Moisand C, Mazarguil H, Mollereau C, Meunier JC (1997) Recognition and
activation of the opioid receptor-like ORL 1 receptor by nociceptin, nociceptin analogs and
opioids. Eur J Pharmacol 321:97-103.

Buwalda B, Felszeghy K, Horvath KM, Nyakas C, de Boer SF, Bohus B, Koolhaas JM (2001)
Temporal and spatial dynamics of corticosteroid receptor down-regulation in rat brain
following social defeat. Physiol Behav 72:349-354.

Calfa G, Volosin M, Molina VA (2006) Glucocorticoid receptors in lateral septum are involved
in the modulation of the emotional sequelae induced by social defeat. Behav Brain Res
172: 324-332.

Canteras NS, Simerly RB, Sawnson LW (1995) Organization of proj sections from the medial
nucleus of the amygdala: a PHAL study in the rat. J Comp Neurol 360: 213-245.

Chaouloff F, Durand M, Mormede P (1997) Anxiety- and activity-related effects of diazepam
and chlordiazepoxide in the rat light/dark and dark/light tests. Behav Brain Res 85:27-35.

Chen Y, Fan Y, Liu J, Mestek A, Tian M, Kozak CA, Yu L (1994) Molecular cloning, tissue
distribution and chromosomal localization of a novel member of the opioid receptor gene
family. FEBS Lett 347:279-283.

Chung KKK, Martinez M, Herbert J (1999) Central serotonin depletion modulates the
behavioral, endocrine and physiological responses to repeated social stress and
subsequent c-fos expression in the brains of male rats. Neuroscience 92:613-625.

Coleman-Mesches K, McGaugh JL (1995a) Differential involvement of the right and left
amygdalae in expression of memory for aversively motivated training. Brain Res 670:75-
81.

Coleman-Mesches K, McGaugh JL (1995b) Muscimol inj ected into the right or left amygdaloid
complex differentially affects retention performance following aversively motivated
training. Brain Res 676:183-188.

Connor M, Vaughan CW, Chieng B, Christie MJ (1996a) Nociceptin receptor coupling to a
potassium conductance in rat locus coeruleus neurones in vitro. Br J Pharmacol 119:1614-
1618.

Connor M, Yeo A, Henderson G (1996b) The effect of nociceptin on Ca2+ channel current and
intracellular Ca2+ in the SH-SY5Y human neuroblastoma cell line. Br J Pharmacol
118:205-207.

Costall B, Jones BJ, Kelly ME, Naylor RJ, Tomkins DM (1989) Exploration of mice in a black
and white test box: Validation as a model of anxiety. Pharmacol Biochem Behav 32:777-
785.









Covington HE, Miczek KA (2001) Repeated social-defeat stress, cocaine or morphine. Effects
on behavioral sensitization and intravenous cocaine self-administration "binges".
Psychopharmacol 158:388-398.

Crawley J, Goodwin FK (1980) Preliminary report of a simple animal behavior model for the
anxiolytic effects of benzodiazepines. Pharmacol Biochem Behav 13:167-170.

Crawley JN (1981) Neuropharmacologic specificity of a simple animal model for the behavioral
actions of benzodiazepines. Pharmacol Biochem Behav 15:695-699.

Crofford LJ (1998) The hypothalamic-pituitary-adrenal stress axis in fibromyalgia and chronic
fatigue syndrome. Z Rheumatol 57: 67-71.

Cullinan WE, Herman JP, Watson SJ (1993) Ventral subicular interaction with the hypothalamic
paraventricular nucleus: Evidence for a relay in the bed nucleus of stria terminalis. J Comp
Neurol 332: 1-20.

Cullinan WE, Helmreich DL, Watson SJ (1996) Fos expression in forebrain afferents to the
hypothalamic paraventricular nucleus following swim stress. J Comp Neurol 368: 88-99.

Danielson PB, Dores RM (1999) Review: Molecular evolution of the opioid/orphanin gene
family. Gen Comp Endocrinol 113:169-186.

Dautzenberg FM, Wichmann U, Higelin J, Py-Lang G, Kratzeisen C, Malherbe P, Kilpatrick GJ,
Jenck F (2001) Pharmacological characterization of the novel nonpeptide orphanin
FQ/nociceptin receptor agonist Ro 64-6198: Rapid and reversible desensitization of the
ORL1 receptor in vitro and lack of tolerance in vivo. J Pharmacol & Exp Ther 298: 812-
819.

de Goeij DC, Kvetnansky R, Whitnall MH, Jezova D, Berkenbosch F, Tilders FJ (1991)
Repeated stress-induced activation of corticotrophin-releasing factor neurons enhances
vasopressin stores and colocalization with corticotrophin-releasing factor in the median
eminence of rats. Neuroendocrinology 53:150-159.

de Goeij DCE, Dijkstra H, Tilders FJH (1992) Chronic psychological stress enhances
vasopressin, but not corticotrophin-releasing factor, in the external zone of the medial
eminence of male rats: Relationship to subordinate status. Endocrinology 13 1:847-853.

Deak T, Nguyen KT, Cotter CS, Fleshner M, Watkins LR, Maier SF, Spencer RL (1999) Long-
term changes in mineralocorticoid and glucocorticoid receptor occupancy following
exposure to an acute stressor. Brain Res 847: 211-220.

Devine DP, Taylor L, Reinscheid RK, Monsma FJ, Civelli O, Akil H (1996) Rats rapidly
develop tolerance to the locomotor-inhibiting effects of the novel neuropeptide orphanin
FQ. Neurochem Res 21:1387-1396.

Devine DP, Watson SJ, Akil H (2001) Nociceptin/orphanin FQ regulates neuroendocrine
function of the limbic-hypothalamic-pituitary-adrenal axis. Neuroscience 102:541-553.










Devine DP, Hoversten MT, Ueda Y, Akil H (2003) Nociceptin/orphanin FQ content is decreased
in forebrain neurones during acute stress. J Neuroendocrinol 15:69-74.

Diorio D, Viau V, Meaney MJ (1993) The role of the medial prefrontal cortex (cingulated gyrus)
in the regulation of hypothalamic-pituitary-adrenal responses to stress. J Neurosci 13:3839-
3847.

Dominguez-Gerpe L, Rey-Mendez M (2001) Alterations induced by chronic stress in
lymphocyte subsets of blood and primary and secondary immune organs of mice. BMC
Immunol 2:7.

Dong HW, Petrovich GD, Watts AG, Swanson LW (2001) Basic organization of proj sections
from the oval and fusiform nuclei of the bed nuclei of the stria terminalis in adult rat brain.
J Comp Neurol 436:430-455.

Dong HW, Swanson LW (2004) Organization of axonal proj sections from the anterolateral area
of the bed nuclei of the stria terminalis. J Comp Neurol 468:277-298.

Dunn JD (1987a) Differential plasma corticosterone responses to electrical stimulation of the
medial and lateral septal nuclei. Neuroendocrinology 46: 406-411.

Dunn JD (1987b) Plasma corticosterone responses to electrical stimulation of the bed nucleus of
the stria terminalis. Brain Res 407:327-331.

Dunn JD, Whitener J (1986) Plasma corticosterone responses to electrical stimulation of the
amygdaloid complex: cytoarchitectural specificity. Neuroendocrinology 42: 211;217.

Ebner K, Wotj ak CT, Landgaf R, Engelmann M (2005) Neuroendocrine and behavioral response
to social confrontation: residents versus intruders, active versus passive coping styles.
Horm Behav 47:14-21.

Elenkov IJ, Wilder RL, Chrousos GP, Vizi ES (2000) The sympathetic nerve--An integrative
interface between two supersystems: The brain and the immune system. Pharmacol Rev.
52: 595-638.

Emmert MH, Herman JP (1999) Differential forebrain c-fos mRNA induction by ether inhalation
and novelty: evidence for distinctive stress pathways. Brain Res 845: 60-67.

Eskandari F, Sternberg EM (2002) Neural-Immune interactions in health and disease. Ann NY
Acad Sci 966:20-27.

Feldman S, Conforti N, Itzik A, Weidenfeld J (1994) Differential effect of amygdaloid lesions of
CRF-41, ACTH, and corticosterone responses following neural stimuli. Brain Res 658: 21-
26.

Fernandez F, Misilmeri MA, Felger JC, Devine DP (2004) Nociceptin/orphanin FQ increases
anxiety-related behavior and circulating levels of corticosterone during neophobic tests of
anxi ety. Neurop sychopharmacology 29:5 9-7 1.










Gavioli EC, Rae GA, Calo G, Guerrini R, DeLima TCM (2002) Central inj sections of nocistatin
or its C-terminal hexapeptide exert anxiogenic-like effect on behaviour of mice in the plus-
maze test. Brit J Pharmacol 136:764-772.

Gavioli EC, Marzola G, Guerrini R, Bertorelli R, Zucchini S, De Lima TCM, Rae GA, Salvadori
S, Regoli D, Calo G (2003) Blockade of nociceptin/orphanin FQ-NOP receptor signalling
produces antidepressant-like effects: Pharmacological and genetic evidences from the
mouse forced swimming test. Eur J Neurosci 17: 1987-1990.

Gavioli EC, V aughan CW, Marzola G, Guerrini R, Mitchell VA, Zucchini S, De Lima TCM,
Rae GA, Salvadori S, Regoli D, Calo G (2004) Antidepressant-like effects of the
nociceptin/orphanin FQ receptor antagonist UFP-101: New evidence from rats and mice.
Naunyn-Schmiedeberg's Arch Pharmacol 369:547-553.

Gavioli EC, Rizzi A, Marzola G, Zucchini S, Regoli D, Calo G (2007). Altered anxiety-related
behavior in nociceptin/orphanin FQ receptor gene knockout mice. Peptides 28: 1229-1239.

Gillies GE, Linton EA, Lowry PJ (1982) Corticotropin releasing activity of the new CRF is
potentiated several times by vasopressin. Nature 299: 355-357.

Gilmer WS, Trivedi MH, Rush AJ, Wisniewski SR, Luther J, Howland RH, Yohanna D, Khan
A, Alpert J (2005) Factors associated with chronic depressive episodes: A preliminary
report from the STAR-D proj ect. Acta Psychiatr Scand 1 12:425-433.

Gold PW, Loriaux DL, Roy A, Kling MA, Calabrese JR, Kellnor CH, Nieman LK, Post RM,
Dicker D, Gallucci W, et al. (1986) Response to corticotrophin-releasing hormone in the
hypercortisolism of depression and Cushing's disease: Pathophysiologic and diagnostic
implications. N Eng J Med 314: 1329-1335.

Goldstein ML (1965) Effects of hippocampal, amygdala, hypothalamic and parietal lesions on a
classically conditioned fear response. Psych Rep 16: 211-219.

Good AJ, Westbrook RF (1995) Effects of a microinj section of morphine into the amygdala on
the acquisition and expression of conditioned fear and hypoalgesia in rats. Behav Neurosci
109:631-641.

Gray TS, Piechowski RA, Yracheta JM, Rittenhouse PA, Bethea CL, Ven de Kar LD (1993)
Ibotenic acid lesions in the bed nucleus of the stria terminalis attenuate conditioned stress-
induced increases in prolactin, ACTH and corticosterone. Neuroendocrinology 57: 517-
524.

Green MK, Barbieri EV, Brown BD, Chen KW, Devine DP (in press) Roles of the bed nucleus
of stria terminalis and of the amygdala in N/OFQ-mediated anxiety and HPA axis
activation. Neuropeptides.

Handley SL, Mithani S (1984) Effects of alpha-adrenoceptor agonists and antagonists in a maze-
exploration model of 'fear'-motivated behaviour. Naunyn Schmiedebergs Arch Pharmacol
327:1-5.










Hasegawa H, Saiki I (2002) Psychological stress augments tumor development through P-
adrenergic activation in mice. Jpn J Cancer Res 93:729-735.

Hauger RL, Lorang M, Irwin M, Aguilera G (1990) CRF receptor regulation and sensitization of
ACTH responses to acute ether stress during chronic intermittent immobilization stress.
Brain Res 532: 34-40.

Heinrichs SC, Pich EM, Miczek KA, Britton KT, Koob GF (1992) Corticotropin-releasing factor
antagonist reduces emotionality in socially defeated rats via direct neurotropic action.
Brain Res 581:190-197.

Henke PG (1984) The bed nucleus of the stria terminalis and immobilization-stress unit activity,
escape behaviour, and gastric pathology in rats. Behav Brain Res 11:35-45.

Herman JP, Schafer MK, Young EA, Thompson R, Douglass J, Akil H, Watson SJ (1989)
Evidence for hippocampal regulation of neuroendocrine neurons of the
hypothalamo-pituitary-adrenocortical axis. J Neurosci 9:3072-3082.

Herman JP, Cullinan WE, Watson SJ (1994) Involvement of the bed nucleus of the stria
terminalis in tonic regulation of paraventricular hypothalamic CRH and AVP mRNA
expression. J Neuroendocrinol 6: 433-442.

Herman JP, Cullinan WE (1997) Neurocircuitry of stress: Central control of the
hypothalamo-pituitary-adrenocortical axis. Trends Neurosci 20:78-84.

Herman JP, Taskers JG, Ziegler DR, Cullinan WE (2002a) Local circuit regulation of
paraventricular nucleus stress integration Glutamate-GABA connections. Pharmacol
Biochem Behav 71:457-468.

Herman JP, Cullinan WE, Ziegler DR, Tasker JG (2002b) Role of the paraventricular nucleus
microenvironment in stress integration. Eur J Neurosci 16:381-385.

Heuser I, Bissette G, Dettling M, Schweiger U, Gotthardt U, Schmider J, Lammers CH,
Nemeroff CB, Holsboer F (1998) Cerebrospinal fluid concentrations of corticotrophin-
releasing hormone, vasopressin, and somatostatin in depressed patients and healthy
controls: Response to amitriptyline treatment. Depress Anxiety 8: 71-79.

Holsboer F, Gerken A, von Bardeleben U, Grimm W, Beyer H, Muller OA, Stalla GK (1986)
Human corticotrophin-releasing hormone in depression-Correlation with thyrotropin
secretion following thyrotropin-releasing hormone. Biol Psychiatry 21: 601-611.

Hughes RN (1972) Chlordiazepoxide modified exploration in rats. Psychopharmacologia
24:462-469.

Ixart G, Szafarczyk A, Belugou JL, Assenmacher I (1977) Temporal relationships between the
diurnal rhythm of hypothalamic corticotrophin releasing factor, pituitary corticotrophin
and plasma corticosterone in the rat. J Endocrinol 72: 113-120.










Izquierdo A, Murray EA (2004) Combined unilateral lesions of the amygdala and orbital
prefrontal cortex impair affective processing in rhesus monkeys. J Neurophysiol 91:2023-
2039.

Jenck F, Moreau JL, Martin JR, Kilpatrick GJ, Reinscheid RK, Monsma FJ, Nothacker HP,
Civelli O (1997) Orphanin FQ acts as an anxiolytic to attenuate behavioral responses to
stress. PNAS 94:14854-14858.

Jenck F, Wichmann J, Dautzenberg FM, Moreau JL, Ouagazzal AM, Martin JR, Lundstrom K,
Cesura AM, Poli SM, Roever S, Kolczewski S, Adam G, Kilpatrick G (2000) A synthetic
agonist at the orphanin FQ/nociceptin receptor ORL1: anxiolytic profile in the rat. Proc
Natl Acad Sci U SA 97:4938-4943.

Johnson JD, O'Connor KA, Deak T, Spencer RL, Watkins LS, Maier SF (2002) Prior stressor
exposure primes the HPA axis. Psychoneuronendocrinology 27: 353-365.

Jordanova V, Stewart R, Goldberg D, Bebbington E, Brugha T, Singleton N, Lindesay JEB,
Jenkins R, Prince M, Meltzer H (2007) Age variation in life events and their relationship
with common mental disorders in a national survey population. Soc Psychiatry Psychiatr
Epidemiol 42:611-616.

Juruena MF, Cleare AJ, Pariante CM (2004) The hypothalamic pituitary adrenal axis,
glucocorticoid receptor function and relevance to depression. Rev Bras Pisquiatr 26: 189-
201.

Kamei J, Matsunawa Y, Miyata S, Tanaka S, Saitoh A (2004) Effects of nociceptin on the
exploratory behavior of mice in the hole-board test. Eur J Pharmacol 489: 77-87.

Kvetnansy R, Bodnar I, Shahar T, Uhereczky G, Krizanova O, Mravec B (2006) Effect of lesions
of A5 and A7 brainstem noradrenergic areas or transaction of brainstem pathways on
sympathoadrenal activity in rats during immobilization stress. Neurochem Res 31: 267-
275.

Kwak SP, Morano MI, Young EA, Watson SJ, Akil H (1993) Diurnal CRH mRNA rhythm in the
hypothalamus: decreased expression in the evening is not dependent on endogenous
glucocorticoids. Neuroendocrinology 57:96-105.

LaBar KS, LeDoux JE (1996) Partial disruption of fear conditioning in rats with unilateral
amygdala damage: Correspondence with unilateral temporal lobectomy in humans. Behav
Neurosci 110:991-997.

Labeur MS, Arzt E, Wiegers GJ, Holsboer F, Reul JMHM (1995) Lon-term
intracerebroventricular corticotrophin-releasing hormone administration induces distinct
changes in rat splenocyte activation and cytokine expression. Endocrinology 136: 2678-
2688.

Lachowicz JE, Shen Y, Monsma FJ, Sibley DR (1995) Molecular cloning of a novel G protein-
coupled receptor related to the opiate receptor family. J Neurochem 64:34-40.










LeDoux JE, Iwata J, Cicchetti P, Reis DJ (1988) Different proj sections of the central amygdaloid
nucleus mediate autonomic and behavioral correlates of conditioned fear. J Neurosci 8:
2517-2529.

Lee Y, Davis M (1997) Role of the hippocampus, the bed nucleus of the stria terminalis, and the
amygdala in the excitatory effect of corticotrophin-releasing hormone on the acoustic
startle reflex. J Neurosci 17: 6434-6446.

Leggett JD, Harbuz MS, Jessop DS, Fulford AJ (2006) The nociceptin receptor antagonist
[Nphe(1),Arg(14),Lys(15)]nociceptin/orphai FQ-NH(2) blocks the stimulatory effects of
nociceptin/orphanin FQ on the HPA axis in rats. Neuroscience 141:2051-2057.

Linthorst ACE, Flachskamm C, Hopkins SJ, Hoadley ME, Labeur MS, Holsboer F, Reul JMHM
(1997) Long-term intracerebroventricular infusion of corticotrophin-releasing hormone
alters neuroendocrine, neurochemical, autonomic, behavioral, and cytokine responses to a
systemic inflammatory challenge. J Neurosci 17: 4448-4460.

Lyons DM, Wang OJ, Lindley SE, Levine S, Kalin NH, Schatzberg AF (1999) Separation
induced changes in squirrel monkey hypothalamic-pituitary-adrenal physiology resemble
aspects of hypercortisolism in humans. Psychoneuroendocrinology 24: 13 1-142.

Manji HK, Drevets WC, Charney DS (2001) The cellular neurobiology of depression. Nature
Med 7: 541-547.

Marin MT, Cruz FC, Planeta CS (2007) Chronic restraint or variable stresses differently affect
the behavior, corticosterone secretion and body weight in rats. Physiol & Behav 90:29-35.

Martin DS, Haywood JR (1992) Sympathetic nervous system activation by glutamate inj sections
into the paraventricular nucleus. Brain Res 577: 261-267.

Martinez M, Phillips PJ, Herbert J (1998) Adaptation in patterns of c-fos expression in the brain
associated with exposure to either single or repeated social stress in male rats. Eur J
Neurosci 10:20-33.

Menard J, Treit D (1996) Lateral and medial septal lesions reduce anxiety in the plus-maze and
probe-burying tests. Physiol & Behav 60: 845-853.

Meunier JC, Mollereau C, Toll L, Suaudeau C, Moisand C, Alvinerie P, Butour JL, Guillemot
JC, Ferrara P, Monsarrat B (1995) Isolation and structure of the endogenous agonist of
opioid receptor-like ORL1 receptor. Nature 377:532-535.

Misilmeri MA, Devine DP (in preparation) N/OFQ activates the hypothalamic-pituitary adrenal
axis after administration into limbic brain sites.

Moghaddam B, Bunney BS (1989) Ionic composition of microdialysis perfusing solution alters
the pharmacological responsiveness and basal outflow of striatal dopamine. J Neurochem
53:652-654.









Mollereau C, Parmentier M, Mailleux P, Butour JL, Moisand C, Chalon P, Caput D, Vassart G,
Meunier JC (1994) ORL1, a novel member of the opioid receptor family. Cloning,
functional expression and localization. FEBS Lett 341:33-38.

Nagaraja TN, Patel P, Gorski M, Gorevic PD (2005) In normal rat, intraventricularly
administered insulin-like growth factor-1 is rapidly cleared from CSF with limited
distribution into brain. Cerebrospinal Fluid Res 2: 5.

Neal CR, Mansour A, Reinscheid R, Nothacker HP, Civelli O, Watson SJ (1999a) Localization
of orphanin FQ (nociceptin) peptide and messenger RNA in the central nervous system of
the rat. J Comp Neurol 406:503-547.

Neal CR, Mansour A, Reinscheid R, Nothacker HP, Civelli O, Akil H, Watson SJ (1999b)
Opioid receptor-like (ORL1) receptor distribution in the rat central nervous system:
Comparison of ORL1 receptor mRNA expression with 125I-[14Tyr]-Orphanin FQ
binding. J Comp Neurol 412:563-605.

Nemeroff CB, Widerlov E, Bissette G, Walleus H, Karlsson I, Eklund K, Kilts CD, Loosen PT,
Vale W (1984) Elevated concentrations of CSF corticotropin-releasing factor-like
immunoreactivity in depressed patients. Science 226:1342-1344.

Nemeroff CB, Bissette G, Akil H, Fink M (1991) Neuropeptide concentrations in the
cerebrospinal fluid of depressed patients treated with electrovonvulsive therapy.
Corticotrophin-releasing factor, beta-endorphin and somatostatin. Br J Psychiatry 158: 59-
63.

Nicholson JR, Akil H, Watson SJ (2002) Orphanin FQ-induced hyperphagia is mediated by
corticosterone and central glucocorticoid receptors. Neuroscience 11 5:63 7-643.

Nikulina EM, Covington HE, Ganschow L, Hammer RP, Miczek KA (2004) Long-term
behavioral and neuronal cross-sensitization to amphetamine induced by repeated brief
social defeat stress: fos in the ventral tegmental area and amygdala. Neuroscience 123:857-
865.

Nothacker HP, Reinscheid RK, Mansour A, Henningsen RA, Ardati A, Monsma FJ, Watson SJ,
Civelli O (1996) Primary structure and tissue distribution of the orphanin FQ precursor.
PNAS 93:8677-8682.

Onaivi ES, Martin BR (1989) Neuropharmacological and physiological validation of a computer-
controlled two-compartment black and white box for the assessment of anxiety. Prog
Neuropsychopharmacol Biol Psychiatry 13:963-976.

Ostrander MM, Ulrich-Lai YM, Choi DC, Richtand NM, Herman JP (2006) Hypoactivity of the
hypothalamo-pituitary-adrenocortical axis during recovery from chronic variable stress.
Endocrinology 147:2008-2017.

Paxinos G, Watson C (1998) The Rat Brain in Stereotaxic Coordinates, 4th Ed. CD-ROM:
Hulasz, P.










Fellow S, Chopin P, File SE, Briley M (1985) Validation of open: closed arm entries in an
elevated plus-maze as a measure of anxiety in the rat. J Neurosci Methods 14: 149-167.

Fellow S, File SE (1986) Anxiolytic and anxiogenic drug effects on exploratory activity in an
elevated plus-maze: A novel test of anxiety in the rat. Pharmacol Biochem Behav 24:525-
529.

Peper M, Karcher S, Wohlfarth R, Reinshagen G, LeDoux JE (2001) Aversive learning in
patients with unilateral lesions of the amygdala and hippocampus. Biol Psychol 58:1-23.

Pomonis JD, Billington CJ, Levine AS (1996) Orphanin FQ, agonist of orphan opioid receptor
ORL 1, stimulates feeding in rats. Neuroreport 20:369-371.

Prewitt CMF, Herman JP (1998) Anatomical interactions between the central amygdaloid
nucleus and the hypothalamic paraventricular nucleus of the rat: a dual tract-tracing
analysis. J Chem Neuroanat 15:173-185.

