UNIVERSITY OF CINCINNATI

Date: 5-Oct-2009

I, Jennifer McGuire , hereby submit this original work as part of the requirements for the degree of: Doctor of Philosophy in Developmental Biology It is entitled: Chronic variable stress as a rodent model of PTSD;

A potential role for neuropeptide Y (NPY)

Student Signature: Jennifer McGuire

This work and its defense approved by: Committee Chair: Floyd Sallee, MD, PhD Floyd Sallee, MD, PhD

Sandra Degen, PhD Sandra Degen, PhD

Renu Sah, PhD Renu Sah, PhD

Steve Danzer, PhD Steve Danzer, PhD

James Herman, PhD James Herman, PhD

11/10/2009 287

Chronic variable stress as a rodent model of PTSD; A potential role for neuropeptide Y (NPY)

A Dissertation submitted to the Division of Research and Advanced Studies of the University of Cincinnati

In Partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

In the Graduate Program in Molecular and Developmental Biology College of Medicine

2009

By Jennifer McGuire

B.S. University of Massachusetts

Thesis Committee Committee Chair: Floyd R Sallee, M.D. Ph.D. Renu Sah, Ph.D. James P Herman, Ph.D. Sandra Degen, Ph.D. Steve Danzer Ph.D.

Abstract

Posttraumatic stress disorder (PTSD) occurs in 20-25% of people who experience trauma.

Eight million people in the United States are treated for PTSD annually and the number is increasing.

Sustained trauma exposure, such as combat, can develop into particularly severe and difficult to treat forms of PTSD. PTSD is thought to be a disorder of learning and memory in which hyperactivity of the amygdala leads to “overconsolidation”, increased accessibility, and enhanced reconsolidation of emotional and fearful memories. Increased amygdalar activity is a consistent finding in PTSD imaging studies. Concurrent suppression of medial prefrontal cortical and hippocampal functioning could further exacerbate amygdalar excitability. There is currently an emphasis in PTSD research to identify the molecular mechanisms behind these changes and to identify “resilience factors” that may help prevent the development of PTSD following trauma. Neuropeptide Y (NPY) is a putative resilience factor based on multiple lines of evidence. NPY is abundantly expressed in PTSD-relevant brain regions, inhibits excitatory neurotransmission, and antagonizes the effects of the pro-stress hormones corticotropin-releasing hormone and norepinephrine. Directly relevant to PTSD, NPY levels are reduced in the cerebrospinal fluid of combat veterans diagnosed with PTSD. The hypothesis tested in these studies is that chronic traumatization, results in depletion of NPY in PTSD-relevant brain regions and the development of PTSD-relevant behaviors.

The emergence of PTSD-like behaviors following trauma was tested in a rodent model of chronic variable stress, a paradigm pertinent to chronic traumatization. The overwhelming majority of current animal models of PTSD focus on the effect of acute trauma. The 7-day CVS paradigm is intended to model multiple single-unit traumas within a traumatization period such as may be experienced during combat. Behavioral outcomes related to fear memory, anxiety and arousal as well as neuroendocrine stress response were measured at early (16hr) and delayed (1 week) post CVS

iii recovery. In the contextual fear conditioning and extinction test, CVS rats allowed to recover for 1 week had a substantially increased freezing response to a trauma reminder than non-stressed rats. This is highly suggestive of enhanced fear recall and consistent with a PTSD-like phenotype. CVS rats allowed a 1-week recovery also had a reduced threshold for fearful arousal when tested on the EPM under aversive bright light. This behavior was not seen under dim light or 1 day after CVS under bright light indicating selective expression in fear-arousing contexts and a progression of physiological changes during recovery. At this same 7-day recovery timepoint, NPY peptide in CVS animals was significantly depleted in amygdala and increased in prefrontal cortex. Both of these changes are consistent with increased amygdalar excitability. Amygdalar NPY depletion persisted at least 15 days after CVS.

In summary, chronic trauma modeled by CVS potentiated fear memory recall and fearful arousal. Consistent with the hypothesis, chronic trauma induces a persistent depletion of NPY in the amygdala, which may contribute to these PTSD-like behaviors. Future studies supplementing NPY before, during and after stress are warranted to further investigate the relevance of NPY in trauma resilience.

iv v Acknowledgements

Thank you to all of my committee members, but especially Dr. Renu Sah, Dr. Jim Herman and

Dr. Randy Sallee for guiding me through.

Thank you also to my husband Matthew for patiently listening to me for months when my

single topic of conversation was neuropeptide Y and PTSD.

I’d like to thank Erica Doczy for generously lending us acoustic startle chambers and all the

rest of the equipment for that experiment, driving it down from Dayton and going way above and

beyond the call of duty. I owe you.

I’d like to acknowledge Anne Christiansen, Rong Zhang, Matia Solomon, Jon Flak, Ryan

Jankord, Amanda Jones, Kenny Jones, Sripana Ghosal and especially Ben Packard for their help on my

experiments, thank you.

Thank you to my parents for always being there.

Finally, I’d like to acknowledge the rats for their cooperation and sacrifice

vi Table of Contents

Abstract……………………………………………………………………………ii

Acknowledgements…………………………………………………………….…vi

Table of Contents………………………………………………….……………...vii

List of Tables and Figures………………………………………………………...x

List of Acronyms and Abbreviations……………………………………………..xii

Chapter 1 Introduction and Review of Literature

1.0 Posttraumatic stress disorder…………………………………………………...1

1.1 DSM-IV diagnostic criteria

1.2 Treatment options for PTSD

1.3 The neurobiology of PTSD and systems regulating fear memory and stress. ... .5

1.31 The amygdala

1.32 The prefrontal cortex

1.33 The hippocampus

1.34 The noradrenergic system

1.35 The hypothalamic-pituitary-adrenocortical axis

1.4 Rodent models of PTSD……………………………………………………..….26

1.5 Stress resilience and neuropeptide Y.………………………………………..….33

1.6 Neuropeptide Y: physiological functions, roles in behavioral regulation and potential in

stress-resilience……………………………………………………………….....35

1.61 Neuropeptide Y in the periphery

1.62 