Redrobe JP, Calo G, Regoli D, Quirion R (2002) Nociceptin receptor antagonists display
antidepressant-like properties in the mouse forced swim test. Naunyn-Schmeideberg's
Arch Pharmacol 365:164-167.

Reinscheid RK, Nothacker HP, Bourson A, Ardati A, Henningsen RA, Bunzow JR, Grandy DK,
Langen H, Monsma FJ, Civelli O (1995) Orphanin FQ: a neuropeptide that activates an
opioidlike G protein-coupled receptor. Science 270:792-794.

Reinscheid RK, Ardati A, Monsma FJ, Civelli O (1996) Structure-activity relationship studies on
the novel neuropeptide orphanin FQ. J Biol Chem 271:14163-14168.

Renner KJ, Smits AW, Quadagno DM, Hough JC (1984) Suppression of sexual behavior and
localization of [3H] puromycin after intracranial inj section in the rat. Physiol Behav 33:
411-414.

Risold PY, Swanson LW (1997) Connections of the rat lateral septal complex. Brain Res Rev
24L 115-195.

Rogan MT, Staubli UV, LeDoux JE (1997) Fear conditioning induces associative long-term
potentiation in the amygdala. Nature 391:604-607.

Rubin RT, Phillips JJ, Sadow TF, McCracken JT (1995) Adrenal gland volume in maj or
depression. Increase during the depressive episode and decrease with successful treatment.
Arch Gen Psychiatry 52: 213-218.

Rygula R, Abumaria N, Flugge G, Fuchs E, Ruther E, Havemann-Reinceke U (2005) Anhedonia
and motivational deficits in rats: Impact of chronic social stress. Behav Brain Res 162: 127-
134.

Sandin J, Georgieva J, Schott PA, Ogren SO, Terenius L (1997) Nociceptin/orphanin FQ
microinj ected into hippocampus impairs spatial learning in rats. Eur J Neurosci 9: 194-197.









Sandin J, Ogren SO, Terenius L (2004) Nociceptin/orphanin FQ modulates spatial learning via
ORL-1 receptors in the dorsal hippocampus of the rat. Brain Res 997:222-233.

Sawchenko PE, Swanson LW (1983) The organization of forebrain afferents to the
paraventricular and supraoptic nuclei of the rat. J Comp Neurol 218: 121-144.

Scicli AP, Petrovich GD, Swanson LW, Thompson RF (2004) Contextual fear conditioning is
associated with lateralized expression of the immediate early gene c-fos in the central and
basolateral amygdalar nuclei. Behav Neurosci 118:5-14.

Selye H (1936) Thymus and adrenals in the response of the organism to injuries and
intoxications. Brit J Exp Path 17:234-248.

Shimohigashi Y, Hatano R, Fujita T, Nakashima R, Nose T, Suj aku T, Saigo A, Shinj o K,
Nagahisa A (1996) Sensitivity of opioid receptor-like receptor ORL 1 for chemical
modification on nociceptin, a naturally occurring nociceptive peptide. J Biol Chem 271:
23642-23645.

Simon P, Dupuis R, Costentin J (1994) Thigmotaxis as an index of anxiety in mice. Influence of
dopaminergic transmissions. Behav Brain Res 61:59-64.

Simpkiss JL, Devine DP (2003) Responses of the HPA axis after chronic variable stress: Effects
of novel and familiar stressors. Neuroendocrinol Lett 24:75-81.

Spampinato S, Baiula M (2006). Agonist-regulated endocytosis and desensitization of the human
nociceptin receptor. NeuroReport, 17: 173-177.

Stefanski R, Palejko W, Kostowski W, Plaznik A (1992) The comparison of benzodiazepine
derivatives and serotonergic agonists and antagonists in two models of anxiety.
Neuropharmacology 31:1251-1258.

Stone KL, Naccarato AM, Devine DP (in preparation) Rats exhibit behavioral despair and
hormonal alterations following social defeat stress: Implications for stress-induced
psychopathology.

Tian JH, Xu W, Fang Y, Mogil JS, Grisel JE, Grandy DK, Han JS (1997) Bidirectional
modulatory effect of orphanin FQ on morphine-induced analgesia: antagonism in brain and
potentiation in spinal cord of the rat. Brit J Pharmacol 120:676-80.

Van de Kar LD, Piechowski RA, Rittenhouse PA, Gray TS (1991) Amygdaloid lesions:
Differential effect on conditioned stress and immobilization-induced increases in
corticosterone and rennin secretion. Neurendocrinology 54: 89-95.

Varty GB, Hyde LA, Hodgson RA, Lu SX, McCool MF, Kazdoba TM, Del Vecchio RA,
Guthrie DH, Pond AJ, Grzelak ME, Xu X, Korftnacher WA, Tulshian D, Parker EM,
Higgins GA (2005) Characterization of the nociceptin receptor (ORL-1) agonist, Ro64-
6198, in tests of anxiety across multiple species. Psychopharmacology 182: 132-143.










Vaughan CW, Christie MJ (1996) Increase by the ORL1 receptor (opioid receptor-likel) ligand,
nociceptin, of inwardly rectifying K conductance in dorsal raphe nucleus neurons. Brit J
Pharmacol 117:1609-1611.

Vaughan CW, Ingram SL, Christie MJ (1997) Actions of the ORL 1 receptor ligand nociceptin on
membrane properties of rat periaqueductal gray neurons in vitro. J Neurosci 17:996-1003.

Vitale G, Arletti R, Ruggieri V, Cifani C, Massi M (2006) Anxiolytic-like effects of
nociceptin/orphanin FQ in the elevated plus maze and in the conditioned defensive burying
test in rats. Peptides 27:2193-2200.

Vyas A, Bernal S, Chattarji S (2003) Effects of chronic stress on dendritic arborization in the
central and extended amygdala. Brain Res 965: 290-294.

Walker DL, Davis M (1997) Double dissociation between the involvement of the bed nucleus of
the stria terminalis and the central nucleus of the amygdala in startle increases produced by
conditioned versus unconditioned fear. J Neurosci 17:93 75 -93 83.

Walker DL, Toufexis DJ, Davis M (2003) Role of the bed nucleus of the stria terminalis versus
the amygdala in fear, stress, and anxiety. Eur J Pharmacol 463:199-216.

Wang JB, Johnson PS, Imai Y, Persico AM, Ozenberger BA, Eppler CM, Uhl GR (1994) cDNA
cloning of an orphan opiate receptor gene family member and its splice variant. FEBS Lett
348:75-79.

Watzl B, Lopez M, Shahbazian M, Chen G, Colombo LL, Huang D, Way D, Watson RR (1993)
Diet and ethanol modulate immune responses in young C57BL/6 mice. Alcohol Clin Exp
Res 17:623-630.

Weisse CS (1992) Depression and immunocompetence: A review of the literature. Psychol Bull
111: 475-489.

Whitnall MH (1993) Regulation of the hypothalamic corticotropin-releasing hormone
neurosecretory system. Prog Neurobiol 40:573-629.

Wick MJ, Minnerath SR, Lin X, Elde R, Law PY, Loh HH (1994) Isolation of a novel cDNA
encoding a putative membrane receptor with high homology to the cloned mu, delta, and
kappa opioid receptors. Brain Res Mol Brain Res 27:37-44.

Wisniewski SR, Rush AJ, Bryan C, Shelton R, Trivedi MH, Marcus S, Husain MM, Hollon SD,
Fava M (2005) Comparison of quality of life measures in a depression population. J Nery
Ment Dis 195:219-225.

Wommack JC, Delville Y (2003) Repeated social stress and the development of agonistic
behavior: Individual differences in coping responses in male golden hamsters. Physiol
Behav 80:303-308.










Ziegler DR, Herman JP (2000) Local integration of glutamate signaling in the hypothalamic
paraventicular region: regulation of glucocorticoid stress responses. Endocrinology 141:
4801-4804.









BIOGRAPHICAL SKETCH

Megan K. Green received her Associate of Arts in August 1998 at Okaloosa Walton

College. In May 2000, she received a dual Bachelor of Arts in psychology and anthropology

from the University of West Florida. Megan began her graduate studies in experimental

psychology at the University of West Florida in August 2001. She continued graduate studies at

the University of Florida in August 2003, where she completed a Master of Science degree in

behavioral neuroscience through the psychology department in December 2005. Currently

Megan is employed as a post-doctoral researcher at St. Charles Pharmaceuticals in Alachua, FL.





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ROLE OF THE NOCICEPTIN/ORPHANIN FQ-NOP RECEPTOR SYSTEM IN STRESS RESPONSES By MEGAN K. GREEN A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2007 1

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2007 Megan K. Green 2

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To Susan L. Menet. She left this world on November 5, 2007, but she will never leave our hearts. 3

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ACKNOWLEDGMENTS I would like to thank my advisor, Dr. Darragh Devine, for all of his guidance, commitment, hard work, and long hours. I w ould also like to thank all of my committee members, Drs. Margaret Bradley, Mohamed Ka bbaj, Henrietta Logan, John Petitto, and Ken Rice, for their time, effort, and input. I th ank Brandon Brown and Emily Barbieri for their contributions to the work presented herein, a nd Amber Muehlmann and Nate Weinstock for their assistance with day-to-day tasks. Finally, I would like to thank Michael Menet for his unwavering support and my mother for all that sh e has done to make my life successes possible. 4

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TABLE OF CONTENTS page ACKNOWLEDGMENTS ............................................................................................................... 4 LIST OF TABLES ...........................................................................................................................7 LIST OF FIGURES .........................................................................................................................8 ABSTRACT ...................................................................................................................... .............11 CHAPTER 1 INTRODUCTION ................................................................................................................ ..13 Stress and Stress Response .................................................................................................... .13 Stress ........................................................................................................................ ........13 HPA Axis Activation Foll owing Stress Exposure ...........................................................14 Anatomy of the Limbic-HPA Axis ..................................................................................15 Autonomic Nervous System ............................................................................................17 Chronic Stress and Disease .....................................................................................................17 Animal Models of Chronic Stress ...........................................................................................19 Chronic Variable Stress ...................................................................................................19 Other Chronic Stress Regimens .......................................................................................20 Social Defeat ...................................................................................................................20 Nociceptin/Orphanin FQ ........................................................................................................22 2 ANATOMICAL ANALYSIS OF ANXI OGENIC BEHAVIORAL EFFECTS OF EXOGENOUS NOCICEPTIN/ORPHANIN FQ ...................................................................26 Background .................................................................................................................... .........26 Methods ..................................................................................................................................27 Animals ....................................................................................................................... .....27 Drugs ......................................................................................................................... ......28 Surgery ....................................................................................................................... .....28 Equipment ..................................................................................................................... ...29 Anxiety-Testing Procedure ..............................................................................................29 Statistical Analyses .......................................................................................................... 31 Results .....................................................................................................................................31 Anxiety-Related Behavior ...............................................................................................31 Circulating Corticosterone ...............................................................................................32 Organ Masses ..................................................................................................................32 Discussion .................................................................................................................... ...........33 Anxiety-Related Behaviors and Corticosterone ..............................................................33 Organ Masses ..................................................................................................................36 Summary ....................................................................................................................... ...36 5

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6 3 DIFFUSION OF NOCICEPTIN/ORPHANIN FQ AFTER INTRACEREBROVENTRICULAR AND INTR APARENCHYMAL INJECTIONS ........46 Background .................................................................................................................... .........46 Methods ..................................................................................................................................46 Animals ....................................................................................................................... .....46 Drugs ......................................................................................................................... ......46 Surgery ....................................................................................................................... .....47 Injection Procedure ..........................................................................................................47 Results .....................................................................................................................................48 Intracerebroventricu lar Injections ...................................................................................48 Intra-Amygdaloid Injections ...........................................................................................49 Intra-BNST Injections .....................................................................................................50 Discussion .................................................................................................................... ...........51 4 NOCICEPTIN/ORPHANIN FQ AND NO P RECEPTOR GENE REGULATION AND NOCICEPTIN/ORPHANIN FQ BINDING TO THE NOP RECEPTOR AFTER SINGLE OR REPEATED SOCI AL DEFEAT EXPOSURE .................................................57 Background .................................................................................................................... .........57 Methods ..................................................................................................................................58 Animals ....................................................................................................................... .....58 Drugs ......................................................................................................................... ......59 Surgery ....................................................................................................................... .....59 Social Defeat Procedure ..................................................................................................59 In Situ Hybridization .......................................................................................................61 Autoradiography ..............................................................................................................6 3 Densitometry and Statistics .............................................................................................64 Results .....................................................................................................................................64 Social Defeats ................................................................................................................ ..64 In Situ Hybridization .......................................................................................................65 Prepro-N/OFQ ..........................................................................................................66 NOP Receptor ..........................................................................................................66 Autoradiography ..............................................................................................................6 7 Organ Masses ..................................................................................................................67 Discussion .................................................................................................................... ...........67 Prepro-N/OFQ mRNA .....................................................................................................68 NOP Receptor mRNA .....................................................................................................69 NOP Receptor Binding ....................................................................................................70 Organ Masses ..................................................................................................................71 5 GENERAL DISCUSSION .....................................................................................................99 LIST OF REFERENCES .............................................................................................................101 BIOGRAPHICAL SKETCH .......................................................................................................114

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LIST OF TABLES Table page 4-1 Social defeat regimen.. ................................................................................................... ....73 7

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LIST OF FIGURES Figure page 2-1 Anxiety-related behaviors following intra-amygdaloid injections of N/OFQ. ..................38 2-2 Anxiety-related behaviors follo wing ICV injections of N/OFQ.. ......................................39 2-3 Concentrations of circulating corticos terone following ICV and intra-amygdaloid injections of N/OFQ. .......................................................................................................... 40 2-4 Photograph of the open field and a diag ram of the zones used for scoring. ......................41 2-5 Anxiety-related behaviors following intra-BNST injections of N/OFQ. ...........................42 2-6 Anatomical map of BNST placements. .............................................................................43 2-7 Concentrations of circulating corticoste rone following intra-BNST injections of N/OFQ................................................................................................................................44 2-8 Analysis of glandular masses. ............................................................................................26 3-1 Representative x-ray images of 3H-N/OFQ diffusion following ICV injections overlaid on the corresponding cresyl violet stained section (A, C, and E) and x-ray images alone (B, D, and F). ...............................................................................................54 3-2 Representative x-ray images of 3H-N/OFQ diffusion following intra-amygdaloid injections overlaid on th e corresponding cresyl viol et stained section. .............................55 3-3 Representative x-ray images of 3H-N/OFQ diffusion following intra-BNST injections overlaid on the co rresponding cresyl vi olet stained section (A, C, and E) and x-ray images alone (B, D, and F). ...............................................................................56 4-1 Photograph of a social defeat interaction.. .........................................................................72 4-2 Photograph of stage 2 of th e social defeat procedure.. ......................................................72 4-3 Prepro-N/OFQ sequence. ...................................................................................................7 3 4-4 NOP receptor sequence.. ................................................................................................... .74 4-5 Number of social de feats per group per day. .....................................................................75 4-6 Total amount of time the rats were defeated per day. ........................................................75 4-7 Representative x-ray images of brain sections treated with sense riboprobes. ..................76 4-8 Prepro-N/OFQ mRNA expr ession in the septum.. ............................................................77 8

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9 4-9 Prepro-N/OFQ mRNA expression in th e bed nucleus of stria terminalis. .........................78 4-10 Representative x-ray images of br ain sections showing prepro-N/OFQ mRNA expression in the septum and BNST. .................................................................................79 4-11 Prepro-N/OFQ mRNA expression in the amygdala. .........................................................80 4-12 Prepro-N/OFQ mRNA expression in the z ona incerta and reticular nucleus of the thalamus.. .................................................................................................................... .......81 4-13 Representative x-ray images of br ain sections showing prepro-N/OFQ mRNA expression in the amygdala, z ona incerta, and reticular nu cleus of the thalamus.. ...........82 4-14 Prepro-N/OFQ mRNA expre ssion in the dorsal raphe.. ....................................................83 4-15 Representative x-ray images of br ain sections showing prepro-N/OFQ mRNA expression in the dorsal raphe. ...........................................................................................83 4-16 NOP rector mRNA expr ession in the septum. ...................................................................84 4-17 Representative x-ray images of br ain sections showing NOP receptor mRNA expression in the septum. ...................................................................................................85 4-18 NOP receptor mRNA expression in the bed nucleus of stria terminalis. ...........................86 4-19 Representative x-ray images of br ain sections showing NOP receptor mRNA expression in the bed nucleus of stria terminalis. ..............................................................87 4-20 NOP receptor mRNA expression in the amygdala. ...........................................................88 4-21 Representative x-ray images of br ain sections showing NOP receptor mRNA expression in the amygdala and the parave ntricular nucleus of the hypothalamus. ..........89 4-22 NOP receptor mRNA expressi on in the paraventricular and ventromedial nuclei of the hypothalamus and in the zona incerta of the thalamus. ...............................................90 4-23 Representative x-ray images of brain sections treated with 1 M N/OFQ plus 125I-[14Tyr]-N/OFQ.. ..........................................................................................................91 4-24 125I-[14Tyr]-N/OFQ binding to the NOP receptor in the septum........................................92 4-25 Representative x-ray images of brain sections showing 125I-[14Tyr]-N/OFQ binding to the NOP receptor in the septum. ....................................................................................93 4-26 125I-[14Tyr]-N/OFQ binding to the NOP recep tor in the ventromedial BNST. ..................94 4-27 Representative x-ray images of brain sections showing 125I-[14Tyr]-N/OFQ binding to the NOP receptor in the BNST. .....................................................................................95

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4-28 125I-[14Tyr]-N/OFQ binding to the NOP receptor in the amygdala. ...................................96 4-29 125I-[14Tyr]-N/OFQ binding to the NOP receptor in the PVN. ..........................................97 4-30 Representative x-ray images of brain sections showing 125I-[14Tyr]-N/OFQ binding to the NOP receptor in the amygdala and the PVN. ..........................................................97 4-31 Gland masses after social defeat.. ......................................................................................9 8 10

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1 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy ROLE OF THE NOCICEPTIN/ORPHANIN FQ-NOP RECEPTOR SYSTEM IN STRESS RESPONSES By Megan K. Green December 2007 Chair: Darragh P. Devine Major: Psychology Intracerebroventricular (ICV) microinjections of the neurop eptide nociceptin/orphanin FQ (N/OFQ) produce elevations in hypothalamic-pituitary-adrenal (HPA ) axis activity and anxietyrelated behaviors. Injections into the amygdala also elevate a nxiety-related behaviors, although to a lesser extent than do ICV injections. Th erefore, the effects of N/OFQ were examined following injections into another limbic structure, the bed nucleus of stria terminalis (BNST). Injections into the BNST produced elevated anxiety-related beha viors and circulating corticosterone. These results suggest that N/OFQ is involved in regulation of affective behaviors and the HPA axis through limbic actions. The po tent effects of N/OFQ following ICV injections may involve additive or synergistic activity at multiple limbic sites. To verify diffusion patterns of N/OFQ following these injections, rats were injected with 3H-N/OFQ into either the lateral ventricle, amygdala, or BNST. Following intraparenchymal injections, diffusion of N/OFQ was localized to the target structures Following the ICV injections, N/OFQ diffused throughout the ventricles and into periventri cular structures. A number of limbic, hypothalamic, and brainstem struct ures were identified th at could contribute to the effects observed following ICV injections.

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1 The possibility that N/OFQ and NOP receptor gene expression and receptor binding are modified by exposure to acute and repeated social stress was also explored. Rats were exposed to social stress (acute, repeated, repeated plus acute, or no stre ss) and then their brains were analyzed for mRNA expression and receptor bind ing. There were no statistically significant differences in prepro-N/OFQ mRNA expression or in receptor binding in any of the groups. However, the rats exposed to acute stress displayed greater NOP receptor mRNA expression in the septum, BNST, amygdala, and PVN, as co mpared to the expression in the unstressed controls. The rats exposed to repeated stre ss displayed greater NOP receptor mRNA expression in the BNST and PVN, and those exposed to repeated plus acute stress displayed greater expression in the amygdala and PVN. Overall, it appears that the N/OFQ-NOP recepto r system is involved in the regulation of affective states through actions in limbic structures. Additionall y, this may be a dynamic system that can be modified by acute and chronic social stress exposure, es pecially in limbic regions and the PVN.

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CHAPTER 1 INTRODUCTION Stress and Stress Response Stress Stress is defined as a physiologi cal response to a change or threat of change, real or perceived, in an organisms environment (Herman and Cullinan, 1997). The physiological responses include activation of systems involve d in preparation for high-energy behavioral responses and the subsequent return to homeostatic balance, such as activation of the autonomic nervous system and the hypothalamic-pituitary-ad renal (HPA) axis. The stimuli that initiate these organismic responses are referred to as st ressors and can be classified as systemic or processive. Systemic stressors involve direct threat s to tissues and organ systems, including events such as cold exposure or food depriv ation (Herman and Cullinan, 1997). These systemic stressors appear to be processed primarily by br ainstem inputs to the hypothalamus, and may not require processing by higher order limbic and corti cal systems. On the other hand, processive stressors, or emotional stressors, do seem to require higher order proces sing involving cortical and limbic inputs to the hypothalamus. These ty pes of stressors do not necessarily represent immediate threats to tissues, but derive their qualitative value rela tive to the organisms previous experience (Herman and Cullinan, 1997). There is anatomical evidence for at least some differentiation between these two stressor classifications. For example, ether exposure, a systemic stressor, and novel open field exposure, a processive stressor, produ ce different patterns of c-fos activation, with open field exposure producing greater c-fos activation in the lateral septum, medial amygdala, and dorsomedial hypothalamus (Emmert and Herman, 1999). Additionall y, lesions of the medial prefrontal cortex (MPFC; Diorio et al., 1993) and of the amygdala (Feldman et al., 1994) alter stress responses to 13

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14 processive stressors, such as restraint, photic, or acoustic stimuli, but not to ether. Rats with MPFC lesions display prolonged elevations in adrenocorticotropic hormone (ACTH) and corticosterone (CORT) following restraint, but no t following ether (Diori o et al., 1993). Rats with central and medial amygdaloid lesions disp lay attenuated corticotro phin releasing hormone (CRH), ACTH, and CORT responses to photic and acoustic stimuli but not to ether (Feldman et al., 1994). These studies provide ev idence that limbic structures respond strongly to processive stressors and less so to systemic stressors. HPA Axis Activation Following Stress Exposure The HPA axis is one important system invol ved in the processing of stressor-related information and in controlling physiological res ponses and emotionally-relevant behaviors. Cortical and limbic structures, as well as brainstem structures, have converging inputs at the paraventricular nucleus of the hypothalamus (PVN) where they are involved in regulation of the HPA axis (Herman et al., 1989). Additionally, th ere are inputs from cortical, limbic, and brainstem regions to the local surround, the peri-PVN. The PVN is comprised of subdivisions that can be differentiated in terms of their cytoarchitecture, neurotransmitter content, and projections. The dorsal parvocellular division, the ventral extent of the medi al parvocellular division, and th e lateral parvocellular division project to the brainstem and the spinal cord and are involved in autonomic regulation. The posterior magnocellular division re leases arginine vasopressin ( AVP) and oxytocin and projects to the posterior pituitary. The dorsolateral extent of the medial parvocellular division primarily contains and releases CRH, although approximately 50% of these cells coexpress AVP. These factors are released into the hypophyseal portal circulation at th e external median eminence and primarily affect the anterior pituitary (for review see Whitn all, 1993; Herman et al., 2002b).

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The dorsolateral extent of the medial parvoc ellular PVN is heavily involved in HPA axis regulation. This region integrates the excitatory and inhibitory inputs from the cortex, limbic structures, brainstem, and peri -PVN to produce a net response (f or reviews see Herman et al., 2002a and b). Net activation of th e dorsolateral extent of the me dial parvocellular PVN results in co-release of CRH and AVP. CRH stimul ates the anterior pituitary to release adrenocorticotropic hormone (ACTH) into the bloodstream, and AVP acts as a powerful synergist in this proce ss (Gillies et al., 1982). This effect may be further potentiated after repeated stress as the number of cells coexpressing CRH and AVP increase (for example de Goeij et al., 1992; Aubry et al., 1999). Once ACTH is released into the bloodstream, it induces the adrenal synthesis and release of glucoc orticoids. The glucocorticoids, including corticosterone (CORT) in ra ts and cortisol in humans, increase energy utilization and cardiovascular tone, shift the immune response fro m pro-inflammatory to anti-inflammatory, and terminate further HPA axis activity. Anatomy of the Limbic-HPA Axis HPA axis activation is affected by inputs from a number of cortical, limbic, and brainstem regions. The hippocampus, MPFC, septum, amygda la, and bed nucleus of stria terminalis (BNST) all project to th e PVN and /or its surround, a region that is dense with glutamatergic and GABA-ergic projections to the PVN (Sawche nko and Swanson 1983; Whitnall, 1993; Boudaba et al., 1996; Boudaba et al., 1997; Ziegler and Herman, 2001; Herman et al., 2002a). The hippocampus is involved in regulation of HPA ax is tone and inputs from this structure provide inhibitory modulation of the PVN (Herman and Cullinan, 1997). The MPFC is involved in negative feedback inhibition of the HPA axis, especially in response to processive stressors (Diorio et al. 1993). The septum provides direct and indirect inputs to the PVN (Sawchenko and Swanson, 1983; Risold and Swanson, 1997). The lateral septum generally provides excitatory 15

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input to the HPA axis while th e medial septal input is inhibitory (Dunn, 1987a). The amygdala has few direct inputs to the PVN (Prewitt and Herman, 1998), although the medial amygdala (MeA) does have some inputs to the peri-P VN (Canteras et al., 1995; Prewitt and Herman, 1998). The amygdalar involvement in HPA axis control is complex. While electrical stimulation of the MeA and the basomedial am ygdala (BMA) increase le vels of circulating CORT and stimulation of the central amygdala (C eA), basolateral amygdala (BLA), and lateral amygdala (LA) decrease CORT (Dunn and White ner, 1986), lesions of both the CeA and MeA decrease the ACTH and CORT response to proce ssive stressors while not affecting basal levels (Feldman et al., 1994). The CeA and MeA have dense projections to the BNST and to hypothalamic nuclei other than the PVN (LeDoux et al., 1988; Canteras et al., 1995; Prewitt and Herman, 1998; Dong et al., 2001). Therefore, the complex actions on HPA axis activity are likely indirect. The BNST is an important lim bic structure as it integrates inputs from the amygdala, the lateral septum, and the hippocampus and has dense inhibi tory and excitatory projections to the PVN and peri-PVN (Saw chenko and Swanson, 1983; LeDoux et al., 1988; Cullinan et al., 1993; Whitnall, 1993; Canteras et al., 1995; Boudaba et al., 1996; Cullinan et al., 1996; Boudaba et al., 1997; Risold and Swans on, 1997; Prewitt and Herman, 1998; Dong et al., 2001; Dong and Swanson, 2004). The inputs to th e PVN from the BNST are also complex. Electrical stimulation of the anterior and medial regions of the BNST increase levels of circulating CORT (Dunn, 1987a,b), stimulation of the lateral re gions of the BNST decrease levels of circulating CORT, and stimulation of the posterior regions produces mixed results (Dunn, 1987b). Additionally, lesions of the anterior regions of the BNST decrease CRH mRNA in the medial parvocellular PVN, while lesi ons of the posterior re gions increase CRH mRNA (Herman et al., 1994). However, lesions of th e lateral BNST attenuate the ACTH and CORT 16

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response to stressors (Gray et al ., 1993). None of these lesions alter the basal ac tivity of ACTH or CORT (Gray et al., 1993; Herman et al., 1994). Autonomic Nervous System Several regions of the PVN, in cluding the dorsal parvocellular, ventromedial parvocellular, and lateral parvocellular neurons project to brainstem autonom ic neurons (for review see Herman et al., 2002b). Sympathetic activation stimulates the re lease of norepinephrine from sympathetic neurons and epinephrine from th e adrenal medulla (Martin and Haywood, 1992; Kvetnansky et al., 2006). This activation further increases card iovascular tone (Martin and Haywood, 1992), as well as inhibi ts the inflammatory immune response and stimulates antiinflammatory responses via innervation at the ly mphoid organs (for review see Elankov et al., 2000; Eskandari and Sternberg, 2002). There is a tight interplay between the HPA axis and the sympathetic nervous system (SNS). Stressors activate both systems (Kvetnansky et al., 2006), and activation of one system typically results in activation of the other (for review see Elenkov et al., 2000). Therefore, there is a dual response from both systems, enhancing the full stress response. Chronic Stress and Disease Responses to acute stressors are thought to be adaptive in the short term by increasing access to energy stores and increasing cardiovascu lar tone. However, the effects of chronic cortisol elevations can be detrimental. In rats chronic stress is associat ed with increased basal CORT (Blanchard et al., 1998), blunted ACTH responses to stressor s (Hauger et al., 1990), altered CRH and AVP levels in the pituitary (Hauger et al., 1990; de Goeij et al., 1991), hippocampal atrophy (Manji et al., 2001), adrenal hype rtrophy (Blanchard et al., 1998; Hauger et al., 1990), thymus involution, and depressive-l ike behaviors such as decreased grooming (Blanchard et al., 1998). 17

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In humans, similar changes are seen in i ndividuals with depression. For example, individuals with depression displa y alterations in HPA axis activit y including elevated levels of CRH in their cerebrospinal fluid (CSF; Nemeroff et al., 1984; Nemeroff et al., 1991; Heuser et al., 1998) and cortisol in their bl ood plasma (Board et al., 1956; Gold et al., 1986; Arborelius et al., 1999). Individuals with depr ession also display blunted ACTH responses to CRH (Gold et al., 1986; Holsboer et al., 1986), but normal to elev ated cortisol responses (Gold et al., 1986; Holsboer et al., 1986; Juruera et al., 2004). There are also increas es in CRH mRNA and in CRH and AVP proteins in the PVN in post-mortem ti ssues of individuals wi th depression (Herman and Cullinan, 1997). In addition, these individuals display hippocampal atrophy (Manji et al., 2001), adrenal hypertrophy (Rubin et al., 1995), a nd immune dysfunction (Weisse, 1992). These symptoms seem to be a function of hypersecretion of CRH (Labeur et al., 1995; Linthorst et al., 1997) as well as impaired negative feedback (Nem eroff et al., 1991; Juruera et al., 2004). This HPA axis dysfunction appears to be a primary f actor in causing depressive symptoms. For example, individuals with depression who re spond well to anti-depressant treatment often display normalization of the HPA axis (Board et al., 1956; Gold et al., 1986; Amsterdam et al., 1988; Nemeroff et al., 1991; Heuser et al., 1998), as well as no rmalization of hippocampal and adrenal size (Manji et al., 2001; Rubin et al., 1995) and immune functioning (Weisse, 1992). Other disorders associated with alterations in HPA axis activity include panic disorder, obsessive compulsive disorder, post-trauma tic stress disorder, Tourettes syndrome, alcoholism and alcohol withdrawal, anorexia, fibromyalgia, and chronic fatigue syndrome (Crofford, 1998; Arborelius et al., 1999; Juruera et al., 2004). These physiological changes and the deve lopment of psychopathology, particularly depression and anxiety disorders, are further associated with chroni c processive stress in humans 18

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(for review see Arborelius et al., 1999). Stress factors associated with chronic depression include unemployment, lack of insurance, lo wer levels of education, low income, general medical conditions, low quality of life, low social adjustment, and generalized anxiety (Gilmer et al., 2005). The quality of life measures involved questions regarding work, economics, and social and familial relationships (Wisniewski et al., 2005). In another study, depression and generalized anxiety disorder were associated wi th recent stressful events including injury or illness and relationship problems (including de ath of a close individual and relationship breakdown). Additionally, both conditions were a ssociated with lifetime stressors, such as a history of bullying or sexual abuse (Jordanova et al ., 2007). Therefore, ch ronic stress exposure leading to HPA axis dysregulation may be implicated in human psychopathology. Animal Models of Chronic Stress Chronic Variable Stress The convergence of the effects of chronic stress in rats a nd of chronic stress in humans suggests that animal studies may provide important insights into the bioche mical basis of stressinduced psychopathology in humans. However, while a number of processive stressors are commonly used in research with rats (e.g., re straint, exposure to a novel environment, and exposure to soiled cages, bright lights, or loud noises), many of these are limited in their longterm effects, because the rats habituate to repe ated exposure to these stimuli (for example see Barnum et al., 2007). One common stress procedure used is the chronic variable stress (CVS) regimen. Many studies have reported increased basal HPA axis activity and the absence of habitutation (for example see Marin et al., 2007). However, as with Marin and colleagues study, often these CVS regimens include at least one stressor with a strong system ic component, such as cold exposure or food/water deprivation. In our lab, when only processive stressors were used in the 19

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CVS regimen, no increases in baseline HPA axis f unctioning, particularly in CORT levels, were observed (Simpkiss and Devine, 2003). Additio nally, Ostrander and colleagues (2006) found that rats displayed habituati on to novel processive stressors after CVS, but maintained responsivity to systemic stressors. Therefore, the utility of these CVS regimens as a model of human chronic processive stress and the develo pment of psychopathology is limited. When only processive stressors are used, CV S does not cause the kinds of chronic, basal changes seen in human populations. Other Chronic Stress Regimens Exposure to predatory stress, social separati on, and inescapable tail shock have produced more substantial and reliable a lterations in behavioral and phys iological activities than the CVS regimen has. For example, repeated visual a nd olfactory exposure to a cat produces increased basal CORT, adrenal hypertrophy and thymus involution, and depressive behaviors such as decreased grooming in rats (Blanchard et al., 1998). Young squirrel monkeys that have been separated from their peers display elevated CORT even several days later (Lyons et al., 1999). Additionally, even a single e xposure to inescapable tail shock produces elevated CORT responses 24 hrs later (Deak et al., 1999) and HPA hyperresponsivity as many as 10 days later (Johnson et al., 2002). These models of stress produce changes that look more like those seen in human chronic stress and depression. Social Defeat Recently, our lab has begun to work with a m odel of social stress, the social defeat procedure. In this procedure a small nave male intruder rat is placed into the cage of a larger conspecific (the resident). This resident has experience with territorial interactions and will typically display dominance behaviors when the in truder is present. These behaviors include pinning the intruder, biting the intr uder, and kicking bedding at the in truder. In response to the 20

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residents dominance behaviors, the intruder typically exhibits submissive behaviors, including freezing and lying on its back with paws up and a bdomen exposed (supine posture). These types of social interactions produce activation in limbic-hypothalamic st ructures of intruder rats as evidenced by increases in c-fos expression in the hypothalamus, septum, BNST, amygdala, and relevant brainstem nuclei such as the locus co eruleus and the nucleus of the solitary tract (Martinez et al., 1998; Chung et al., 1999; Nikulina et al., 2004). Additionally, several studies have reported activation of the HPA axis in intruder rats following social defeat exposure, as evidenced by increases in circulating ACTH (H einrichs et al., 1992; Ebner et al., 2005) and CORT (Heinrichs et al., 1992; Covington and Miczek, 2001; Wommack and Delville, 2003; Ebner et al., 2005). Also, unlike CVS, social def eat does not result in habituation of the HPA axis response, or even of the body temperature re sponse (i.e., induction of fever; Barnum et al., 2007). Additionally, rats exposed to repeated soci al defeat display increases in basal CORT (de Goeij et al., 1992; Barnum et al., 2007; Stone et al., in preparation). Repeated social defeat also causes other long-term changes, including decreases in thymus and seminal vesicle masses (Buwalda et al., 2001). These results suggest that social defeat affects the functioning of the HPA axis and HPA axis regulation, as well as other organs involved in immune response and reproduction. In addition to physiological changes, repeated social defeat also induces long-term behavioral changes. Rats that are exposed to social defeat display increased anxiety-related behaviors in the elevated plus maze (Heinrichs et al., 1992; Calfa et al. 2006) and increased behavioral despair in the Porsolt test (Rygula et al., 2005; Stone et al., in preparation). All of these results suggest that repeated social defeat may be a valuable tool in studying potential long-t erm effects of chronic stress exposure. 21

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Nociceptin/Orphanin FQ One neurotransmitter system that has been shown to be important in the neural response to stress is nociceptin/orphanin FQ (N/OFQ). N/OFQ and its cognate receptor NOP constitute a highly conserved (Danielson and Dores, 1999) pep tide neurotransmitter system that affects an interesting range of very important behavioral and physiological activities. N/OFQ is a 17 amino acid peptide that is structural ly similar to the endogenous opi oids, particularly dynorphin A (Meunier et al., 1995; Reinsche id et al., 1995). However, N/OFQ does not bind to the or opioid receptors with high affinity (Shimohigashi et al., 1996), but does bind with high affinity to the NOP receptor (Reinscheid et al., 1995; Shimohi gashi et al., 1996; Butour et al., 1997). The NOP receptor is a 7-transmembrane, G-protein coupled receptor (Bunzow et al., 1994; Chen et al., 1994; Wang et al., 1994; Wick et al., 1994; L achowicz et al., 1995; Re inscheid et al., 1996) that is negatively linked to ad enylate cyclase (Mollereau et al., 1994; Lachowicz et al., 1995; Reinscheid et al., 1995; Reinscheid et al., 1996), increases inward rectifying K+ channel conductance (Connor et al., 1996a ; Vaughan and Christie, 1996; Vaughan et al., 1997), and inhibits Ca2+ conductance (Connor et al., 1996b). The NOP receptor shows high structural homology with the opioid receptors (Bunzow et al., 1994; Chen et al., 1994; Mollereau et al., 1994; Wang et al., 1994; Wick et al., 1994; Lachowicz et al., 1995), although it does not bind any of the opioids with high affinity (Bunzow et al., 1994; Wang et al., 1994; Lachowicz et al., 1995; Butour et al., 1997). This low affi nity between N/OFQ and opioid receptors and between NOP receptors and opioid peptides suggests that the N/OFQ-NOP receptor system is functionally distinct from the opioid system. N/OFQ, the NOP receptor, and their mRNAs ar e widely distributed throughout the brain, spinal cord, and periphery (Bunz ow et al., 1994; Chen et al., 1994; Mollereau et al., 1994; Wang et al., 1994; Wick et al., 1994; Lachowicz et al., 1995; Nothacker et al., 1996; Neal et al., 1999a 22

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and b; Devine et al., 2003), consistent with a wide range of functions including pain modulation (Meunier et al., 1995; Reinscheid et al., 1995; Tian et al., 1997), motor performance (Reinscheid et al., 1995; Devine et al., 1996) spatial learning (Sandin et al ., 1997; Sandin et al., 2004), and feeding (Pomonis et al., 1996; Nicholson et al., 2002). However, N/OFQ and NOP receptor expression are relatively high in some limbi c regions including the hypothalamus, septum, BNST, and amygdala (Bunzow et al., 1994; Wa ng et al., 1994; Lachowicz et al., 1995; Nothacker et al., 1996; Neal et al ., 1999a and b; Devine et al., 2003). This limbic localization is consistent with an additional role of N/OFQ in emotional regulation, particularly stress and anxiety responses. For example, Devine and colleagues (2003) found that N/OFQ is released from forebrain neurons following ex posure to acute restraint stress. Exogenous N/OFQ administration has also been found to increase anxiety-related behaviors and HPA-axis activa tion in response to stressful/a nxiety-provoking stimuli. To examine anxiety-related behaviors, standard neophobic tests are generally used, such as the open field test, elevated plus maze, and light-dark test These tests take advantage of rats motivation to explore during foraging activities, while, on th e other hand, displaying a natural aversion to brightly lit and open spaces. For example, rats show thigmotaxis in the open field (for example see Simon et al., 1994), and they show a preference for the enclosed arms of the elevated plus maze (Handley and Mithani, 1984; Pe llow et al., 1985) and for the dark box of the light-dark test (Crawley and Goodwin, 1980; Costall et al., 1989; Onaivi and Martin, 1989; Chaouloff et al., 1997). The balance between exploration a nd avoidance can be manipulated in a highly reproducible manner by anxiolytic drugs (i.e., drugs that humans report to be anxiety-reducing, such as diazepam) and by anxiogenic drugs (i.e., drugs that humans report to be anxiety-inducing, such as FG 7142). Rats incr ease their exploration of open or lit spaces 23

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following administration of anxiolytic compounds and decrease their exploration following administration of anxiogenic compounds (Hughe s, 1972; Crawley and Goodwin, 1980; Crawley, 1981; Handley and Mithani, 1984; Pellow et al ., 1985; Pellow and File, 1986; Costall et al., 1989; Onaivi and Martin, 1989; Stefanski et al., 1992; Simon et al., 1994; Chaouloff et al., 1997; Fernandez et al., 2004). In our lab, a modified version of the open field test is use d. A start box was attached to one wall of the open field, which allows the use of latency to enter the open field and time spent in the open field as measures of anxiety-related behavior (in addition to thigmotactic behavior that has been reported in previous versions of the open field). The test has been calibrated (lighting, handling, etc.) so that vehicle-treated rats spend approximately 25% of the test time in the open field, allowing observation of both increases and decreases in anxiety-related behaviors. Under these conditions, rats that have been tr eated with diazepam ge nerally show shorter latencies to enter the open field, more total tim e spent in the open field, and more exploration away from the start box and into the middle of the open field, as compared to vehicle-treated rats. On the other hand, rats treated with FG 7142 generally show longer latencies to enter the open field, less total time spent in the open field, and less exploration away from the start box and into the center of the open field (Fernandez et al., 2004). These data provide evidence that the modified open field is a valid and sensitive tool for measuring changes in the expression of anxiety-related behaviors. Intracerebroventricular (ICV) injections of N/OFQ have been shown to increase anxiety-related behaviors in this open field test, as well as the elevated plus maze and the light-dark test (Fernandez et al. 2004 ; Green et al., in press). N/OF Q-treated rats, as compared to vehicle-treated rats, display longer latencies to enter and spend less total time in the open area of 24

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25 the open field test, the open arms of the elevated plus maze, and the lit box in the light-dark test. These behaviors resemble the effects following inj ections of other anxiogenic drugs, such as FG 7142 and are opposite from the effects following in jections of anxiolytic drugs, such as diazepam. These results suggest that N/OFQ has an anxiogenic action after ICV administration. In addition to these behavioral effects, ICV injections of N/OFQ increase HPA axis activity in rats. When rats are injected into the lateral ventricl e under unstressed conditions (i.e., the rats are allowed to recover from the stress of handling and cannula implantation prior to the delivery of the drug), and mildly stressed conditions (N/OFQ injec tions are administered without allowing rats to recover from the stress of hand ling) they exhibit substantial elevations in circulating ACTH and CORT (Dev ine et al., 2001; Nicholson et al., 2002; Legget et al., 2006). Elevations of ACTH and CORT are also observed wh en rats are injected and then exposed to the open field test (Fernandez et al., 2004). Additiona lly, injections of N/OFQ affect gene regulation in this system, producing increases in CRH mRNA in the PVN and increases in proopiomelanocortin (POMC; the precursor to AC TH) mRNA in the pitu itary (Legget et al., 2006). This suggests that N/OFQ is importa nt in the regulation of the HPA axis. In the following experiments, the role of N/OFQ in stress and anxiety is further explored. The potential role of limbic structures in produc ing increased anxiety-re lated behaviors and HPA axis activation is examined, and the diffusi on characteristics of N/OFQ following ICV and intraparenchymal injections ar e verified. Finally, the endoge nous regulation of N/OFQ mRNA and NOP receptor mRNA following acute and repeat ed social defeat exposure is examined.

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CHAPTER 2 ANATOMICAL ANALYSIS OF ANXIOGE NIC BEHAVIORAL EFFECTS OF EXOGENOUS NOCICEPTIN/ORPHANIN FQ Background The N/OFQ-induced elevations in circulating hormone concentr ations described in Chapter 1 are mediated by limbic inputs, including th e septum, BNST, and am ygdala (Misilmeri and Devine, in preparation). Specifi cally, unstressed injec tions into these limbic structures produce elevations in circulating ACTH and CORT. Accordingly, limbic st ructures are implicated in the HPA-axis modulating effects of N/OFQ under unstressed conditions. In light of the observations that N/OFQ produces anxiogenic beha vioral effects and activation of the HPA axis, and that the hormona l alterations produced by N/OFQ are at least partially mediated by limbic struct ures, we previously explored th e role of one limbic structure, the amygdala, in affecting anxiety-related behavior s and HPA axis activation in rats (Green et al., in press). The rats that were injected with N/OFQ into the amygdala displayed longer latencies to enter the open field (Figure 2-1). Ho wever, the behavioral eff ects of these injections into the amygdala were not as potent as the eff ects after injections into the lateral ventricle (Figure 2-2). Additionally, there were no signifi cant between-groups differences in circulating CORT concentrations when rats were given in tra-amygdaloid injections of N/OFQ or vehicle prior to the open field exposure (Figure 2-3). This lack of a hormonal effect might seem surprising at first, since there are elevations in circulating CORT con centrations after intraamygdaloid N/OFQ when rats are tested under un stressed conditions (Misilmeri and Devine, in preparation). However, in that unstressed expe riment, the controls exhibited low basal CORT levels, and the effects of the N/OFQ injections we re small. In our behavioral experiment, the rats were injected under stressed conditions (h andling) and were exposed to the additional stressor of the novel open field. In this instan ce the circulating CORT concentrations in the 26

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27 vehicle-treated rats were much higher. Thus, the stress-induced elevations in CORT could have obscured any HPA axis-activating effect of the amygdaloid injections of N/OFQ. This finding concurs with a previous report in which the mild stress of a novel environment partially obscured the hormonal effects of N/OFQ after an ICV injection (Devine et al., 2001). Because of these less potent effects, the BNST was examined as a potential site for the anxiogenic effects of N/OFQ. The BNST is anot her limbic structure known to participate in the regulation of behavioral and hor monal responses to anxiety-induc ing stimuli (for examples see Henke, 1984; Rogan et al., 1997; Walker and Da vis, 1997). The amygdala and BNST can be differentiated in terms of their roles in fear and anxiety (Lee a nd Davis 1997; Walker and Davis, 1997; for review see Walker et al., 2003). Altho ugh the distinctions are not entirely clear, the amygdala appears to play a larger role in fear-related behaviors (such as startle responses to a specific, usually conditioned, stimulus), and the BNST appears to be more important in responding to anxiety-provoking stimuli (primarily those stimuli that are long in duration, nonspecific, and non-conditioned). For example, lesions of the BNST, but not the amygdala, block light-enhanced startle, while amygdaloid lesions but not BNST lesions, block fear-potentiated startle (Walker and Davis, 1997). The modified ope n field test used in the present experiment resembles tests of generalized anxiety more than it resembles tests of fear, as there is no specific or conditioned fear stimulus. In this respect, the BNST may be more involve d in the behavioral responses during neophobic tests of anxiety such as the open field test and may be an important potential mediator of the anxioge nic effects that were seen af ter ICV injections of N/OFQ. Methods Animals Male Long Evans rats (n = 34, Harlan, Indi anapolis, IN) were hous ed in polycarbonate cages (43 x 21.5 x 25.5 cm) on a 12hr-12hr light-dark cy cle (lights on at 7:00 am). The rats were

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pair-housed in a clim ate-controlled vivarium (temperature 21-23 C humidity 55-60%) until surgery. After surgery, the rats were singlyhoused in the same environment. Standard laboratory chow and tap water were available ad libitum throughout the experiment. All procedures in this and subsequent experiments were pre-approved by the University of Floridas Institutional Animal Care and Use Committ ee, and the experiments were conducted in compliance with the National Research Councils Guide for the Care and Use of Laboratory Animals. Drugs Ketamine and xylazine were both obtained from Henry Schein (Melville, NY) at concentrations of 100 mg/ml. Ketamine-xylazi ne was mixed by adding 2 ml of xylazine to 10 ml of ketamine yielding a 12 ml solution of 83.3 mg/ml ketamine and 16.7 mg/ml xylazine. Ketorolac tromethamine (30 mg/ml) and AErrane (99.9 % isoflurane) were also purchased from Henry Schein. N/OFQ was obtained from Sigma-Aldrich (St. Louis, MO) and was dissolved in artificial extracellular fluid (aECF) com posed of 2.0 mM Sorensons phos phate buffer (pH 7.4) containing 145 mM Na+, 2.7 mM K+, 1.0 mM Mg2+, 1.2 mM Ca2+, 150 mM Cl-, and 0.2 mM ascorbate. These ion concentrations replicate the concentrations found in ex tracellular fluid in the brain (Moghaddam and Bunney, 1989). N/ OFQ was prepared at concentrations of 0.01, 0.1, and 1.0 nmole per 0.5 l aECF. Surgery Each rat (265-407g) was implanted with a guide cannula under ketamine-xylazine anesthesia (62.5 mg/kg ketamine + 12.5 mg/kg xylazine, i.p. in a volume of 0.75 ml/kg). Ketorolac tromethamine (2 mg/kg, s.c.) was inje cted for analgesia at the time of surgery. AErrane was administered as supplemental surgi cal anesthesia as necessary. Once the rat was 28

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anesthetized, a stainles s steel guide cannula (11mm, 22 gauge ) was implanted into the right BNST (0.3 mm posterior to bregma 3.0 mm lateral from the midline, and 5.4 mm ventral from dura; n = 28). Cannulae were implanted at a 14o angle to minimize intrus ion of the cannula into the ventricle. Six additional rats were each im planted with a cannula aimed at extra-BNST sites (anatomical controls). Each cannula was secured w ith dental cement anchored to the skull with stainless steel screws (0.80 x 3/32). An obturator that extended 1.2 mm beyond the guide cannula tip was inserted at the tim e of surgery and removed on the day of the experiment at the time that an intracranial injection was administer ed. Following surgery, each rat was given at least 7 days to recover from surgery. Equipment Anxiety-related behavior was measured in an open field test. The open field was composed of a 90 x 90 x 60 cm field with a 20 x 30 x 60 cm start box attach ed to the outside of the open field, at the midpoint of one side (see Figure 2-4). The bottom and sides were constructed of black acrylic. A black acrylic guillotine door separated the start box and the open field. This door was attached to a rope and pulley system, which allowe d the door to be opened from outside the testing room. The tops of th e start box and open field were open and a camera was mounted on the ceiling above the testing apparatus to re cord the rats behaviors. Illumination of the start box and open fiel d were approximately equal (14-30 lux). Anxiety-Testing Procedure After the surgical recovery period, each rat was handled for 5 minutes on each of 3 consecutive days, and then given one day with no disturbance. On the 5th day, each rat was fitted with a 28-gauge stainless steel injector c onnected with polyethylene (PE20) tubing to a 1 l Hamilton syringe. Each rat then received a 0.5 l injection of aECF vehicle or aECF containing 0.01, 0.1, or 1.0 nmole N/OFQ by an experi menter blind to the dose. The injections 29

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were administered over a 2-minute period using a syringe pump, and the injector was left in place for 3 additional minutes to allow diffusion of the injection volume. Each rat was freely moving in its home cage during the injection procedure. These injections and the subsequent behavior al tests were comple ted 90-210 minutes after the vivarium lights were turned on, the time during which th e HPA axis is at its daily nadir (Ixart et al., 1977; Kwak et al., 1993). Five minutes after the injec tion, each rat was individually placed in the start box of the open field test, and the door to the testing room was closed, isolating the rat from the experimenter and any other disruptive influences. The rat was then given 1 minute to acclimate to the novel envi ronment of the start box. After 1 minute the guillotine door was opened remotely, and it remained open throughout the test period. The rat was given 5 minutes to explore th e start box and open field. Each rat was then returned to its home cage until 30 minutes elapsed after the start of the injection. At this time, the rat was rapidly decapitated. Immediately after decapitation, 6 ml of trunk blood was collected into polypropylene tubes containing 600 l of Na2EDTA (20 g/l) on ice. The tube s were centrifuged at 1000x gravity. The plasma fraction was collected, aliquotted, and frozen at -80C. Later, RIA was performed for quantification of plasma concentrations of CORT using a kit from Diagnostic Products Corp. (Los Angeles, CA). The inte rassay variability for this kit increased with sample CORT concentrations, ranging from less than 5% for lo wer plasma CORT concen trations to less than 15% for higher plasma CORT concentrations. Additionally, each brain was removed, frozen in 2-methylbutane at -40C, and stored at -80C. Later, each brain was sectioned at 30 m and stained with cr esyl violet for cannula 30

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placement verification. Adrenal glands, thymus glands, and spleens were dissected out and weighed for verification of the health status of the rats. An observer who was blind to the treatment conditions scored the expl oratory behavior of the rats from the videotapes, using a grid that wa s superimposed on the video monitor. This grid divided the open field into 25 equal squares. The outer 16 squares defined an outer zone and the inner 9 squares defined an inner zone (Fig. 2-4) Between-groups differences in the latency to enter the open field, total time spent in the open fi eld, latency to enter and time spent in the inner zone, and the number of entries in to the open field and the inner zone were used as measures of anxiety. An entry into the open field or moveme nt from the periphery to the inner zone was counted when all 4 paws of the rat le ft one zone and entered a new zone. Statistical Analyses In order to analyze differences between the N/OFQ-treated groups and the vehicle-treated group, one-way ANOVAs were calculated for latency to enter the open field and the inner zone, total time spent in the open field and the inner zone, number of entries into the open field and inner zone, plasma CORT concentrations, a nd organ masses. All significant effects ( p< 0.05) were further analyzed usi ng Fishers LSD post-tests. Differences between anatomical controls and in tra-BNST vehicle contro ls for each of the listed measures were analyzed by individual t -tests. Results Anxiety-Related Behavior The intra-BNST N/OFQ-treated rats showed greater expression of anxiety-related behaviors than the aECF vehicletreated rats did. The N/OFQ-trea ted rats displayed significantly longer latencies to enter the ope n field (Fig. 2-5a; F(3,24) = 4.009, p<0.05) than did the vehicle-treated rats, but the groups did not differ significantly in latency to enter the inner zone 31

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(Fig. 2-5b; F(3,24) = 2.593, p>0.05). There were also no significant differences between N/OFQ-treated and vehicle-treated rats in tim e spent in the open fiel d (Fig. 2-5c; F(3.24) = 2.075, p>0.05) or the inner zone (Fig. 2-5d; F(3,24) = 2.669, p>0.05). However, the N/OFQ-treated rats displayed significantly fewer entries into the open fi eld (Fig. 2-5e; F(3,24) = 3.055, p<0.05) and significantly fewer entries into the inner zone (Fig. 2-5f; F(3,24) = 3.321, p<0.05) than did the vehicle-treated rats. The BNST placements were fairly evenly distributed throughout the medial and lateral divisions (Fig.2-6). The effects of vehicle injections into th ese BNST sites were compared against the effects of N/OFQ injections into extra-BNST sites (anatomical controls). Comparisons between the vehicle-treated rats and the N/OFQ-treated anatomical controls showed no significant differences on any measure of latency (Fig. 2-5a and b), time (Fig. 2-5c and d), or entries (Fig. 2-5e and f). Circulating Corticosterone Injections of N/OFQ into the BNST produced significant elevations in circulating CORT (Fig. 2-7; F(3,22) = 3.171, p<0.05) as compared to the CORT concentrations in the vehicle-treated rats. Concentr ations of circulating CORT we re not significantly different between N/OFQ-treated anatomical c ontrols and vehicle-treated rats. Organ Masses There were no significant diffe rences in adrenal weights (Fig. 2-8A), thymus gland weights (Fig. 2-8B), or spleen weights (F ig. 2-8C) between the vehicle-treated and N/OFQ-treated rats and between the vehicletreated rats and anatomical controls. 32

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Discussion Anxiety-Related Behaviors and Corticosterone We previously found that injections of N/OFQ into the right lateral ventricle produce dose orderly elevations in all meas ured anxiety-related behaviors in the modified open field and elevations in circulating CORT (Fernandez et al., 2004; Green et al ., in press). In jections into the right amygdala also elevated anxiety-re lated behaviors, alt hough the behavioral and hormonal effects were less potent than those seen following ICV injections. Similarly, injections into the right BNST produced el evations in anxiety-related behaviors and elevations in circulating CORT, and once again, these effects were less potent than those observed after ICV injections. The behavioral results are somewhat surprising as the amygda la, and especially the BNST appeared to be ideal candidates for media ting the behavioral actions of N/OFQ. Both structures are involved in respons es to emotionally-salient stre ssors, and both structures have relatively high levels of NOP receptor mRNA expression and binding (Neal et al., 1999b). It is possible, however, that ICV injections are havi ng effects at multiple sites, accounting for the greater potency of this route of administration. Because injections into the amygdala and into the BNST produce partial effects on a nxiety-related behaviors, there ma y be additive or synergistic effects at these and other limbic, cortical, and br ainstem structures following ICV injections. For example, the lateral septum may be another st ructure involved. Like the amygdala and the BNST, the lateral septum is involved in responses to stressors and has at least moderate levels of NOP receptor binding. The hormonal effects following intraparenc hymal injections are less surprising. The modest effects following amygdaloid injections can be expected cons idering there are only sparse connections between the amygdaloid complex and the PVN (Prewitt and Herman, 1998). Unlike the amygdala, though, the BNST does have many connections to the PVN (for example 33

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see Prewitt and Herman, 1998; Dong et al., 2001 ; Dong and Swanson, 2004). This anatomical difference is consistent with the greater elevations in circulating CORT observed in stressed (the present experiment) and unstressed rats (Misilmeri and Devine, in preparation) following intraBNST injections versus intra-amygdaloid injections. One concern with the smaller effects of our intraparenchymal injections versus ICV injections is the fact that they were done unilate rally (injections were into the right hemisphere in all groups). Previous analysis of the diffusion of N/OFQ af ter unilateral ICV injections suggested that there was at leas t some bilateral distribution, prim arily at midline periventricular structures (D. P. Devine, personal communication). It is not clear if bilateral injections into the BNST or the amygdala would have increased the effects. To our knowledge, the effects of bilateral BNST injections of a nxiolytic or anxiogenic compounds have not been reported with any standard tests of fear or a nxiety. Thus it is possible that injections of N/OFQ into the right and left BNST might produce more potent effects. However, in the case of the amygdala, the right hemispheric structure is generally more dominantly involved in emotionally-relevant behavioral responses (Coleman-Mesches and McGaugh, 1995a and b; Andersen and Teicher, 1999; Adamec et al., 2001; Peper et al., 2001; Scicli et al., 2004), or is at least no less involved than the left amygdala (Good and Westbrook, 1995; LaBar and LeDoux, 1996; Izquierdo and Murray, 2004). In fact, b ilateral injections of drugs in to the amygdal ae may add little in terms of changes in emotionally-relevant behaviors when compared to the effects of unilateral injections into the right am ygdala (for example see Coleman-Mesches and McGaugh, 1995b). Therefore, in our previous analysis of the eff ects of unilateral amygdaloid injections it does not seem likely that bilateral injections w ould have produced more potent effects. 34

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There are reports that N/OFQ and its synthetic analogue Ro64-6198 each exert anxiolytic actions in rats and mice (Jenck et al., 1997; Je nck et al., 2000; Gavioli et al., 2002; Varty et al. 2005). Additionally, some researchers find opposing effects depending on the dose of drug used (Kamei et al., 2004), the number of times the drug is administered (Vital e et al., 2006), or the anxiety tests used (in this cas e with NOP receptor knockouts; Gavioli et al., 2007). The reasons for these discrepancies are currently unclear; although, there are methodologi cal differences that may account for the contrasting results (see Fern andez et al 2004 for a full discussion of the potential explanations). It is possible that N/OFQ may produce differing anxiogenic and anxiolytic actions depending upon th e prior history and/or the current stress status of the animals, but we, in the Devine lab, have so far been unable to detect anxiolytic effects in our studies. It has been argued that the increases in anxi ety-related behaviors may be due to locomotor impairment produced by N/OFQ. At higher dos es of N/OFQ there are profound motoric effects that result in postural changes and reduced lo comotion (Devine et al., 1996). Recently, Vitale and colleagues (2006) reported reduc ed exploration of the open arms of the elevated plus maze after a single dose of N/OFQ administered ICV. However, a second dose given 2 hours later resulted in a reversal of this effect. They interpreted the reduced e xploration as a locomotor effect, and concluded that the lack of this e ffect following the second administration was due to tolerance (rats develop tolerance to the locomoto r-inhibiting effects of N/OFQ; see Devine et al., 1996). However, anxiogenic actions have been observed at doses that do not produce locomotor effects (Devine et al., 1996; Fernandez et al., 2004; Green et al., in press). Additionally, locomotor controls show that motoric effects ar e not the likely source of the reduced exploration following N/OFQ injections (Fernandez et al., 2004; Green et al., in press). For example, when the threatening stimulus of a brightly lit enviro nment in the dark-light box is removed, thus 35

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creating a dark-dark test, N/OFQtreated rats and vehicle-treate d rats explored the boxes in a comparable manner (Fernandez et al., 2004). Likewi se, when the threateni ng stimulus of a large open field is removed and replaced with an unders ized open field equal in size to the start box, N/OFQ-treated rats and vehicle-tr eated rats again behaved in a co mparable manner (Green et al., in press). The only difference in these conditions is the presence or absence of a threatening stimulus. Thus, it seems likely that behavioral differences observed in the open field test and light-dark box are due to differenc es in the anxiety states of the rats and not due to locomotor impairment. The results of the present experiment, as well as the results following injections into the amygdala, provide additional evidence of an anxi ety-related effect as opposed to a locomotor effect. The intraparenchymal injections in these experiments were highly specific, with no significant effects observed in th e anatomical controls. Given the known roles of the BNST and amygdala, injections into these structures would not be expected to affect locomotor behaviors, but would be expected to affect anxiety-related behaviors. Organ Masses In the present study, the adrenal, thymus, and spleen masses were measured to establish that there were no systematic differences in hea lth status or stress e xposure between the various groups of rats. Since there were no significant differences in adrenal gland masses, thymus gland masses, or spleen masses between any of th e groups tested, it can be concluded that there were no apparent differences in the health or st ress history of the rats that can account for the behavioral or hormonal differences. Summary The results of the present study show that N/OFQ injections affect anxiety and HPA axis activity through limbic structures including the BN ST. In addition, previous work has shown a 36

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role for the amygdala. This suggests the possi bility that limbic N/OFQ neurotransmission may be involved in regulation of affect. However, because ICV injections of N/OFQ produced greater, more potent effects than did injections into either the BNST or the amygdala, there may be additive or synergistic actions between the BNST and the amygdala. Additionally, the contralteral structures as well as other relevant limbic, cortical, and brainstem structures may be involved. 37

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0.0 0.01 0.1 1.0 AC 0 100 200 300 ** A *Dose N/OFQ (nmoles)Time (seconds) 0.0 0.01 0.1 1.0 AC 0 100 200 300 BDose N/OFQ (nmoles)Time (seconds) 0.0 0.01 0.1 1.0 AC 0 100 200 300 CDose N/OFQ (nmoles)Time (seconds) 0.0 0.01 0.1 1.0 AC 0 1 2 3 4 5 6 7 8 9 10 DDose N/OFQ (nmoles)Time (seconds) 0 0.01 0.1 1.0 AC 0 1 2 3 4 5 6 7 8 9 10 EDose N/OFQ (nmoles)Number of Entries 0 0.01 0.1 1.0 AC 0 1 2 3 4 5 6 7 8 9 10 FDose N/OFQ (nmoles)Number of Entries Figure 2-1. Anxiety-related behaviors following intra-amygdaloid injections of N/OFQ. (A) N/OFQ-treated rats exhibited longer late ncies to enter the open field. However, injections of N/OFQ into the right amygdala did not significantly alter (B) the latency to enter the inner zone, (C) the time spent in the open field, (D) the time spent in the inner zone, (E) the number of entries into the open field, (F) or the number of entries into the inner zone. Values expressed are group means SEM (n = 9-10 rats per group). Significant differences between the N/OFQ-treated rats and the aECF-treated controls are expressed as p < 0.05 and ** p < 0.01. AC =anatomical controls. 38

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0.0 0.01 0.1 1.0 0 100 200 300** ** ADose N/OFQ (nmoles)Time (seconds) 0.0 0.01 0.1 1.0 0 100 200 300* B **Dose N/OFQ (nmoles)Time (seconds) 0.0 0.01 0.1 1.0 0 100 200 300** ** C *Dose N/OFQ (nmoles)Time (seconds) 0.0 0.01 0.1 1.0 0 1 2 3 4 5 6 7 8 9 10** D **Dose N/OFQ (nmoles)Time (seconds) 0 0.01 0.1 1.0 0 1 2 3 4 5 6 7 8 9 10** ** E *Dose N/OFQ (nmoles)Number of Entries 0 0.01 0.1 1.0 0 1 2 3 4 5 6 7 8 9 10** F ** *Dose N/OFQ (nmoles)Number of Entries Figure 2-2. Anxiety-related behavi ors following ICV injections of N/OFQ. N/OFQ-treated rats exhibited: (A) longer latencies to enter the open field and (B) the inner zone, (C) less total time in the open field and (D) the inne r zone, and (E) fewer entries into the open field and (F) the inner zone. Values expressed are group means SEM ( n = 7-8 rats per group). Significant differences between the N/OFQ-treated rats and the aECFtreated controls are expressed as p < 0.05 and ** p < 0.01. 39

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0.0 0.01 0.1 1.0 0 200 400 600** ** A **Dose N/OFQ (nmoles)CORT (ng/ml) 0.0 0.01 0.1 1.0 AC 0 200 400 600 BDose N/OFQ (nmoles)CORT (ng/ml) Figure 2-3. Concentrations of circulating corticosterone following ICV and intra-amygdaloid injections of N/OFQ. (A) Injections of N/OFQ into the right lateral ventricle produced elevations in circulating CORT. (B) N/OFQ administration into the right amygdala did not significantly alter the leve ls of circulating corticosterone.Values expressed are group means SEM ( n = 7-10 rats per group). Significant differences between the N/OFQ-treated rats and the aE CF-treated controls are expressed as ** p < 0.01. AC = anatomical controls 40

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Start Box Outer Zone Inner Zone Figure 2-4. Photograph of the open field and a diagram of the zone s used for scoring. The rat is placed in the start box for the first minute of the test. After the door opens, the rat may move between the start box and the open field freely. The outer zone represents the periphery of the open field. The inner zo ne represents the central region of the open field. 41

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0 0.01 0.1 1.0 AC 0 100 200 300 ** ADose N/OFQ (nmoles)Time (seconds) 0 0.01 0.1 1.0 AC 0 100 200 300 BDose N/OFQ (nmoles)Time (seconds) 0 0.01 0.1 1.0 AC 0 100 200 300 CDose N/OFQ (nmoles)Time (seconds) 0 0.01 0.1 1.0 AC 0 1 2 3 4 5 6 7 8 9 10 DDose N/OFQ (nmoles)Time (seconds) 0 0.01 0.1 1.0 AC 0 1 2 3 4 5 6 7 8 9 10 EDose N/OFQ (nmoles)Number of Entries 0 0.01 0.1 1.0 AC 0 1 2 3 4 5 6 7 8 9 10* FDose N/OFQ (nmoles)Number of Entries Figure 2-5. Anxiety-related behaviors follo wing intra-BNST injections of N/OFQ. N/OFQ-treated rats exhibited (A) longer latencies to enter th e open field and (E) fewer entries into the open field and (F ) the inner zone. However, there were no significant differences in (B) latency to enter the inner zone or in (C) time spent in the open field or (D) the inner zone. Values expressed are group means SEM (n = 6-8 rats per group). Significant differences between the N/OFQ-treated rats and the aECF-treated controls are expressed as p < 0.05 and ** p < 0.01. AC = anatomical controls. 42

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Figure 2-6. Anatomical map of BNST placements. Th e injector tip placements are illustrated for each rat, including anatomical controls (adapted from Paxinos and Watson, 1998.). =1.0 nmole doses; =0.1 nmole doses; =0.01 nmole doses; =aECF controls. Anatomical controls are identified by a black dot placed within the marker. 43

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BNST Circulating Corticosterone 0 0.01 0.1 1.0 AC 0 200 400 600 **Dose N/OFQ (nmoles)CORT (ng/ml) Figure 2-7. Concentrations of circulating corticosterone following intra-BNST injections of N/OFQ. Injections of N/OFQ into the BNST produced elevations in CORT at the highest dose administered. Values expressed are group means SEM ( n = 6-8 rats per group). Significant differences betw een the N/OFQ-treated rats and the aECF-treated controls are expressed as ** p < 0.01. AC = anatomical controls 44

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45 0.0 0.01 0.1 1.0 AC 0 10 20 30 40 50 60 70 80 90 100 ADose N/OFQ (nmoles)Weight (mg) 0.0 0.01 0.1 1.0 AC 0 50 100 150 200 250 300 350 400 450 500 BDose N/OFQ (nmoles)Weight (mg) 0.0 0.01 0.1 1.0 AC 0 100 200 300 400 500 600 700 800 900 1000 CDose N/OFQ (nmoles)Weight (mg) Figure 2-8. Analysis of glandular masses. (A) Ad renal gland masses, (B) thymus gland masses, and (C) spleen masses showed no signifi cant differences between groups. Values expressed are group means SEM ( n = 6-8 rats per group).

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CHAPTER 3 DIFFUSION OF NOCICEPTIN/ORPHANIN FQ AFTER INTRACEREBROVENTRICULAR AND INTRAPARENCHYMAL INJECTIONS Background The behavioral and hormonal effects following in jections of N/OFQ into the right lateral ventricle, the amygdala, the BNST, and other limbi c structures have been described (Fernandez et al., 2004; Green et al., in press; Missilmeri a nd Devine, in preparation) However, the extent of the diffusion of N/OFQ following these injecti ons is not clear. Data from the anatomical controls in Chapter 2 suggest th at diffusion following injections into the BNST and the amygdala was limited to the target structure. To confir m this and compare it w ith the penetration of N/OFQ into the tissue following ICV injections, 3H-N/OFQ was injected into the lateral ventricle, the BNST, an d the amygdala. Methods Animals Male Long Evans rats (n = 15, Harlan, Indi anapolis, IN) were hous ed in polycarbonate cages (43 x 21.5 x 25.5 cm) on a 12hr-12hr light-dark cy cle (lights on at 7:00 am). The rats were pair-housed in a climat e-controlled vivarium (temperature 21-23 C humidity 55-60%) until surgery. After surgery, the rats were singlyhoused in the same environment. Standard laboratory chow and tap water were available ad libitum throughout the experiment. Drugs Ketamine and xylazine were both obtained fr om Henry Schein (Mel ville, NY) and were mixed as described in Chapter 2. Ketorolac tromethamine (30 mg/ml) and AErrane (99.9 % isoflurane) were also purc hased from Henry Schein. N/OFQ was obtained from SigmaAldrich (St. Louis, MO) and was dissolved in artificial extracellular fluid (aECF), as described in Chapte r 2. N/OFQ was prepared at concentrations of 46

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47 1.0 nmole per 1.0 l aECF for ICV injections or per 0.5 l aECF for intra-amygdaloid and intra-BNST injections. 3H-N/OFQ was prepared by adding 0.01 nmoles 3H-N/OFQ (Phoenix Pharmaceuticals, Belmont, CA) to the 1.0 nmole N/OFQ doses. Surgery Each rat (268-319g) was implanted with a guide cannula under ketamine-xylazine anesthesia (62.5 mg/kg ketamine + 12.5 mg/kg xylazine, i.p. in a volume of 0.75 ml/kg). Ketorolac tromethamine (2 mg/kg, s.c.) was inje cted for analgesia at the time of surgery. AErrane was administered as supplemental surgi cal anesthesia as necessary. Once the rat was anesthetized, a stainles s steel guide cannula (6 or 11mm, 22 gauge) was imp lanted into the right lateral ventricle (ICV; 0.8 mm posterior to breg ma, 1.4 mm lateral from the midline, and 2.7 mm ventral from dura; n = 5), the right amygdala (1 .8 mm posterior to bregma, 3.9 mm lateral, and 6.2 mm ventral; n = 5) or the ri ght BNST (0.3 mm posterior to br egma, 3.0 mm lateral from the midline, and 5.4 mm ventral from dura; n = 5). The ICV and amygdaloid cannulae were each implanted vertically, and the BNST cannulae were implanted at a 14o angle. Each cannula was secured with dental cement anchor ed to the skull with stainless steel screws (0.80 x 3/32). An obturator that extended 1.2 mm be yond the guide cannula tip was inse rted at the time of surgery and removed on the day of the experiment at the time that an intracranial injection was administered. Following surgery, each rat was gi ven at least 7 days to recover from surgery. Injection Procedure Following recovery from surgery, each of the rats was handled for 5 minutes on one day. Two days later the rats were given 3H-N/OFQ injections. Each rat received a 1.0 l (ICV, n= 5) or 0.5 l (intra-amygdala, n= 5, and intra-BNST, n= 5) injection of aE CF containing 1.0 nmole N/OFQ plus 0.01 nmole 3H-N/OFQ. These injections were administered over a 2-minute period, and the injector was left in place for 3 additiona l minutes to allow for diffusion. Each rat was

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rapidly decapitated 12 minutes following the start of the 3H-N/OFQ injection. This time point corresponded with the time at which the rats in Experiment 1 were removed from the open field. Immediately after decapitation (approxima tely 15 min following the start of the 3H-N/OFQ injection), each brain was removed, frozen in 2-me thylbutane at -40C, an d stored at -80C. Later, each brain was sectioned at 20 m and placed on x-ray film (Kodak X-Omat) at -80C for 12 weeks. Following film exposure, the brain sections were stained with cres yl violet for further cannula placement verification and anatomi cal evaluation of the film images. Results Intracerebroventricular Injections Injections of 3H-N/OFQ into the righ t lateral ventricle pr oduced differing diffusion patterns depending on whether there was a successful ventricular placement or not. Of the 5 ICV placements, cresyl violet staining revealed 1 clear h it, 3 marginal hits, and 1 miss. In the rat with the clear ventricular placement (Fig. 3-1), 3H signal was seen in all aspects of the right lateral ventricle (Fig. 3-1a and b), conti nuing in more posterior regions into the dorsal and ventral 3rd ventricle (Fig. 3-1a-d), through the cerebral aqueduct, and into the 4t h ventricle (Fig. 3-1e and f). From these locations there was visible diffu sion approximately 0.5-1mm into the tissues, reaching the lateral septum; medial and latera l subnuclei of the BNST, striatum; subfornical organ; numerous thalamic nuclei; paraventricular, suprachiasmatic, posterior, and arcuate nuclei of the hypothalamus; medial preopt ic area; fimbria; periaqueductal gray; some cerebellar regions (particularly those that emerge near the 4th ventricle); tegmental nuc lei; and locus coeruleus. In general, signal was visible around the ventricula r spaces throughout the anterior to posterior extent. In the rats with marginal ventricular hits (e.g., the cannula tip was located at the edge of the corpus callosum near the ventri cular wall) there was some visible 3H signal in the right lateral 48

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ventricle, but only faint signal in the dorsal and ventral 3rd ventri cle. No signal was visible in the more posterior cerebral aque duct or 4th ventricle. In th ese cases the diffusion from the ventricular regions was much lighter, primarily reaching the lateral septum and striatum. In the rat with a clear ventri cular miss (the cannula tip was lo cated in the corpus callosum and did not pierce the ventricle) 3H signal is very faint in the right lateral ventricle and only located in a very limited anterior to posterior span (less than 1mm). In this rat, as well as those with marginal hits, the 3H-N/OFQ was visible primarily as an injection bolus dispersed over the corpus callosum. In all cases, there was subs tantial signal along the edges of the cannula track, suggesting that the drug diffused up along the sides of the cannula implant. This generally led to signal affecting the cingulate, M1, and M2 cort ices, lateral septum, and striatum. Intra-Amygdaloid Injections All 5 of the intra-amygdaloid placements di splayed visible signal over the amygdala. In general, substantial signal wa s visible along the edges of the cannula track with an injection bolus of approximately 1-1.5mm radius ventral to the cannula tip. This bolus was typically visible over the central amygda la, medial amygdala, basola teral amygdala, basomedial amygdala, and internal capsule (Fig. 3-2a). Cons idering some variability in placement, other structures that occasionally displayed signal included the BN ST-intraamygdaloid nucleus, anterior amygdala, reticular thalamus, latera l hypothalamus, and substantia innominata. As with the ICV placements, all amygdaloid placements displayed signal along the edges of the cannula track, affecting structures such as the S1 cortex and the striatum. To verify that this was occurring following the injections (and not as an effect of the removal of the cannula following decapitation), one intra-amygdaloid im planted brain was rapidly frozen prior to removing the cannula. In this instance there was still substantial si gnal visible around the 49

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cannula track, suggesting that this was, in fact, occurring duri ng and following the injections (Fig. 3-2b). Intra-BNST Injections All 5 of the intra-BNST placements displa yed visible signal over the BNST. However, they also all displayed some ve ntricular involvement, even when the cannula did not apparently pierce the ventricular wall. Observation follo wing cresyl violet staining revealed 1 placement that did not appear to pierce the ventricle, 1 with marginal entry into the right lateral ventricle, and 3 that fully passed through the right lateral ventricle. With the one placement that did not pierce the ventricle, there was vi sible signal over the medial BNST (Fig. 3-3a). As was the case with the ICV and intra-amygdaloid placements, there was visible signal along the cannula track edges, r eaching structures such as the S1 cortex, the striatum, and some thalamic nuclei. As menti oned above, despite no obvious penetration into the ventricles, there was still visible signal in the lateral and 3rd ventricles (Fig. 3-3b). From the ventricular spaces there was faint diffusion visibl e into the subfornical organ; septum; and the paraventricular, suprachiasmatic, and arcuate nuclei of the hypothalamus; and the median eminence. In the case of the more marginal ventricu lar penetration, there wa s visible signal over the medial and lateral BNST, anterior commissure diagonal band, ventral pallidum, substantia innominata, and preoptic areas (Fig. 3-3c). The visible signal al ong the cannula track was evident over the M1 cortex, the lateral and medial septum, and the striatum. There was signal visible in the right lateral ventri cle; however, in more posterior regions the signal was very faint (Fig. 3-3d). Here there was slight visible signal in the cerebral aqueduct with some diffusion into the periaqueductal gray. 50

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In the cases where the cannula passed through th e right lateral ventricle, there was visible signal over the medial and lateral BNST and ante rior commissure (Fig. 3-3e). In some cases there was signal over the preoptic areas, hypothalamic nuclei (PVN, ar cuate, lateral), and ventral pallidum. The signal along the edges of the can nula track was generally visible over the M1 cortex and the striatum. Additional signal greatly resembled ICV in jections, with signal visible in the right lateral ventricle, dor sal and ventral 3rd ventricle, cereb ral aqueduct, and 4th ventricle. Diffusion from these ventricular regions affected structures such as the septum; subfornical organ; thalamic nuclei; periventri cular, paraventricular, arcuate, ventromedial, dorsomedial, and anterior nuclei of the hypothalamus; and the medi al preoptic area. In more posterior regions, there was greater variability in the signal displa yed. Two of the 3 placements displayed visible signal in the cerebral aqueduct with diffusion into the periaqueductal gray (Fig. 3-3f). One of these continued to display clear signal into th e 4th ventricle with di ffusion over the emerging cerebellum, the tegmental nuclei, and the locus coeruleus. Discussion Injections of 3H-N/OFQ into the right lateral ventricl e revealed differing diffusion patterns depending on the accuracy of the placement. In our behavioral experiments, only rats that had a cannula placement that was clearly within the lateral ve ntricle were used. This was verified by cresyl violet staining and visualization of the can nula tip. Therefore, the diffusion pattern that is depicted in Figure 3-1 is the best representation of the diffusion th at occurred in the rats in the behavioral studies. Accordingly, the potent hormonal and behavioral effects seen following ICV injections were likely due to ipsilateral e ffects in rostral brain regions, with potential bilateral involvement of periventricular, midline structures at more caudal sites. The sites that were labeled with 3H-N/OFQ and are most likely to be involved in anxiety-related responses include the lateral septum, the BNST, the medial preoptic area, the periaqueductal gray, the 51

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tegmental nuclei, and the locus co eruleus. Interestingly, substa ntial diffusion into the amygdala from the ventricles was not observed, although our behavioral study sugge sts that the amygdala is involved in anxiogenic responses to N/ OFQ. It is important to note that 3H produces a faint signal, requiring months of film exposure. Thus the possibility cannot be ruled out that a small amount of the ICV-injected N/OFQ might diffuse as far as the amygdala. The size and shape of the diffusion patterns af ter lateral ventricle, BNST, and amygdala injections are in agreement w ith previous analyses using ot her compounds (puromycin, CORT, insulin-like growth facto r) that were injected into the hypotha lamus, frontal cortex, or lateral ventricle (see Renner et al., 1984; Diorio et al., 1993; Nagaraja et al., 2005). The localization observed following intraparenchymal injections suggests that the behavioral and hormonal effects are primarily due to actions within the target structures. However, with all of the injections (ICV, intra-BNST, and intra-amygda loid), there was significant diffusion up the cannula track. Again, this is in agreement with previous diffusion studies (for example see Renner et al., 1984). This does not appear, however to contribute to the behavioral or hormonal effects observed in chapter 2. In those experiment s, anatomical controls were included. In all the anatomical controls, there would be diffusi on back up the cannula track affecting the same general extra-amygdaloid and extra-BNST sites as the targeted injectio ns did. The lack of behavioral and hormonal effects in the anatomical controls confirms that the effects of the BNST and amygdaloid injections were indeed mediated at the target structures. On the other hand, the possibility cannot be ru led out that the BNST injections of N/OFQ described in chapter 2 produced some effects via diffusion into the ventricles, since some ventricular diffusion was apparent even when th e cannula did not pierce th e lateral ventricle. While our analyses of anxiety-re lated behaviors or circulating CO RT did not include data from 52

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rats in which clear penetration of the ventricle was observed, this may not have eliminated all ventricular involvement. 53

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F E D C B A Figure 3-1. Representative x-ray im a g es of 3H-N/OFQ diffusion following ICV injections overlaid on the corresponding cresyl violet st ained section (A, C, and E) and x-ray im ages alone (B, D, and F). (A, B) Injections in to the r i ght lateral v entr i cle resulted in strong 3H s i gnal in the ipsila ter al ve ntricle with som e diff usion into th e c ontra late ral ventricle and the 3rd ventri cle. Further diffusion from these ventricles is m o st prom inent in the periventricular structur es within approximately 0.5-1mm from the ventricular wall. Diffusion c ontinues posteriorly through (C, D) the 3rd ventricle and (E,F) into the 4th ventricle. 54

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B A Figure 3-2. Representative x-ray im a g es of 3H-N/OFQ diffusion following intra-am ygdaloid injections overlaid on the co rresponding cresyl vi olet stained section. (A) Diffusion of 3H-N/OFQ from these placem ents is generally lo calized to th e in jection site with diffusion throughout the am ygdaloid com plex. (B) In addition there is substantial diffusion along the cannula track that occu rs during the injection procedure, as evidenced b y freezing th e tissu e prio r to cannu la rem oval. 55

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56 C D E F B A Figure 3-3. Representative x-ray images of 3H-N/OFQ diffusion following intra-BNST injections overlaid on the corresponding cresyl violet stained section (A, C, and E) and x-ray images alone (B, D, and F). (A,B) Diffusion of 3H-N/OFQ from successful BNST placements, with no ventricular piercing, results in 3H signal that is generally localized to the injection s ite with diffusion across numerous BNST sub-nuclei and along the cannula track. With these placements there is slight diffusion into the ventricles. (C, D) Cannulae that pierce th e lateral ventricle or (E, F) fully pass through it result in injections w ith diffusion into the posterior 4th ventricle and surrounding structures. In our anxiety experiments, rats with placements piercing the ventricle were removed from the experiments.

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CHAPTER 4 NOCICEPTIN/ORPHANIN FQ AND NOP RECEPTOR GENE REGULATION AND NOCICEPTIN/ORPHANIN FQ BINDING TO THE NOP RECEPTOR AFTER SINGLE OR REPEATED SOCIAL DEFEAT EXPOSURE Background Administration of the NOP receptor antagonists J-113397, [Nphe1]-nociceptin (1-13)-NH2 and UFP-101 have antidepressant effects in animal models of depression (Redrobe et al. 2002; Gavioli et al. 2003; 2004). This raises the possi bility that the N/OFQ-NOP system may play an important role in stress-associated psychopat hology. If N/OFQ neur otransmission is truly implicated in these disorders, it seems reasonable to expect that severe or repeated stress might cause dysregulation of the expression of gene s and/or proteins in the N/OFQ-NOP receptor system. In fact, Devine and colleagues (2003) fo und that the system is tightly regulated. Acute restraint stress caused a d ecrease in the N/OFQ content in the forebrain of rats. This decrease occurred rapidly and was replenished within a 24 hour period. Thus, it ap pears that there is a release of N/OFQ from basal forebrain neurons following acute stress expo sure and the content is restored rapidly in those ne urons. Following from this, the pr imary aim of this study was to examine the gene regulation of N/OFQ and the NOP receptor in response to acute and repeated social stressors. N/OFQ is cleaved from a precu rsor protein, prepro-N/OFQ. In rats, prepro-N/OFQ is 181 amino acids, containing one copy of N/OFQ at the 135-151 amino acid positions (Nothacker et al., 1996). Therefore, the gene regulati on of prepro-N/OFQ was examined using in situ hybridization with a radiolabelle d riboprobe complimentary to the prepro-N/OFQ mRNA. In addition, repeated N/OFQ release might affect gene regulation of the NOP receptor, a 367 amino acid protein (Wang et al., 1994). Therefore, in situ hybridization was conducted using a 57

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58 radiolabelled riboprobe aimed at the NOP recepto r mRNA in order to explore changes in the receptors gene expression. Changes in receptor mRNA expression, however, may not accurately represent changes in protein synthesis. Post-transcriptional proc esses could destroy the mRNA, blocking protein synthesis. Additionally, post-tr anslational mechanisms could re sult in the break ing down of the protein or in the packaging of receptors in vesicles for later use, but not resulting in insertion of the receptors into the membrane. To determine if changes in receptor mRNA expression results in changes in receptor insertion and functionality, a preliminary examination of the binding of N/OFQ to the NOP receptor was cond ucted using autoradiography with 125I-N/OFQ. In order to expose rats to social stressors, the social defeat procedure described in chapter 1 was used. This allowed the examination of cha nges in gene regulation in response to a single, acute social defeat, repeated social defeat with no acute exposure, and repeat ed social defeat with an acute exposure. Methods Animals Forty-eight male Long Evans ra ts and 24 female Long Evans rats (Harlan, Indianapolis) were used in this experiment. Twenty-four of th e male rats and the 24 female rats were used as resident pairs in the social def eat procedure. These male rats weighing 400-500g at arrival (600700g at the time of the experiment), were pair-housed prior to vasectomy surgery, and then singly housed for 1 week during recovery. The females (200-225g at arrival) were pair-housed until the males recovered from surgery, at which time the males and females were housed together. Four of the male rats were used as intruders for the purposes of training the resident males. The remaining 20 males, weighing a pproximately 300g, were used as experimental intruder rats. The intruder rats were pair-housed throughout the cour se of the experiment. All of

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the rats were housed in 43 x 21.5 x 25.5 cm pol ycarbonate cages on a 12h r-12hr light-dark cycle (lights on at 7:00 am) in a climate-controlled vi varium (temperature 21-23C, humidity 55-60%). Standard laboratory chow and tap water were available ad libitum Drugs Ketamine and xylazine were both obtained fr om Henry Schein (Mel ville, NY) and mixed as described in Chapter 2. Ketorolac tromethamine (30 mg/ml) and AErrane (99.9 % isoflurane) were also purchased from Henry Schein. Surgery Each of the male resident rats was vasectomized under ketamine-xylazine anesthesia (62.5 mg/kg ketamine + 12.5 mg/kg xylazine, i.p. in a vol ume of 0.75 ml/kg). Ketorolac tromethamine (2 mg/kg, s.c.) was injected for analgesia at the time of surgery. AErrane was administered as supplemental surgical anesthesia as necessar y. Vasectomy was completed by making a 1 cm ventral midline incision just rostra l to the penis. Each vas defe rens was isolated using forceps and a 0.5 cm section of each duct was removed using a mini cautery tool. The abdominal wall was sutured with absorbable 4-0 Ethilon monofilament nylon non-wicking suture (Ethicon Inc.), and the external incision was closed with stainl ess steel wound clips (9mm, World Precision Instruments Inc.). Following surgery, each rat was returned to its home cage and allowed 7-10 days for recovery, at which time the wound clips were removed. Social Defeat Procedure The resident male and female rats were pai r-housed for at least 10 days and each of the male resident rats was trained and tested fo r territorial behavior prior to introducing the experimental intruder rats. In each training session, resident females were removed from the home cage 10 minutes before resident-intruder en counters. After 10 minutes, the intruder was placed into the cage with the resident. The en counter continued for 5 minutes or until the 59

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intruder displayed submissive behavior defined as entering into a supine posture (Fig. 4-1) 3 times for at least 2 seconds each or freezing for a total of 90 seconds. Encounters were also terminated if either rat was severely bitte n, which rarely occurred. The resident rat was considered dominant if the intruder submitted as defined above. The 14 residents that most consistently displayed dominance behaviors at th e end of the training period were used for the experiment. The social defeat procedure with the experiment al intruder rats occurred in 2 stages. The first stage was identical to the training session and was terminated by the same criteria (3 submissions, 90 seconds of freezing, 5 minutes ma ximum, or severe biting). Immediately following termination of stage 1, each intruder rat was placed, individu ally, into a 10cm x 10cm x 15cm (inner dimensions) double-walled wire mesh cage and placed back into the residents cage to allow further stress exposure while protecti ng the rat from potential injury (Fig. 4-2). The intruder remained in the cage until 10 minutes had passed from the start of stage 1. This allowed for equalization of the dur ation of stress exposure of the intruder rats re gardless of how quickly stage 1 was terminated. Following the 10-mi nute session, the intruder rat was returned to its home cage and the female was returned to the residents cage. The social defeat procedure was conducted over 6 days wherein specific treatment groups were exposed to differing amounts of repeated and/or acute exposure (Table 1). Group 1 experienced no social defeats on any of the 6 days (No Stress Controls). Group 2 experi enced a social defeat encounter on the 6th day only (No Repeated, Acute). Group 3 experienced a social defeat encounter on each of the first 5 days, but did not experience an encounter on the 6th day (Repeated, No Acute), and group 4 experienced social defeat encounters on all 6 days (Repeated, Acute). On the 6th day, all of the 60

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intruder rats were rapidly decapit ated at 3 hours following the start of the defeat session, or at the equivalent time in the groups that were not acutely defeated before termination. Immediately after terminati on of the rats, each brain was removed, frozen in 2-methylbutane at -40C, and stor ed at -80C until use. The th ymus glands, adrenal glands, and spleens were dissected out and weighed for examina tion of the health/stress status of each rat. The social defeat interactions were recorded and subsequently scored for the number of defeats and total time spent in submissive posture s (total time defeated) for each interaction. There was an uneven number of rats per group, with 4 rats included in groups 1 and 4 and 6 rats included in groups 2 and 3. The in situ hybridization procedure has limitations in the number of slides that can be processed per experiment; ther efore, the number of ra ts was reduced to 4 per group in all 4 groups. In group 4, there was variabili ty in the number of defeats that each rat received; therefore, the groups were balanced such that each group included some rats that received a high number of defeats and some rats that had a low number of defeats. In Situ Hybridization Each brain was sectioned into 15 m slices and mounted onto polyl ysine-subbed slides. Every third section was processed for prepro -N/OFQ, NOP receptor mRNA, or NOP receptor autoradiography. The 504 base cDNA fragment corre sponding to the 5' end of the coding region of the rat prepro-N/OFQ (Fig. 4-3) was clone d into plasmid pAMP and was provided by Dr. Olivier Civelli. The 529 base cDNA fragment co rresponding to 102 bases in the 3' untranslated region through 427 bases of the open reading fram e of the NOP receptor (Fig. 4-4) was cloned into BSSK and was provided by Dr. Huda Akil. Antisense and sense riboprobes were generated and labeled with 35S-UTP (>1000 Ci/nmole; GE Healthcare). The pAMP plasmid was linearized with EcoR1 and transcribed with SP6 polymeras e to generate the an tisense prepro-N/OFQ riboprobe. This plasmid was also linearized with HindIII and transcribed with T7 to yield the 61

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sense probe. The BSSK plasmid was linearized with EcoR1 and transcribed with T7 to generate the antisense riboprobe for NOP receptor mRNA, a nd this plasmid was also linearized with Xho1 and transcribed with T3 to yield the sense probe for the NOP receptor. The brain sections were prepared for hybridization by soaking in 4% formaldehyde for 1 hour, then rinsing in 3 washes of 2x SSC and one wash of ddH2O all at room temperature (RT). The sections were soaked in 0.1M Triethanolamine plus 0.25% vol/ vol acetic anhydride for 10 min., then rinsed in ddH2O and dried using graded ethanols (50, 70, 80, 95, 95, 100, and 100%). The radiolabelled riboprobes were prepared in 50% hybridization buffer and were hybridized to the mounted sections. The slides were coverslipped and in cubated overnight in humidified boxes at 55C. Coverslips were then removed and the sections were treated with RNAse A (to remove singlestranded, nonspecific label), then washed in 2x SSC (RT), 1x SSC (RT), and 0.1x SSC (67C) to further remove excess label. The sections were dried in graded ethanols from 50% to 100%. Once the sections and slides were completely dried they were placed on Kodak X-OMAT film in x-ray cassettes and exposed at room temperature for 1 week (NOP receptor) or 2 weeks (N/OFQ). The films were developed then photog raphed and digitized with an MCID camera and software system (Imaging Research Inc., Canada). Due to the limitations in the total number of slides that can be processed in any single in situ hybridization experiment, separate experiments were conducted for the anterior and posterior halves of the brain for each mRNA probe. This division was between -3.6 (last anterior section) and -4.8 (first posterior section) mm posterior to bregma, according to the Paxinos and Watson (1998) rat brain atlas. Optical densities were m easured in selected brai n regions that are known to participate in physiol ogical stress responses and/or emoti onal regulation. Those regions were the MPFC; the dorsolateral, interomediolateral, and ventrolateral septum (DLS, ILS, andVLS, 62

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respectively); the anteromedial BNST, the posteromedial BNST, the lateral BNST, and the ventromedial BNST; the CeA, Me A, BMA, BLA, and LA; the PVN, arcuate, and ventromedial (VMH) hypothalamus; the MPOA; the reticular nucle us and zona incerta of the thalamus; CA1, CA2, CA3, and dentate gyrus region s of the hippocampus; the substa ntia nigra pars reticularis, pars lateralis, and pars comp acta; the periaqueductal gray; the dorsal raphe; and the locus coeruleus. Autoradiography All sections from the 4 groups were processe d together for autoradiographic analysis of N/OFQ binding to the NOP receptor so that identical radioligand binding conditions were used across groups. Slides were incubated with 125I-[14Tyr]-N/OFQ such that there were approximately 6000 counts per minute per section. Controls for nonspecific binding were also incubated with 1 M N/OFQ. The iodinated probe was applied in an incubation chamber (RT, 60-80% humidity) in a buffer of 50 mM Tris HCl (pH 7.0), 1.0 mM EDTA, 0.1% BSA with proteinase inhibitor consisting of 0.1 mM phenylmethylsulfonyl fluoride, 1 g/ml aprotinin, 1 g/ml leupeptin, 1.0 g/ml pepstatin, and 1.0 mM iodoacetamide. The incubation was terminated after 1 hour with four 4-minute wa shes in 50 mM Tris HCl on ice (pH 7.0). The sections were then rinsed in ddH2O to remove excess salt. Dry sections were placed on Kodak X-OMAT film in x-ray cassettes and exposed at room temperature for 5 days. The films were developed then photographed and digitized with the MCID camera and software system. The total number of slides did not exceed the number that could be processed at one time, so all sections were processed t ogether. Optical densit ies were measured in the same regions as in the in situ hybridization experiments. 63

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Densitometry and Statistics Potential differences between the repeated a nd the repeated plus acute groups in the number of defeats and the tota l time defeated over the first 5 days were analyzed by a 2x5 repeated measures two-way AN OVA. All significant effects ( p< 0.05) were further analyzed using Bonferroni post-tests. Additionally, potential differences between the acute and repeated plus acute groups in the number of def eats and the total time defeated on the 6th day were analyzed using T-tests. Densitometric analysis of radioactive signal wa s analyzed using the MCID Basic software. A standard outline, using the Paxinos and Wats on Rat Brain Atlas (1998), was determined for each region analyzed. Bilateral optical density measures were sampled from each region. If multiple sections were used per re gion, the optical densities for thes e sections were integrated to create one data point per rat per region per pr obe. Background measures were taken from the striatum and cerebellum since these are regions with very low levels of expression of N/OFQ mRNA, NOP receptor mRNA, and NOP receptor pr otein. Background values were subtracted from values obtained in the analyzed regions to control for potential diffe rences in non-specific binding. The group means were compared by a one-way ANOVA for each region analyzed. All significant effects ( p< 0.05) were further analyzed using Dunnets post-tests. In order to analyze differences in organ masses between the stressed and non-stressed groups, group means were compared by one-way ANOVAs for adrenal gla nd weights, thymus gland weights, and spleen weights. Results Social Defeats Whereas I attempted to balance the stress-expos ed groups by including the data from some rats that were defeated multiple times and some rats that were defeated fewer times, there was 64

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still variability in the nu mber of defeats between groups (Fig. 45). In general, over the first 5 days the rats experiencing repeated defeats a nd those experiencing repeat ed plus acute defeats were exposed to, on average, 11.25 defeats and 9.75 defeats, respectively. However, these differences were not statistically significant (F(1,24)= 1.385, p>0.05). Additionally, there were no significant differences in the number of defeats between days 1 through 5 (F(4,24)= 1.068, p>0.05) or in the interaction between stress group and day (F(4, 24)= 1.748, p>0.05). On the 6th day the acutely defeated group and the repeatedly plus acutely defeated group received, on average, 3 defeats and 1.25 defeats, respectively. Again, though, these differences were not statistically significant (t(6)= 1.849, p>0.05). There was also some observed variability in the total time defeated (Fig. 4-6). The repeatedly stressed group experienced an averag e total of 104.75 sec of defeat over the first 5 days, and the repeatedly plus acutely stressed group experien ced an average total of 136.75 sec of defeat over the first 5 days. However, thes e differences were not statistically significant (F(1,24)= 2.796, p>0.05). There were also no significant differences in the time defeated between days 1 through 5 (F(4,24)= 1.286, p>0.05) or in the interacti on between stress group and day (F(4,24)= 1.016, p>0.05). On day 6, the acutely stressed gr oup was defeated for an average 20 sec while the repeatedly plus acutely stressed group was defeated for an average of 12.5 sec. Again, these differences were not sta tistically signifi cant (t(5)= 0.756, p>0.05). In Situ Hybridization Sense controls for prepro-N/OFQ and NOP receptor mRNAs displayed no specific signal. The background levels were approximately equal to those treated with antisense probes, and none of the sampled regions expressed signal substantially higher than background after hybridization with the sense probe s (Fig. 4-7). Therefore, signal measured from antisensetreated sections can be cons idered specific for prepro-N /OFQ or NOP receptor mRNA. 65

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Prepro-N/OFQ There were no significant differences across groups in prepro-N/OFQ mRNA expression in any of the regions examined. However, there were cons istent trends toward elevated expression in several limbic regions fo llowing acute social de feat exposure. These elevations occured in the dorsolateral and ventrolateral septal nuclei (Fig. 4-8, 4-10), all the nuclei of the BNST (Fig. 4-9, 4-10), the CeA a nd MeA (Fig. 4-11, 4-13), the zona incerta and reticular nucleus (Fig. 4-12, 4-13), and the dorsa l raphe (Fig. 4-14, 4-15) Signal was low and there were no visible trends in the MPFC, BMA, BLA, LA, PVN, arcuate, VMH, MPOA CA1, CA2, CA3, DG, SNpr, SNpl, SNpc, P AG, or LC (data not shown). NOP Receptor There were significant stress-induced increases in NOP receptor mRNA expression in several important limbic regions. In the septum (Fig. 4-16, 4-17), there were significant elevations in NOP r eceptor mRNA expression following acute stress exposure in both the DLS (Fig. 4-16a; F(3, 15)= 7.134, p<0.01), and the VLS (Fig. 4-16c; F(3, 15)= 3.656, p<0.05). The ANOVA for the ILS revealed a significant overa ll effect that appears to result from a trend toward increases in NOP receptor mRNA expression in the acutely defeated groups, (Fig. 4-16b; F(3, 15)= 3.52, p<0.05). However, the post-tests revealed no significant differences between any of the stressed groups and the control group for NOP receptor mRNA expression in the ILS. In the BNST, there was a significant elevation in NOP receptor mRNA expression in the ventromedial region following repeated so cial defeat exposure (Fig. 4-18, 4-19; F(3, 14)= 6.132, p<0.05). In the amygdala, NOP receptor mRNA was elevated in several regions, particularly following acute social defeat exposure (Fig. 4-20). In the CeA, there were elevations in NOP receptor mRNA following exposure to acute and repeat ed plus acute social defeat exposure (Fig. 4-20a, 4-21; F(3, 15)= 6.449, p<0.01). In the MeA, these elevations occured following acute social defeat (Fig. 4-20b; F(3, 15)= 5.174, p<0.05). There were also si gnificant elevations in NOP 66

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receptor mRNA following acute social defeat in the BMA (Fig. 4-20c, 4-19; F(3, 15)= 6.02, p<0.01), the BLA (Fig. 4-20d, 4-21; F(3, 15)= 8.66, p<0.01), and the LA (Fig. 4-20e; F(3, 15)= 9.802, p<0.01). In the PVN there were significant elevations in NOP receptor mRNA in all stress-exposed groups (Fig. 4-21, 4-22a; F(3, 15)= 10.47, p<0.01). Differences in NOP receptor mRNA expression approached sign ificance in the VMH (Fig. 22b, F(3, 15)= 3.398, p=0.0536) and in the zona incerta (Fig. 22c, F(3, 15)= 3.25, p=0.0599). These differences appear to be due to elevations following exposure to acute and to rep eated plus acute social defeat. There were no significant differences in NOP receptor mRNA e xpression in the reticular nucleus of the thalamus, in any of the hippocampal regions, or in any of the midbrain or brainstem regions. Autoradiography Controls for non-specific binding displayed little to no signal and background levels were approximately equivalent to those treated with 125I-[14Tyr]-N/OFQ (Fig. 4-21) Therefore, signal measured from 125I-[14Tyr]-N/OFQ -treated sections can be considered specific for binding to the NOP receptor. However, there were no significa nt differences in N/OF Q binding between any of the stress-exposed groups fo r any of the regions analy zed (Figs. 4-24 to 4-30). Organ Masses There were no significant diffe rences in adrenal weights (Fig. 4-31a), thymus gland weights (Fig. 4-31b), or spleen weights (Fig. 4-31c) between th e non-stressed controls and the socially defeated rats. Discussion In general, basal expression of prepro -N/OFQ and NOP receptor mRNAs, as well as NOP receptor binding agrees with previous anatomi cal examinations (Neal et al., 1999a and b). Stress exposure appears to alter th ese basal levels, particularly in the expression of NOP receptor mRNA. In the BNST, it appears that the N/OF Q-NOP receptor system plays a role in limbic 67

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plasticity following repeated, se vere stress exposure. Additionally, in the PVN, NOP receptor regulation appears to be sensitiv e to acute and repeated social stressors. This suggests that stressor exposure might produce significan t changes in HPA responses to N/OFQ. Prepro-N/OFQ mRNA Devine and colleagues (2003) previously f ound release of N/OFQ following acute stress exposure, and the N/OFQ content was replenished within 24 hours. In order for the peptide content to be replenished, transc ription and protein synthesis need to occur. Therefore, it was expected that acute and repeated plus acute social defeat woul d produce increases in prepro-N/OFQ mRNA. While ther e were not statistically significant changes, there were consistent trends toward elev ated mRNA expression following acute social defeat exposure. These trends were repeated over multiple limbic structures, including the septum, the BNST, and the amygdala. All of these regions are involve d in responses to fea rand anxiety-provoking stimuli (for example see Goldstein, 1965; Van de Kar et al., 1991; Gray et al., 1993; Calfa et al., 2006; Menard and Treit, 1996; Lee and Davis, 1997; Walker and Davis, 1997; Vyas et al., 2003). Several of the regions where trends were presen t are in the basal forebrain, the same region where Devine and colleagues (2003) found releas e of N/OFQ. This concordance and the consistency of the trends over multiple limbic structures suggest that the effect is a real one. One potential reason for the lack of statistical significance is the variability in the amount of stress exposure, with some rats experiencing fewer so cial defeats and less total time in submissive postures. While between groups differences in th e number and total duration of defeats were not statistically significant, we do not know if these variables are cr itical in determining the affective and physiological responses of the intruders (or if the mere presence of the resident is the important factor). It is possibl e that the variability in resident s behaviors could contribute to variability in the mRNA expression of intr uders, reducing statistical power. Another 68

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consideration is that Devine and colleagues (2003) f ound that N/OFQ content was replenished 24 hours later. It is possible that in the current study the timepoint selected was not during the peak of the mRNA upregulation. It is also possible that there is a relatively small upregulation of mRNA (as we observed in the consistent trend across many forebrain regions), and that N/OFQ is slowly replenished over an exte nded portion of that 24 hour period. NOP Receptor mRNA There were significant changes in NOP recepto r mRNA expression in several brain regions following social defeat exposure. NOP recepto r mRNA expression was si gnificantly increased in several limbic-hypothalamic regions, includ ing the septum, the BNST, the amygdala, and the PVN. These changes primarily occurred following acute social defeat exposure, although in the amygdala there were also increases in NOP recep tor mRNA in the repeated plus acute group and in the BNST there were increases in mRNA in the repeated (but no acute) group. This increase in the BNST following repeated stress is intere sting given the most recent hypothesis about the roles of the amygdala and the BNST. It has been shown that the amygdala and the BNST can be differentiated in terms of their roles in fea rand anxiety-like responses, with the amygdala primarily involved in fear-like behaviors and the BNST involved in anxiety-like behaviors (Walker and Davis, 1997; for review see Walker et al., 2003). It has been proposed that the involvement of these structures in the response to stressor exposure is related to the length of the exposure, with the amygdala involved in the early, immediate response and the BNST involved in the later, long-term responses (Walker et al., 2003). Our findi ngs are consistent with this interpretation, with the amygdala showing changes in response to an acute social defeat and the BNST showing changes in response to repeated de feat sessions. These results suggest that the N/OFQ-NOP receptor system may respond differe ntly, depending on the type and amount of stress exposure and the region of the brain. 69

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NOP receptor mRNA was also expressed in greater amounts in the PVN following all social defeat regimens (acute, repeated, and repe ated plus acute), as compared to the expression in the no stress control group. It is known that administration of N/OF Q into the ventricles produces activation of the HPA axis in unstresse d rats and prolongs the activation in mildly stressed rats (Devine et al., 2001). One likely si te for this activation is the PVN, since this structure contains the neurons that synthesize and release CRH, activating the HPA axis. Therefore, NOP receptor upregulation in the PVN could suggest an increased sensitivity of the HPA axis. The upregulation of NOP receptor mRNA in re sponse to stress exposure is somewhat surprising given the increased release of N/OFQ following stressful events (Devine et al., 2003). In some receptor systems, an increase in neurotransmitter release (or the overconsumption of a drug that acts at the receptor) re sults in long-term desensitizat ion of the receptora mechanism that accounts for many tolerance e ffects. It has already been obs erved that rats display rapid tolerance to the locomotor-inhibiting effects of N/OFQ (Devine et al., 1996) ; thus, the system is capable of tolerance and the receptors may desens itize in response to N/OFQ release. There is evidence that the NOP receptor do es, in fact, undergo rapid desens itization (Dautzenberg et al., 2001; Spampinato and Baiula, 2006). However, Spampinato and Baiula (2006) demonstrated that at higher levels of N/OFQ exposure, th e NOP receptor undergoes internalization and recycling resulting in resensitiz ation and, even, supersentization. These experiments, however, are in artificial systems and it is not clear how these results compare to stress-induced release of N/OFQ in vivo NOP Receptor Binding There were no significant incr eases in N/OFQ binding in an y of the regions analyzed, despite the upregulation of NOP receptor mRNA in several regions. There may be some post70

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transcriptional or post-translational mechanism inte rfering with the synthesis of the receptors or with the insertion of th e receptors into the cell membrane. However, it may be more likely that the time point selected to examine binding was not the ideal time point In the acute groups, binding was examined 3 hours after stressor exposure. At this tim e point, protein synthesis and receptor insertion may not have yet occurred. In the repeated but no acute groups, binding was examined 27 hours after the last stressor exposure. At this point, receptor levels may have returned to baseline. Devine and colleagues ( 2003) have already shown the peptide content is replenished within 24 hours. Therefore, th e N/OFQ-NOP receptor systems appears to be a tightly regulated system in which changes in response to stress ors are transient. To fully examine the potential changes in re ceptor synthesis and insertion and potential changes in binding, a more thorough analysis w ould be required. This would involve a full Scatchard analysis to examine binding, prot ein analysis by immunohistochemistry under nonpermeabilizing and permeabilizi ng conditions (to assay membra ne insertion), and protein quantification by Western blot. Organ Masses There were no statistically significant changes in adrenal, thymus, or spleen masses in any of the stress-treated groups. Chronic stress exposure tends to produce thymus and spleen involution and adrenal hypertrophy (for examples see Selye, 1936; Bryant et al., 1991; Watzl et al., 1993; Blanchard et al., 1998; Dominguez-Ge rpe and Rey-Mendez, 2001; Hasegawa and Saiki, 2002). The thymus glands appear to be most sensitive to stress exposure. Selye (1936) found that even single exposures to potent system ic stressors caused rapid thymus involution. There was a trend toward decreased thymus wei ghts in all of the groups that received social defeat, including the acute group. Therefore, there is some evidence in this experiment that the social defeat procedure was potent enough to produce changes in thymus glands. 71

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72 Figure 4-1. Photograph of a social defeat interaction. The intrude r is engaging in submissive behaviors by displaying a supine posture while the resident is displaying dominant behaviors by standing over the intruder. One defeat was counted when the rats engaged in this interaction for at least 2 seconds prior to disengaging. This first stage of the social interaction continued until 3 defeats occurred, the intruder froze for 90 total seconds, or until 5 minutes had elapsed. Figure 4-2. Photograph of stage 2 of the social defeat procedure. Following 3 defeats or 5 minutes, the intruder was placed into a doublewalled wire mesh cage placed into the residents cage. The resident s continued to display domi nant behaviors by climbing atop the cage and kicking bedding at the intruder. The intruder remained in this cage until 10 minutes had elapsed from the start of the procedure.

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Table 1: Groups of intruder rats Groups Repeated stress Acute stress Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Group 1 Group 2 Group 3 Group 4 Table 4-1. Social defeat regime n. Four groups of rats were exposed to differing amounts of social defeat. Group 1 was not exposed to a ny social defeat but was left undisturbed in their cages (No stress c ontrols). Group 2 was exposed to a single social defeat session on day 6 (Acute). Group 3 was exposed to one social defeat session on each of 5 days, but no social defeat on day 6 (Repeated, No Acute). Group 4 was exposed to one social defeat session on each of the 6 days (Repeated, Acute). 1 cctcttgctt cccacggctg caccatgaaa atcctgtttt gtgatgtcct gctgctcagc 61 ctgctctcca gcgtgttcag cagctgtccc gaggactgcc tcacctgcca ggagaggctc 121 cacccggctc cgggcagctt caacctgaag ctgtgcatcc tccagtgtga agagaaggtc 181 ttcccccgcc ctctctggac tctttgcacc aaagccatgg ccagtgactc tgagcagctc 241 agccctgctg atccagagct cacgtccgct gctctttacc agtcgaaagc ctcggagatg 301 cagcacctga agagaatgcc gcgtgtcagg agtgtggtgc aagcccgaga cgcagagcct 361 gaggcagatg cagagcctgt cgcagatgag gccgatgagg tggagcagaa gcagctgcag 421 aaaaggtttg ggggcttcac tggggcccgg aagtcagccc ggaagttggc caaccagaag 481 cggttcagtg agtttatgag gcagtacctg gtcctgagca tgcagtcaag ccaacgccgg 541 cgcactctgc accagaatgg taatgtgtag ccagaaggag cccctcccag ctgcaccggc 601 cactgcaacc catgagcatc caggtgagcc cccgtacagc atgtgtccac accaagacct 661 gcaggccggg agtcaggatt cctccttccc tgaggcactg aacacccgcg gcacctcccc 721 acagcatgtc tcaccacaat cctgttgcta catcagagtg tatttttgta attcctccag 781 ctaacatttt aatggcccca tcttcttgct catcctctgc cctctcgtag ggccaggtga 841 gaggaacatg aaatcagacc tggggttttg cctcaccact gccataactg gtttgtaaag 901 gagctgttct ttttgactga ttgtttgaaa caactttctc cattaaactt ctactgagca 961 aaatggttaa taaa translation=MKILFCDVLLLSLLSSVFSSCPEDCLTCQERLHPAPGSFNLKLCILQCEEKVFPRPLWTLCTKAM ASDSEQLSPADPELTSAALYQSKASEMQHLKRMPRVRSVVQARDAEPEADAEPVADEADEVEQKQLQKRFGGFTGAR KSARKLANQKRFSEFMRQYLVLSMQSSQRRRTLHQNGNV Figure 4-3. Prepro-N/OFQ sequence. The 972 base s for the prepro-N/OFQ gene are displayed (Accession # NM_013007). The highlighted re gion represents the portion of the sequence that was included in the prepro-NOFQ plasmid insert used for the in situ hybridization. Sequencing was performe d by the University of Florida DNA Sequencing Core, Interdisciplinary Center for Biotechnology Research. 73

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1 gcggccgcct ttctgctaag cattggggtc tattttggcc cagcttctga agaggctgtg 61 tgtgccgttg gaggaactgt actgagtggc tttgcagggt gacagcatgg agtccctctt 121 tcctgctcca tactgggagg tcttgtatgg cagccacttt caagggaacc tgtccctcct 181 aaatgagacc gtaccccacc acctgctcct caatgctagt cacagcgcct tcctgcccct 241 tggactcaag gtcaccatcg tggggctcta cttggctgtg tgcatcgggg ggctcctggg 301 gaactgcctc gtcatgtatg tcatcctcag gcacaccaag atgaagacag ctaccaacat 361 ttacatattt aatctggcac tggctgatac cctggtcttg ctaacactgc ccttccaggg 421 cacagacatc ctactgggct tctggccatt tgggaatgca ctctgcaaga ctgtcattgc 481 tatcgactac tacaacatgt ttaccagcac ttttactctg accgccatga gcgtagaccg 541 ctatgtggct atctgccacc ctatccgtgc ccttgatgtt cggacatcca gcaaagccca 601 ggctgttaat gtggccatat gggccctggc ttcagtggtt ggtgttcctg ttgccatcat 661 gggttcagca caagtggaag atgaagagat cgagtgcctg gtggagatcc ctgcccctca 721 ggactattgg ggccctgtat tcgccatctg catcttcctt ttttccttca tcatccctgt 781 gctgatcatc tctgtctgct acagcctcat gattcgacga cttcgtggtg tccgtctgct 841 ttcaggctcc cgggagaagg accgaaacct gcggcgtatc actcgactgg tgctggtagt 901 ggtggctgtg tttgtgggct gctggacgcc tgtgcaggtg tttgtcctgg ttcaaggact 961 gggtgttcag ccaggtagtg agactgcagt tgccatcctg cgcttctgca cagccctggg 1021 ctatgtcaac agttgtctca atcccattct ctatgctttc ctggatgaga acttcaaggc 1081 ctgctttaga aagttctgct gtgcttcatc cctgcaccgg gagatgcagg tttctgatcg 1141 tgtgcggagc attgccaagg atgttggcct tggttgcaag acttctgaga cagtaccacg 1201 gccagcatga ctaggcgtgg acctgcccat ggtgcctgtc agcccacaga gcccatctac 1261 acccaacacg gagctcacac aggtcactgc tctctaggtt gaccctgaac cttgagcatc 1321 tggagccttg aatggctttt cttttggatc aggatgctca gtcctagagg aagacctttt 1381 agcaccatgg gacaggtcaa agcatcaagg tggtctccat ggcctctgtc agattaagtt 1441 ccctccctgg tataggacca gagaggacca aaggaactga atagaaacat ccacaacaca 1501 gtggacatgc ctggtgagcc catgtaggta ttcatgcttc acttgactct tctctggctt 1561 ctccctgctg ccctggctct agctgggctc aacctgaggt attgtagtgg tcatgtagtc 1621 actcttgtga ctacatgttg tgtgctgttg ctctcggcct ttcagtattt ccacaggact 1681 gctgaacata cctggtattg cagtggggag cattaatttt cttttaaagt gagactggcc 1741 cttaagcttg gcgttgcctt ggagcgtctt ctacttctga cttcactgat gcagtcagat 1801 tacccgaggg tgagcatcag tggtttcttg gatggctgtt ttctgaagat tcttcccatc 1861 cagtacatgg agtctatgaa ggggagtcac aattcatctg gtactgccac tacctgctct 1921 ataatcctgg gctatcttct tggcaagatg acagtggggg agacaagaca cagagcttcc 1981 ctaaggctct ttccctccaa aaccactgtg aactcttatc ctacagactg ttcggcaagc 2041 actgcttcta ggtgtgtggg aggtaatcag gagaaagctt tgtggcctct gtaggctgct 2101 cacaacatgg aggcaccaca tgctggtctt gcctgcttag tacaggcagg acagagcaga 2161 atatgctctc tctcgattct ctacaaactc cctcagttct ccagcagagt ctcttttact 2221 tgctatcaga ggtcaggagt tgtactgcta gaagcatact tgtagcttgg gaagagtggc 2281 agtcaggatg tgttctactc tatatccaca gtgaccacct gcttcatata tagggttagg 2341 acatatctga gtaaggcctg agtgtgctgc caaattggag gttggtatga gagctgatgc 2401 ctaaagtggc tcatttgcaa ggactattat ggtttggaat agcaatgggg ggcatgggaa 2461 gaagagtcta taccttggag atctatttga tggttcacag aagaggtttt gtaaacgccc 2521 tttctatggg tcagatatca aaataccagc aacgttggat agattctgac cttttactga 2581 gacctcggtc agatggtttc atgtcatgca gagaacctag gctggttcct gtgtcagaga 2641 gacctgggct tctggggagg ccagggttct tcctttgaca cttgtgcggg agccgttagc 2701 tctaga translation=MESLFPAPYWEVLYGSHFQGNLSLLNETVPHHLLLNASHSAFLPLGLKVTIVGLYLAVCIGGLLGNCLVMYVIL RHTKMKTATNIYIFNLALADTLVLLTLPFQGTDILLGFWPFGNALCKTVIAIDYYNMFTSTFTLTAMSVDRYVAICHPIRALDVRT SSKAQAVNVAIWALASVVGVPVAIMGSAQVEDEEIECLVEIPAPQDYWGPVFAICIFLFSFIIPVLIISVCYSLMIRRLRGVRLLS GSREKDRNLRRITRLVLVVVAVFVGCWTPVQVFVLVQGLGVQPGSETAVAILRFCTALGYVNSCLNPILYAFLDENFKACFRKFCC ASSLHREMQVSDRVRSIAKDVGLGCKTSETVPRPA Figure 4-4. NOP receptor sequence. The 2706 bases for NOP receptor gene are displayed (Accession # NM_031569.2). The highlighted regi on represents the portion of the sequence that was included in the NOP receptor plasmid insert used for the in situ hybridization. Sequencing was performe d by the University of Florida DNA Sequencing Core, Interdisciplinary Center for Biotechnology Research. 74

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Mean Number of Defeats per Day One T wo Three Fou r Five Si x 0 1 2 3 4 5A R R+A DayNumber of Defeats Figure 4-5. Number of social defeats per group per day. Values shown are group means SEM (n = 4 rats per group). A = acute stre ss group, R = repeated stress group, R+A = repeated plus acute stress group. Total Time Defeated per Day One T wo Three Fou r Five Si x 0 10 20 30 40 50 60A R R+A DayTime (sec) Figure 4-6. Total amount of time the rats were defeated per day. Values shown are group means SEM (n = 4 rats per group). A= acute stress, R= repeat ed stress, R+A= repeated plus acute. 75

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C D A B Figure 4-7. Representative x-ray im ages of brain sections treated with sense riboprobes. Sections treated with (A,B) N/OFQ sense strands and (C,D) NOP receptor sense strands did not display any specific signal, and background levels were comparable to those of antisense-treated sections. 76

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77 NS A R R+A 0.00 0.05 0.10Stress ExposureOptical DensityA B NS A R R+A 0.00 0.05 0.10 Stress ExposureOptical Density Figure 4-8. Prepro-N/OFQ mRNA expression in the septum. There were trends toward greater mRNA expression in (A) the dorsolateral septum and (B) the ventrolateral septum following exposure to acute social defeat Values expressed are group means in optical density SEM (n = 4 rats per gr oup). NS= no stress, A= acute stress, R= repeated stress, R+A= repeated plus acute.

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NS A R R+A 0.00 0.05 0.10Stress ExposureOptical Density NS A R R+A 0.00 0.05 0.10Stress ExposureOptical Density NS A R R+A 0.00 0.05 0.10Stress ExposureOptical Density NS A R R+A 0.00 0.05 0.10Stress ExposureOptical DensityA B C D Figure 4-9. Prepro-N/OFQ mRNA expression in the bed nucleus of stria terminalis. There were trends toward greater mRNA expression in the (A) anteromedial BNST, (B) posteromedial BNST, (C) ventromedial BNST, and (D) lateral BNST following exposure to acute social def eat. Values expressed are gr oup means in optical density SEM (n = 4 rats per group). NS= no stress, A= acute stress, R= repeated stress, R+A= repeated plus acute. 78

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B A Figure 4-10. Representative x-ray images of brain sections show ing prepro-N/OFQ mRNA expression in the septum and BN ST. Sections were exposed to 125I-labelled riboprobes complimentary to segments of prepro-N/OFQ mRNA. (A) No stress control rats display low to moderate signal in the dorsolateral septum, intermediolateral septum, ventromedial se ptum, and lateral BNST. (B) Rats exposed to acute social defeat displayed trends to ward increased expression in these regions. 79

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NS A R R+A 0.00 0.05 0.10Stress ExposureOptical Density NS A R R+A 0.00 0.05 0.10Stress ExposureOptical DensityA B Figure 4-11. Prepro-N/OFQ mRNA expression in the amygdala. There were trends toward greater mRNA expression in (A) the central amygdala and (B) the medial amygdala following exposure to acute social defeat Values expressed are group means in optical density SEM (n = 4 rats per gr oup). NS= no stress, A= acute stress, R= repeated stress, R+A= repeated plus acute. 80

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NS A R R+A 0.00 0.05 0.10Stress ExposureOptical Density NS A R R+A 0.00 0.05 0.10Stress ExposureOptical DensityA B Figure 4-12. Prepro-N/OFQ mRNA e xpression in the zona incerta and reticular nucleus of the thalamus. There were trends toward greater mRNA expression in (A) the zona incerta and (B) the reticular nucleus following e xposure to acute social defeat. Values expressed are group means in optical density SEM (n = 4 rats per group). NS= no stress, A= acute stress, R= repeated stress, R+A= repe ated plus acute. 81

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B A Figure 4-13. Representative x-ray images of brain sections show ing prepro-N/OFQ mRNA expression in the amygdala, zona incerta, and reticular nucleu s of the thalamus. Sections were exposed to 125I-labelled riboprobes complimentary to segments of prepro-N/OFQ mRNA. (A) No stress contro l rats display moderate signal in the amygdala and low signal in the zona incerta and reticular nucleus. (B) Rats exposed to acute social defeat displayed trends to ward increased expression in these regions. NS= no stress, A= acute stress, R= repeated stress, R+A= repeated plus acute. 82

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Prepro-N/OFQ mRNA Expression in the Dorsal Raphe NS A R R+A 0.00 0.05 0.10Stress ExposureOptical Density Figure 4-14. Prepro-N/OFQ mRNA expression in the dorsal raphe. There were trends toward greater mRNA expression in the dorsal raph e following exposure to acute social defeat. Values expressed are group means in optical density SE M (n = 4 rats per group). NS= no stress, A= acute stress, R= re peated stress, R+A= repeated plus acute. B A Figure 4-15. Representative x-ray images of brain sections show ing prepro-N/OFQ mRNA expression in the dorsal raphe. Sections were exposed to 125I-labelled riboprobes complimentary to segments of prepro-N/O FQ mRNA. (A) No stress control rats display moderate signal dorsal raphe. (B) Rats exposed to acute social defeat displayed trends toward increased expre ssion in these regions. NS= no stress, A= acute stress, R= repeated stress, R+A= repeated plus acute. 83

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NS A R R+A 0.00 0.05 0.10**Stress ExposureOptical Density NS A R R+A 0.00 0.05 0.10Stress ExposureOptical Density NS A R R+A 0.00 0.05 0.10*Stress ExposureOptical DensityA B C Figure 4-16. NOP rector mRNA expression in the septum. mR NA expression was significantly greater in the (A) dorsolateral septum, (B) intermediolateral septum, and (C) ventrolateral septum following exposure to ac ute social defeat. Values expressed are group means in optical density SEM (n = 4 rats per group). Significant differences between the stress-exposed rats and the no stress controls ar e expressed as p < 0.05, ** p < 0.01. NS= no stress, A= acute stress, R= repeated stress, R+A= repeated plus acute. 84

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B A Figure 4-17. Representative x-ray images of brain sections showing NOP receptor mRNA expression in the septum. Sections were exposed to 125I-labelled riboprobes complimentary to segments of NOP recep tor mRNA. (A) No stress control rats display low signal in the dorsolateral, interm ediolateral, and ventrolateral septum. (B) Rats exposed to acute social defeat disp layed significantly gr eater mRNA expression in these regions regions. 85

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NOP mRNA Expression in Ventromedial BNST NS A R R+A 0.00 0.05 0.10**Stress ExposureOptical Density Figure 4-18. NOP receptor mRNA expression in the bed nucleus of stria terminalis. mRNA expression was significantly greater in th e ventromedial BNST following exposure to repeated social defeat. Values expressed ar e group means in optical density SEM (n = 4 rats per group). Significant differences between the stress-exposed rats and the no stress controls are expressed as ** p < 0.01. NS= no stress, A= acute stress, R= repeated stress, R+A= repeated plus acute. 86

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B A Figure 4-19. Representative x-ray images of brain sections showing NOP receptor mRNA expression in the bed nucleus of stria terminalis. Sections were exposed to 125Ilabelled riboprobes complimentary to segments of NOP receptor mRNA. (A) No stress control rats display low signal in the ventromedial BNST. (B) Rats exposed to repeated social defeat displayed signi ficantly greater mRNA expression in the ventromedial BNST. 87

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NS A R R+A 0.00 0.05 0.10** *Stress ExposureOptical Density NS A R R+A 0.00 0.05 0.10*Stress ExposureOptical Density NS A R R+A 0.00 0.05 0.10**Stress ExposureOptical Density NS A R R+A 0.00 0.05 0.10**Stress ExposureOptical DensityA C B D NS A R R+A 0.00 0.05 0.10**Stress ExposureOptical DensityE Figure 4-20. NOP receptor mRNA expression in the amygdala. mRNA expression was significantly greater in (A) the central amygdala, (B) the medial amygdala, (C) the basomedial amygdala, (D) the basolatera l amygdala, and (E) the lateral amygdala following exposure to acute social defeat. Additionally, there was significantly greater mRNA expression in the central am ygdala following repeated plus acute social defeat. Values expressed are group m eans in optical density SEM (n = 4 rats per group). Significant differences between the stress-exposed rats and the no stress controls are expressed as p < 0.05 and ** p < 0.01. NS= no stress, A= acute stress, R= repeated stress, R+A= repeated plus acute. 88

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ab c d A B D CFigure 4-21. Representative x-ray images of brain sections showing NOP receptor mRNA expression in the amygdala and the parave ntricular nucleus of the hypothalamus. Sections were exposed to 125I-labelled riboprobes complimentary to segments of NOP receptor mRNA. (A) No stress control rats display low to moderate signal in the amygdala and PVN. Rats exposed to (B) acu te and (D) repeated plus acute social defeat displayed greater mR NA expression in the amygdala. Rats exposed to (B) acute, (C) repeated, and (D) repeated plus acute social defeat displayed greater mRNA expression in the PVN. 89

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NS A R R+A 0.00 0.05 0.10** ** *Stress ExposureOptical Density NS A R R+A 0.00 0.05 0.10Stress ExposureOptical DensityA B NS A R R+A 0.00 0.05 0.10Stress ExposureOptical DensityC Figure 4-22. NOP receptor mRNA e xpression in the paraventricular and ventromedial nuclei of the hypothalamus and in the zona incerta of the thalamus. (A) mRNA expression was significantly greater in the PVN following any exposure to social defeat. In (B) the ventromedial hypothalamus and (C) the zona incerta elevations in mRNA expression following social defeat approached signifi cance. Values expressed are group means in optical density SEM (n = 4 rats per gr oup). Significant differences between the stress-exposed rats and the no stress controls are expressed as p < 0.05 and ** p < 0.01. NS= no stress, A= acute stress, R= rep eated stress, R+A= re peated plus acute. 90

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a b B AFigure 4-23. Representative x-ray images of brain sections treated with 1 M N/OFQ plus 125I-[14Tyr]-N/OFQ. Sections treated with 1 M N/OFQ plus 125I-[14Tyr]-N/OFQ did not display any specific signal, and bac kground levels were comparable to those sections treated with only 125I-[14Tyr]-N/OFQ. 91

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NS A R R+A 0.00 0.05 0.10Stress ExposureOptical Density NS A R R+A 0.00 0.05 0.10Stress ExposureOptical Density NS A R R+A 0.00 0.05 0.10Stress ExposureOptical DensityA B C Figure 4-24. 125I-[14Tyr]-N/OFQ binding to the NOP receptor in the septum. There were no significant changes in binding following any stress exposure in (A) the dorsolateral septum, (B) the intermediolateral septum, or (C) the ventrolateral septum. Values expressed are group means in optical density SEM (n = 4 rats per group). NS= no stress, A= acute stress, R= repeated stress, R+A= repeated plus acute. 92

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a b A BFigure 4-25. Representative x-ray im ages of brain sections showing 125I-[14Tyr]-N/OFQ binding to the NOP receptor in the septum. Despit e an increase in NOP receptor mRNA, (B) the acutely stressed group did not disp lay a significant incr ease in binding as compared to (A) the no stress controls. 93

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N/OFQ Binding to the NOP Receptor in the Ventromedial BNST NS A R R+A 0.00 0.05 0.10Stress ExposureOptical Density Figure 4-26. 125I-[14Tyr]-N/OFQ binding to the NOP recepto r in the ventromedial BNST. There were no significant changes in binding following a ny stress exposure in the ventromedial BNST. Values expressed are group means in optical density SEM (n = 4 rats per group). NS= no stress, A= acu te stress, R= repeated stress, R+A= repeated plus acute. 94

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a b B AFigure 4-27. Representative x-ray im ages of brain sections showing 125I-[14Tyr]-N/OFQ binding to the NOP receptor in the BNST. Despite an increase in NOP receptor mRNA, (B) the repeatedly stressed group did not displa y a significant incr ease in binding as compared to (A) the no stress controls. 95

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NS A R R+A 0.00 0.05 0.10Stress ExposureOptical Density NS A R R+A 0.00 0.05 0.10Stress ExposureOptical Density NS A R R+A 0.00 0.05 0.10Stress ExposureOptical Density NS A R R+A 0.00 0.05 0.10Stress ExposureOptical Density NS A R R+A 0.00 0.05 0.10Stress ExposureOptical DensityAB C D E Figure 4-28. 125I-[14Tyr]-N/OFQ binding to the NOP recep tor in the amygdala. There were no significant changes in binding following a ny stress exposure in (A) the central amygdala, (B) the medial amydala, (C) the ba somedial amygdala, (D) the basolateral amygdala, or (E) the lateral amygdala. Valu es expressed are group means in optical density SEM (n = 4 rats pe r group). NS= no stress, A= acute stress, R= repeated stress, R+A= repeated plus acute. 96

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N/OFQ Binding to the NOP Receptor in the PVN NS A R R+A 0.00 0.05 0.10Stress ExposureOptical Density Figure 4-29. 125I-[14Tyr]-N/OFQ binding to the NOP r eceptor in the PVN. There were no significant changes in binding in the PVN following any stress exposure. Values expressed are group means in optical density SEM (n = 4 rats per group). NS= no stress, A= acute stress, R= repeated stress, R+A= repeated plus acute. b d C D B AFigure 4-30. Representative x-ray images of brain sections showing 125I-[14Tyr]-N/OFQ binding to the NOP receptor in the amygdala and the PVN. Despite an increase in NOP receptor mRNA in these regions, none of the stressed groups displayed increases in binding. Shown are representa tive images from (A) the no stress groups, (B) the acutely stressed group, (C) the re peatedly stressed group, and (D) the repeatedly plus acutely stressed group. 97

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98 NS A R R+A 0 10 20 30 40 50 60 70 80 90 100Stress ExposureWeight (mg) NS A R R+A 0 50 100 150 200 250 300 350 400 450 500 550 600Stress ExposureWeight (mg) NS A R R+A 0 100 200 300 400 500 600 700 800 900 1000Stress ExposureWeight (mg)A B C Figure 4-31. Gland masses after social defeat. (A) Adrenal gl and masses, (B) thymus gland masses, and (C) spleen masses showed no significant differences between groups. Values expressed are group means SEM ( n = 4 rats per group).

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CHAPTER 5 GENERAL DISCUSSION There is mounting evidence that N/OFQ is i nvolved in the expression of anxiety-related behaviors and in the activation of the HPA axis. Injections of N/OFQ into the lateral ventricles and into limbic structures increa se circulating ACTH and CORT (Devine et al., 2001; Nicholson et al., 2002; Fernandez et al., 2004 ; Legget et al., 2006; Green et al., in press; Misilmeri and Devine, in preparation) and alter anxiety-related behaviors (Jenck et al., 1997; Jenck et al., 2000; Gavioli et al., 2002; Fernandez et al., 2004; Kamei et al., 2004; Vart y et al. 2005; Vitale et al., 2006; Gavioli et al., 2007; Green et al., in pre ss). Additionally, exposure to acute stressors results in release of N/OFQ from forebrain neur ons (Devine et al., 2003) and in increases in NOP receptor mRNA expression (present experiments). However, most of these effects are modest and are generally evident in mildly to moderately stressful environments. For example, Devine and colleagues (2001) found that when rats are more profoundly stressed (e.g., restraint), N/OFQ does not alter the HPA axis activation. This has also been observed recently with restraint and with unhandled rats (unpublished). However, there is some evidence of changes in the regulation of the N/OFQ-NOP receptor system in response to severe chronic stress, part icularly in the BNST an d the PVN, two regions that are important in affective responses. Th e upregulation of NOP receptor mRNA in the BNST following chronic stress is consistent with the role of the BNST in long-term responses to ongoing stressors. Additionally, HPA axis regul ation in the PVN by the N/OFQ-NOP system may be altered by chronic social stressor exposure. It remains evident that N/OFQ is an importa nt neuromodulator in a number of functions, including stress response, pain m odulation (Meunier et al., 1995; Re inscheid et al., 1995; Tian et al., 1997), motor performance (Reinscheid et al ., 1995; Devine et al., 1996), spatial learning 99

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100 (Sandin et al., 1997; Sandin et al ., 2004), and feeding (Pomonis et al., 1996; Nicholson et al., 2002). In general, it appears to be a fairly tightly regulated system that is generally resistant to dysregulation. Thus it seems important in the f unctioning of normal, nonpathological behavior. Its potential involvement in stress-induced ps ychopathology is suggested by stress-induced changes in NOP receptor mRNA (especially in the BNST and PVN of chronically-stressed rats). However, the lack of changes in receptor autora diography indicates that this possibility will need to be studied in more detail.

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LIST OF REFERENCES Adamec RE, Blundell J, Collins A (2001) Neural plasti city and stress induced changes in defense in the rat. Neurosci Biobehav Rev 25:721-744. Amsterdam JD, Maislin G, Winokur A, Berwis h N, Kling M, Gold P (1988) The oCRH stimulation test before and after clinical re covery from depression. J Affect Disord 14: 213-222. Andersen SL, Teicher MH (1999) Serotonin latera lity in amygdala predicts performance in the elevated plus maze in rats. NeuroReport 10:3497-3500. Arborelius L, Owens MJ, Plotsky PM, Nemeroff CB (1999) The role of co rticotrophin-releasing factor in depression and anxiety disorders. J Endocrinol 160:1-12. Aubry JM, Bartanusz V, Jezova D, Belin D, Ki ss JZ (1999) Single stress induces long-lasting elevations in vasopressin mRNA levels in CRF hypophysiotrophic neurons, but repeated stress is required to modify AVP i mmunoreactivity. J Neuroendocinol 11:377-384. Barnum CJ, Blandino P, Deak T (2007) Adaptation in the corticosterone and hyperthermic responses to stress following repeated st ressor exposure. J Ne uroendocinol 19:632-642. Blanchard RJ, Nikulina JN, Sakai RR, McKitt rick C, McEwan B, Blanchard DC (1998) Behavioral and endocrine cha nge following chronic predatory stress. Physiol Behav 63: 561-569. Board F, Persky H, Hambur DA (1956) Psychol ogical stress and endoc rine functions; blood levels of adrenocortical and thyroid hormones in acutely di sturbed patients. Psychosom Med 18: 324-333. Boudaba C, Szabo K, Tasker JG (1996) Physiolo gical mapping of local inhi bitory inputs to the hypothalamic paraventricular nucl eus. J Neurosci 16: 7151-7160. Boudaba C, Schrader LA, Tasker JG (1997) Physiological evidenc for local excitatory synaptic circuits in the ra t hypothalamus. J Ne urophysiol 77: 3396-3400. Bryant HU, Bernton EW, Kenner JR, Holaday JW (1991) Role of adrenal cortical activation in the immunosuppressive effects of chroni c morphine treatment. Endocrinology 128:32533258. Bunzow JR, Saez C, Mortrud M, Bouvier C, Williams JT, Low M, Grandy DK (1994) Molecular cloning and tissue distribution of a putative member of the rat opioid receptor gene family that is not a mu, delta or kappa opi oid receptor type. FEBS Lett 347: 284-288. 101

PAGE 102

102 Butour JL, Moisand C, Mazarguil H, Moller eau C, Meunier JC (1997) Recognition and activation of the opioid receptor-like ORL 1 r eceptor by nociceptin, nociceptin analogs and opioids. Eur J Pharmacol 321:97-103. Buwalda B, Felszeghy K, Horvath KM, Nyakas C, de Boer SF, Bohus B, Koolhaas JM (2001) Temporal and spatial dynamics of corticoste roid receptor down-regulation in rat brain following social defeat. Physiol Behav 72:349-354. Calfa G, Volosin M, Molina VA (2006) Glucocorti coid receptors in lateral septum are involved in the modulation of the emotional sequelae induced by social def eat. Behav Brain Res 172: 324-332. Canteras NS, Simerly RB, Sawnson LW (1995) Or ganization of projecti ons from the medial nucleus of the amygdala: a PHAL study in the rat. J Comp Neurol 360: 213-245. Chaouloff F, Durand M, Mormde P (1997) Anxiet yand activity-related effects of diazepam and chlordiazepoxide in the rat light/dark a nd dark/light tests. Behav Brain Res 85:27-35. Chen Y, Fan Y, Liu J, Mestek A, Tian M, Kozak CA, Yu L (1994) Molecular cloning, tissue distribution and chromosomal localization of a novel member of the opioid receptor gene family. FEBS Lett 347:279-283. Chung KKK, Martinez M, Herbert J (1999) Ce ntral serotonin depletion modulates the behavioural, endocrine and phys iological responses to re peated social stress and subsequent c-fos expression in the brai ns of male rats. Neuroscience 92:613-625. Coleman-Mesches K, McGaugh JL (1995a) Differen tial involvement of the right and left amygdalae in expression of memory for aver sively motivated trai ning. Brain Res 670:7581. Coleman-Mesches K, McGaugh JL (1995b) Muscimol injected into the right or left amygdaloid complex differentially affects retention pe rformance following aversively motivated training. Brain Res 676:183-188. Connor M, Vaughan CW, Chieng B, Christie MJ (1996a) Nociceptin receptor coupling to a potassium conductance in rat locus coeruleu s neurones in vitro. Br J Pharmacol 119:16141618. Connor M, Yeo A, Henderson G (1996b) The effect of nociceptin on Ca2+ channel current and intracellular Ca2+ in the SH -SY5Y human neuroblastoma cell line. Br J Pharmacol 118:205-207. Costall B, Jones BJ, Kelly ME, Naylor RJ, Tomk ins DM (1989) Exploration of mice in a black and white test box: Validation as a model of anxiety. Pharmacol Biochem Behav 32:777785.

PAGE 103

Covington HE, Miczek KA (2001) Rep eated social-defeat stress, cocaine or morphine. Effects on behavioral sensitization and intravenous cocaine self-administration "binges". Psychopharmacol 158:388-398. Crawley J, Goodwin FK (1980) Pr eliminary report of a simple animal behavior model for the anxiolytic effects of benzodiazepines. Pharmacol Biochem Behav 13:167-170. Crawley JN (1981) Neuropharmacologic specificity of a simple anim al model for the behavioral actions of benzodiazepines. Pharmacol Biochem Behav 15:695-699. Crofford LJ (1998) The hypothalamic-pituitary-adre nal stress axis in fibromyalgia and chronic fatigue syndrome. Z Rheumatol 57: 67-71. Cullinan WE, Herman JP, Watson SJ (1993) Ventra l subicular interaction with the hypothalamic paraventricular nucleus: Evidence for a relay in the bed nucleus of stria terminalis. J Comp Neurol 332: 1-20. Cullinan WE, Helmreich DL, Watson SJ (1996) Fo s expression in forebrain afferents to the hypothalamic paraventricular nucleus following swim stress. J Comp Neurol 368: 88-99. Danielson PB, Dores RM (1999) Review: Molecu lar evolution of the opioid/orphanin gene family. Gen Comp E ndocrinol 113:169-186. Dautzenberg FM, Wichmann U, Higelin J, Py-Lang G, Kratzeisen C, Malherbe P, Kilpatrick GJ, Jenck F (2001) Pharmacological charact erization of the novel nonpeptide orphanin FQ/nociceptin receptor agonist Ro 64-6198: Rapid and revers ible desensitization of the ORL1 receptor in vitro and lack of tolera nce in vivo. J Pharmacol & Exp Ther 298: 812819. de Goeij DC, Kvetnansky R, Whitnall MH, Jezo va D, Berkenbosch F, Tilders FJ (1991) Repeated stress-induced activation of cortic otrophin-releasing factor neurons enhances vasopressin stores and colocaliz ation with corticotrophin-releasing factor in the median eminence of rats. Neuroendocrinology 53:150-159. de Goeij DCE, Dijkstra H, Tilders FJH (1992) Chronic psychological stress enhances vasopressin, but not corticotrophin-releasing factor, in the ex ternal zone of the medial eminence of male rats: Relationship to s ubordinate status. En docrinology 131:847-853. Deak T, Nguyen KT, Cotter CS, Fleshner M, Wa tkins LR, Maier SF, Spencer RL (1999) Longterm changes in mineralocorticoid and gl ucocorticoid receptor occupancy following exposure to an acute stre ssor. Brain Res 847: 211-220. Devine DP, Taylor L, Reinscheid RK, Monsma FJ, Civelli O, Akil H (1996) Rats rapidly develop tolerance to the loco motor-inhibiting effects of th e novel neuropeptide orphanin FQ. Neurochem Res 21:1387-1396. Devine DP, Watson SJ, Akil H (2001) Nocicep tin/orphanin FQ regulates neuroendocrine function of the limbic-hypothalamic-pituit ary-adrenal axis. Neuroscience 102:541-553. 103

PAGE 104

Devine DP, Hoversten MT, Ueda Y, Akil H (2003) Nociceptin/orphanin FQ content is decreased in forebrain neurones during acute stress. J Neuroendocrinol 15:69-74. Diorio D, Viau V, Meaney MJ (1993) The role of the medial prefrontal cortex (cingulated gyrus) in the regulation of hypothalamic-pituitary-adren al responses to stre ss. J Neurosci 13:38393847. Dominguez-Gerpe L, Rey-Mendez M (2001) Al terations induced by chronic stress in lymphocyte subsets of blood and primary and secondary immune organs of mice. BMC Immunol 2:7. Dong HW, Petrovich GD, Watts AG, Swanson LW (2001) Basic organization of projections from the oval and fusiform nuclei of the bed nucle i of the stria terminalis in adult rat brain. J Comp Neurol 436:430-455. Dong HW, Swanson LW (2004) Organization of axona l projections from th e anterolateral area of the bed nuclei of the stria te rminalis. J Comp Neurol 468:277-298. Dunn JD (1987a) Differential plasma corticosterone responses to electrical stimulation of the medial and lateral septal nuc lei. Neuroendocrinology 46: 406-411. Dunn JD (1987b) Plasma corticosterone responses to electrical stimulation of the bed nucleus of the stria terminalis. Brain Res 407:327-331. Dunn JD, Whitener J (1986) Plasma corticosterone responses to electrical stimulation of the amygdaloid complex: cytoarchitectural specificity. Neuroendocrinology 42: 211;217. Ebner K, Wotjak CT, Landgaf R, Engelmann M ( 2005) Neuroendocrine and behavioral response to social confrontation: residents versus intruders, active versus passive coping styles. Horm Behav 47:14-21. Elenkov IJ, Wilder RL, Chrousos GP, Vizi ES (2000) The sympathetic nerveAn integrative interface between two supersystems: The brai n and the immune system. Pharmacol Rev. 52: 595-638. Emmert MH, Herman JP (1999) Differential fore brain c-fos mRNA induction by ether inhalation and novelty: evidence for distinctive stress pathways. Brain Res 845: 60-67. Eskandari F, Sternberg EM (2002) Neural-Immune interactions in health and disease. Ann NY Acad Sci 966:20-27. Feldman S, Conforti N, Itzik A, Weidenfeld J (1 994) Differential effect of amygdaloid lesions of CRF-41, ACTH, and corticosterone responses followinf neural stimuli. Brain Res 658: 2126. Fernandez F, Misilmeri MA, Felger JC, Devine DP (2004) Nociceptin/orphanin FQ increases anxiety-related behavior and ci rculating levels of corticos terone during neophobic tests of anxiety. Neuropsychopharmacology 29:59-71. 104

PAGE 105

Gavioli EC, Rae GA, Calo G, Guerrini R, DeLi ma TCM (2002) Central injections of nocistatin or its C-terminal hexapeptide exert anxiogenic-like effect on behaviour of mice in the plusmaze test. Brit J Pharmacol 136:764-772. Gavioli EC, Marzola G, Guerrini R, Bertorelli R, Zucchini S, De Lima TCM, Rae GA, Salvadori S, Regoli D, Calo G (2003) Blockade of nociceptin/orphanin FQ-NOP receptor signalling produces antidepressant-like effects: Pharm acological and genetic evidences from the mouse forced swimming test Eur J Neurosci 17:1987-1990. Gavioli EC, V aughan CW, Marzola G, Guerrini R, Mitchell VA, Zucchini S, De Lima TCM, Rae GA, Salvadori S, Regoli D, Calo G (2004) Antidepressant-like effects of the nociceptin/orphanin FQ receptor antagonist U FP-101: New evidence from rats and mice. Naunyn-Schmiedebergs Arch Pharmacol 369:547-553. Gavioli EC, Rizzi A, Marzola G, Zucchini S, Regoli D, Calo G (2007). Altered anxiety-related behavior in nociceptin/orphanin FQ recep tor gene knockout mice. Peptides 28: 1229-1239. Gillies GE, Linton EA, Lowry PJ (1982) Corticot ropin releasing activity of the new CRF is potentiated several times by vasopressin. Nature 299: 355-357. Gilmer WS, Trivedi MH, Rush AJ, Wisniewski SR, Luther J, Howland RH, Yohanna D, Khan A, Alpert J (2005) Factors associated with chronic depressive episodes: A preliminary report from the STAR-D project. Acta Psychiatr Scand 112:425-433. Gold PW, Loriaux DL, Roy A, Kling MA, Calabrese JR, Kellnor CH, Nieman LK, Post RM, Dicker D, Gallucci W, et al. (1986) Response to corticotrophin-releasing hormone in the hypercortisolism of depression and Cushings disease: Pathophysiologic and diagnostic implications. N Eng J Med 314: 1329-1335. Goldstein ML (1965) Effects of hippocampal, amygdala, hypothalamic and parietal lesions on a classically conditioned fear response. Psych Rep 16: 211-219. Good AJ, Westbrook RF (1995) Effects of a microinjection of morphine into the amygdala on the acquisition and expression of conditioned f ear and hypoalgesia in rats. Behav Neurosci 109:631-641. Gray TS, Piechowski RA, Yracheta JM, Rittenh ouse PA, Bethea CL, Ven de Kar LD (1993) Ibotenic acid lesions in the bed nucleus of the stria terminalis attenuate conditioned stressinduced increases in prolactin, ACTH and corticosterone. Neuroendocrinology 57: 517524. Green MK, Barbieri EV, Brown BD, Chen KW, Devi ne DP (in press) Roles of the bed nucleus of stria terminalis and of the amygdala in N/OFQ-mediated anxiety and HPA axis activation. Neuropeptides. Handley SL, Mithani S (1984) Effects of alpha-a drenoceptor agonists and antagonists in a mazeexploration model of fear-m otivated behaviour. Naunyn Schmiedebergs Arch Pharmacol 327:1-5. 105

PAGE 106

Hasegawa H, Saiki I (2002) Psychological stress augments tumor development through adrenergic activation in mice. Jpn J Cancer Res 93:729-735. Hauger RL, Lorang M, Irwin M, Aguilera G (19 90) CRF receptor regulation and sensitization of ACTH responses to acute ether stress duri ng chronic intermittent immobilization stress. Brain Res 532: 34-40. Heinrichs SC, Pich EM, Miczek KA, Britton KT, K oob GF (1992) Corticotropin-releasing factor antagonist reduces emotionality in socially de feated rats via direct neurotropic action. Brain Res 581:190-197. Henke PG (1984) The bed nucleus of the stria te rminalis and immobilization-stress unit activity, escape behaviour, and gastric pathology in rats. Behav Brain Res 11:35-45. Herman JP, Schafer MK, Young EA, Thompson R, Douglass J, Akil H, Watson SJ (1989) Evidence for hippocampal regulation of neuroendocrine neurons of the hypothalamo-pituitary-adrenocortical axis. J Neurosci 9:3072-3082. Herman JP, Cullinan WE, Watson SJ (1994) Involvement of the bed nuc leus of the stria terminalis in tonic regula tion of paraventricular hy pothalamic CRH and AVP mRNA expression. J Neuroendo crinol 6: 433-442. Herman JP, Cullinan WE (1997) Neurocircuit ry of stress: Cent ral control of the hypothalamo-pituitary-adrenocortical ax is. Trends Neurosci 20:78-84. Herman JP, Taskers JG, Ziegler DR, Cullinan WE (2002a) Local circuit regulation of paraventricular nucleus st ress integration Glutamate GABA connections. Pharmacol Biochem Behav 71:457-468. Herman JP, Cullinan WE, Ziegler DR, Tasker JG (2002b) Role of the paraventricular nucleus microenvironment in stress integration. Eur J Neurosci 16:381-385. Heuser I, Bissette G, Dettling M, Schweiger U, Gotthardt U, Schmider J, Lammers CH, Nemeroff CB, Holsboer F (1998) Cerebrospinal fluid concentrations of corticotrophinreleasing hormone, vasopressin, and somatost atin in depressed patients and healthy controls: Response to amitr iptyline treatment. Depr ess Anxiety 8: 71-79. Holsboer F, Gerken A, von Bardeleben U, Gri mm W, Beyer H, Muller OA, Stalla GK (1986) Human corticotrophin-releasing hormone in depressionCorrelation with thyrotropin secretion following thyrot ropin-releasing hormone. Biol Psychiatry 21: 601-611. Hughes RN (1972) Chlordiazepoxide modified exploration in rats. Psychopharmacologia 24:462-469. Ixart G, Szafarczyk A, Belugou JL, Assenmacher I (1977) Temporal relationships between the diurnal rhythm of hypothalamic corticotrophin releasing factor, pitu itary corticotrophin and plasma corticosterone in the rat. J Endocrinol 72:113-120. 106

PAGE 107

Izquierdo A, Murray EA (2004) Combined unilate ral lesions of the am ygdala and orbital prefrontal cortex impair affective processi ng in rhesus monkeys. J Neurophysiol 91:20232039. Jenck F, Moreau JL, Martin JR, Kilpatrick GJ, Reinscheid RK, Monsma FJ, Nothacker HP, Civelli O (1997) Orphanin FQ acts as an anxiol ytic to attenuate behavioral responses to stress. PNAS 94:14854-14858. Jenck F, Wichmann J, Dautzenberg FM, Moreau JL, Ouagazzal AM, Martin JR, Lundstrom K, Cesura AM, Poli SM, Roever S, Kolczewski S, Adam G, Kilpatrick G (2000) A synthetic agonist at the orphanin FQ/nociceptin recepto r ORL1: anxiolytic profile in the rat. Proc Natl Acad Sci U S A 97:4938-4943. Johnson JD, OConnor KA, Deak T, Spencer RL, Watkins LS, Maier SF (2002) Prior stressor exposure primes the HPA axis. Ps ychoneuronendocrino logy 27: 353-365. Jordanova V, Stewart R, Goldberg D, Bebbingt on E, Brugha T, Singleton N, Lindesay JEB, Jenkins R, Prince M, Meltzer H (2007) Age va riation in life events and their relationship with common mental disorders in a nationa l survey population. Soc Psychiatry Psychiatr Epidemiol 42:611-616. Juruena MF, Cleare AJ, Pariante CM (2004) The hypothalamic pituitary adrenal axis, glucocorticoid receptor functi on and relevance to depressi on. Rev Bras Pisquiatr 26:189201. Kamei J, Matsunawa Y, Miyata S, Tanaka S, Saitoh A (2004) Effects of nociceptin on the exploratory behavior of mice in the hol e-board test. Eur J Pharmacol 489: 77-87. Kvetnansy R, Bodnar I, Shahar T, Uhereczky G, Krizanova O, Mravec B ( 2006) Effect of lesions of A5 and A7 brainstem noradrenergic area s or transaction of brainstem pathways on sympathoadrenal activity in rats during i mmobilization stress. Neurochem Res 31: 267275. Kwak SP, Morano MI, Young EA, Watson SJ, Akil H (1993) Diurnal CRH mRNA rhythm in the hypothalamus: decreased expression in th e evening is not dependent on endogenous glucocorticoids. Neuroe ndocrinology 57:96-105. LaBar KS, LeDoux JE (1996) Partial disruption of fear conditioning in rats with unilateral amygdala damage: Correspondence with unilatera l temporal lobectomy in humans. Behav Neurosci 110:991-997. Labeur MS, Arzt E, Wiegers GJ, Hols boer F, Reul JMHM (1995) Lon-term intracerebroventricular corticot rophin-releasing hormone admi nistration induces distinct changes in rat splenocyte activation and cytokine expression. Endocrinology 136: 26782688. Lachowicz JE, Shen Y, Monsma FJ, Sibley DR (1995) Molecular cloning of a novel G proteincoupled receptor related to the opiat e receptor family. J Neurochem 64:34-40. 107

PAGE 108

LeDoux JE, Iwata J, Cicchetti P, Reis DJ (1988) Different projections of the central amygdaloid nucleus mediate autonomic and behavioral corr elates of conditioned fear. J Neurosci 8: 2517-2529. Lee Y, Davis M (1997) Role of the hippocampus, th e bed nucleus of the st ria terminalis, and the amygdala in the excitatory e ffect of corticotr ophin-releasing hormone on the acoustic startle reflex. J Ne urosci 17: 6434-6446. Leggett JD, Harbuz MS, Jessop DS, Fulford AJ (2006) The nociceptin receptor antagonist [Nphe(1),Arg(14),Lys(15)]nociceptin/orphanin FQ -NH(2) blocks the stimulatory effects of nociceptin/orphanin FQ on the HPA ax is in rats. Neuroscience 141:2051-2057. Linthorst ACE, Flachskamm C, Hopkins SJ, Hoadley ME, Labeur MS, Holsboer F, Reul JMHM (1997) Long-term intracerebroven tricular infusion of cor ticotrophin-releasing hormone alters neuroendocrine, neurochemical, autonomic behavioral, and cytokine responses to a systemic inflammatory challe nge. J Neurosci 17: 4448-4460. Lyons DM, Wang OJ, Lindley SE, Levine S, Kalin NH, Schatzberg AF (1999) Separation induced changes in squirrel monkey hypothalamic-pituitary-adrenal physiology resemble aspects of hypercortisolism in humans. Psychoneuroendocrinology 24: 131-142. Manji HK, Drevets WC, Charney DS (2001) The cellular neurobiology of depression. Nature Med 7: 541-547. Marin MT, Cruz FC, Planeta CS (2007) Chronic rest raint or variable stresses differently affect the behavior, corticosterone secretion and body weight in rats. Physiol & Behav 90:29-35. Martin DS, Haywood JR (1992) Sympathetic nervous system activation by glutamate injections into the paraventricular nucleus. Brain Res 577: 261-267. Martinez M, Phillips PJ, Herbert J (1998) Adaptation in patterns of c-fos expression in the brain associated with exposure to either single or repeated social stress in male rats. Eur J Neurosci 10:20-33. Menard J, Treit D (1996) Lateral and medial sept al lesions reduce anxiet y in the plus-maze and probe-burying tests. Physiol & Behav 60: 845-853. Meunier JC, Mollereau C, Toll L, Suaudeau C, Moisand C, Alvinerie P, Butour JL, Guillemot JC, Ferrara P, Monsarrat B (1995) Isolation and structure of the endogenous agonist of opioid receptor-like ORL1 receptor. Nature 377:532-535. Misilmeri MA, Devine DP (in preparation) N/OFQ activates the hypothalamic-pituitary adrenal axis after administration into limbic brain sites. Moghaddam B, Bunney BS (1989) Ionic composition of microdialysis perfusing solution alters the pharmacological responsiveness and basal ou tflow of striatal dopamine. J Neurochem 53:652-654. 108

PAGE 109

Mollereau C, Parmentier M, Mailleux P, Butour JL, Moisand C, Chalon P, Caput D, Vassart G, Meunier JC (1994) ORL1, a novel member of the opioid receptor family. Cloning, functional expression and local ization. FEBS Lett 341:33-38. Nagaraja TN, Patel P, Gorski M, Gorevic PD (2005) In normal rat, intraventricularly administered insulin-like growth factor-1 is rapidly cleared from CSF with limited distribution into brain. Cere brospinal Fluid Res 2: 5. Neal CR, Mansour A, Reinscheid R, Nothacker HP, Civelli O, Watson SJ (1999a) Localization of orphanin FQ (nociceptin) pe ptide and messenger RNA in th e central nervous system of the rat. J Comp Neurol 406:503-547. Neal CR, Mansour A, Reinscheid R, Nothack er HP, Civelli O, Akil H, Watson SJ (1999b) Opioid receptor-like (ORL1) receptor distri bution in the rat centr al nervous system: Comparison of ORL1 receptor mRNA expr ession with 125I-[14Tyr]-Orphanin FQ binding. J Comp Neurol 412:563-605. Nemeroff CB, Widerlov E, Bissette G, Walleus H, Karlsson I, Eklund K, Kilts CD, Loosen PT, Vale W (1984) Elevated concentrations of CSF corticotropin-releasing factor-like immunoreactivity in depressed patients. Science 226:1342-1344. Nemeroff CB, Bissette G, Akil H, Fink M ( 1991) Neuropeptide concentrations in the cerebrospinal fluid of depressed patients treated with elec trovonvulsive therapy. Corticotrophin-releasing factor, beta-endorphin and somatostatin. Br J Psychiatry 158: 5963. Nicholson JR, Akil H, Watson SJ (2002) Orpha nin FQ-induced hyperphagia is mediated by corticosterone and central glucocor ticoid receptors. Neuroscience 115:637-643. Nikulina EM, Covington HE, Ganschow L, Hammer RP, Miczek KA (2004) Long-term behavioral and neuronal cross-sensitization to amphetamine induced by repeated brief social defeat stress: fos in the ventral te gmental area and amygdala. Neuroscience 123:857865. Nothacker HP, Reinscheid RK, Mansour A, He nningsen RA, Ardati A, Monsma FJ, Watson SJ, Civelli O (1996) Primary structure and tissue distribution of the or phanin FQ precursor. PNAS 93:8677-8682. Onaivi ES, Martin BR (1989) Neuropharmacological and physiological validation of a computercontrolled two-compartment black and white box for the assessment of anxiety. Prog Neuropsychopharmacol Biol Psychiatry 13:963-976. Ostrander MM, Ulrich-Lai YM, Choi DC, Richta nd NM, Herman JP (2006) Hypoactivity of the hypothalamo-pituitary-adrenocortical axis during recovery from chronic variable stress. Endocrinology 147:2008-2017. Paxinos G, Watson C (1998) The Rat Brain in Stereotaxic Coordinates, 4th Ed. CD-ROM: Hulasz, P. 109

PAGE 110

Pellow S, Chopin P, File SE, Briley M (1985) Va lidation of open: closed arm entries in an elevated plus-maze as a measure of anxiet y in the rat. J Neurosci Methods 14:149-167. Pellow S, File SE (1986) Anxiolytic and anxioge nic drug effects on exploratory activity in an elevated plus-maze: A novel test of anxiety in the rat. Pharmacol Biochem Behav 24:525529. Peper M, Karcher S, Wohlfarth R, Reinshagen G, LeDoux JE (2001) Av ersive learning in patients with unilateral lesions of the am ygdala and hippocampus. Biol Psychol 58:1-23. Pomonis JD, Billington CJ, Levine AS (1996) Orphanin FQ, agonist of orphan opioid receptor ORL1, stimulates feeding in rats. Neuroreport 20:369-371. Prewitt CMF, Herman JP (1998) Anatomical in teractions between th e central amygdaloid nucleus and the hypothalamic paraventricular nucleus of the rat: a dual tract-tracing analysis. J Chem Neuroanat 15:173-185. Redrobe JP, Calo G, Regoli D, Quirion R (2002) Nociceptin receptor antagonists display antidepressant-like properties in the mouse forced swim test. Naunyn-Schmeidebergs Arch Pharmacol 365:164-167. Reinscheid RK, Nothacker HP, Bourson A, Ar dati A, Henningsen RA, Bunzow JR, Grandy DK, Langen H, Monsma FJ, Civelli O (1995) Orpha nin FQ: a neuropeptide that activates an opioidlike G protein-coupled receptor. Scie nce 270:792-794. Reinscheid RK, Ardati A, Monsma FJ, Civelli O (1996) Structure-activity relationship studies on the novel neuropeptide orphanin FQ. J Biol Chem 271:14163-14168. Renner KJ, Smits AW, Quadagno DM, Hough JC (1984) Suppression of sexual behavior and localization of [3H] puromycin after intracranial injecti on in the rat. Physiol Behav 33: 411-414. Risold PY, Swanson LW (1997) Connections of the rat lateral septal complex. Brain Res Rev 24L 115-195. Rogan MT, Staubli UV, LeDoux JE (1997) Fear conditioning induces associative long-term potentiation in the am ygdala. Nature 391:604-607. Rubin RT, Phillips JJ, Sadow TF, McCracken JT (1995) Adrenal gland volume in major depression. Increase during the depressive episode and decrease with successful treatment. Arch Gen Psychiatry 52: 213-218. Rygula R, Abumaria N, Flugge G, Fuchs E, Ruther E, Havemann-Reinceke U (2005) Anhedonia and motivational deficits in ra ts: Impact of chronic social stress. Behav Brain Res 162:127134. Sandin J, Georgieva J, Schott PA, Ogren SO, Terenius L (1997) Nociceptin/orphanin FQ microinjected into hippocampus impairs spatia l learning in rats. Eu r J Neurosci 9:194-197. 110

PAGE 111

Sandin J, Ogren SO, Terenius L (2004) Nocicep tin/orphanin FQ modulates spatial learning via ORL-1 receptors in the dorsal hippocam pus of the rat. Brain Res 997:222-233. Sawchenko PE, Swanson LW (1983) The organi zation of forebrain afferents to the paraventricular and supraoptic nuclei of the rat. J Comp Neurol 218: 121-144. Scicli AP, Petrovich GD, Swanson LW, Thomps on RF (2004) Contextual fear conditioning is associated with lateralized expression of the immediate early gene c-fos in the central and basolateral amygdalar nuclei. Behav Neurosci 118:5-14. Selye H (1936) Thymus and adre nals in the response of th e organism to injuries and intoxications. Brit J Exp Path 17:234-248. Shimohigashi Y, Hatano R, Fujita T, Nakashima R, Nose T, Sujaku T, Saigo A, Shinjo K, Nagahisa A (1996) Sensitivity of opioid re ceptor-like receptor ORL1 for chemical modification on nociceptin, a naturally occurri ng nociceptive peptide. J Biol Chem 271: 23642-23645. Simon P, Dupuis R, Costentin J (1994) Thigmotaxis as an index of anxiety in mice. Influence of dopaminergic transmissions. Behav Brain Res 61:59-64. Simpkiss JL, Devine DP (2003) Responses of the HPA axis after chronic variable stress: Effects of novel and familiar stressors. Neuroendocrinol Lett 24:75-81. Spampinato S, Baiula M (2006). Agonist-regulated endocytosis a nd desensitization of the human nociceptin receptor. NeuroReport, 17: 173-177. Stefanski R, Palejko W, Kostowski W, Plazn ik A (1992) The comparison of benzodiazepine derivatives and serotonergic agonists and antagonists in two models of anxiety. Neuropharmacology 31:1251-1258. Stone KL, Naccarato AM, Devine DP (in preparation) Rats exhibit behavioral despair and hormonal alterations following social defeat stress: Implications for stress-induced psychopathology. Tian JH, Xu W, Fang Y, Mogil JS, Grisel JE, Grandy DK, Han JS (1997) Bidirectional modulatory effect of orphanin FQ on morphine-induced analgesi a: antagonism in brain and potentiation in spinal cord of the rat. Brit J Pharmacol 120:676-80. Van de Kar LD, Piechowski RA, Rittenhouse PA Gray TS (1991) Amygdaloid lesions: Differential effect on conditioned stress a nd immobilization-induced increases in corticosterone and rennin secr etion. Neurendocrinology 54: 89-95. Varty GB, Hyde LA, Hodgson RA, Lu SX, McCool MF, Kazdoba TM, Del Vecchio RA, Guthrie DH, Pond AJ, Grzelak ME, Xu X, Korfmacher WA, Tulshian D, Parker EM, Higgins GA (2005) Characterization of the nociceptin receptor (ORL-1) agonist, Ro646198, in tests of anxiety across multiple species. Psychopharmacology 182:132-143. 111

PAGE 112

Vaughan CW, Christie MJ (1996) Increase by the OR L1 receptor (opioid receptor-like1) ligand, nociceptin, of inwardly rectifying K conductan ce in dorsal raphe nucleus neurons. Brit J Pharmacol 117:1609-1611. Vaughan CW, Ingram SL, Christie MJ (1997) Actions of the ORL1 receptor ligand nociceptin on membrane properties of rat periaqueductal gr ay neurons in vitro. J Neurosci 17:996-1003. Vitale G, Arletti R, Ruggieri V, Cifani C, Massi M (2006) Anxiol ytic-like effects of nociceptin/orphanin FQ in the elevated plus maze and in the conditioned defensive burying test in rats. Peptides 27:2193-2200. Vyas A, Bernal S, Chattarji S (2003) Effects of chronic stress on dendritic arborization in the central and extended amygdala. Brain Res 965: 290-294. Walker DL, Davis M (1997) Doubl e dissociation between the involve ment of the bed nucleus of the stria terminalis and the central nucleus of the amygdala in startle increases produced by conditioned versus unconditioned fear. J Neurosci 17:9375-9383. Walker DL, Toufexis DJ, Davis M (2003) Role of the bed nucleus of the stria terminalis versus the amygdala in fear, stress, and anxiety. Eur J Pharmacol 463:199-216. Wang JB, Johnson PS, Imai Y, Persico AM, Oze nberger BA, Eppler CM, Uhl GR (1994) cDNA cloning of an orphan opiate receptor gene family member and its splice variant. FEBS Lett 348:75-79. Watzl B, Lopez M, Shahbazian M, Chen G, Colombo LL, Huang D, Way D, Watson RR (1993) Diet and ethanol modulate immune responses in young C57BL/6 mice. Alcohol Clin Exp Res 17:623-630. Weisse CS (1992) Depression and immunocompetenc e: A review of the literature. Psychol Bull 111: 475-489. Whitnall MH (1993) Regulation of the hypothalamic corticotropin-releasing hormone neurosecretory system. Prog Neurobiol 40:573-629. Wick MJ, Minnerath SR, Lin X, Elde R, Law PY, Loh HH (1994) Isolation of a novel cDNA encoding a putative membrane receptor with high homology to the cloned mu, delta, and kappa opioid receptors. Brain Res Mol Brain Res 27:37-44. Wisniewski SR, Rush AJ, Bryan C, Shelton R, Trivedi MH, Marcus S, Husain MM, Hollon SD, Fava M (2005) Comparison of quality of life measures in a depre ssion population. J Nerv Ment Dis 195:219-225. Wommack JC, Delville Y (2003) Repeated social stress and the development of agonistic behavior: Individual differences in coping responses in male golden hamsters. Physiol Behav 80:303-308. 112

PAGE 113

Ziegler DR, Herman JP (2000) Local integrat ion of glutamate signaling in the hypothalamic paraventicular region: regulat ion of glucocorticoid stress responses. Endocrinology 141: 4801-4804. 113

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114 BIOGRAPHICAL SKETCH Megan K. Green received her Associate of Arts in August 1998 at Okaloosa Walton College. In May 2000, she received a dual Bach elor of Arts in psychology and anthropology from the University of West Florida. Mega n began her graduate st udies in experimental psychology at the University of West Florida in August 2001. She continued graduate studies at the University of Florida in August 2003, where she completed a Master of Science degree in behavioral neuroscience through the psychol ogy department in December 2005. Currently Megan is employed as a post-doctoral researcher at St. Charles Pharmaceuticals in Alachua, FL.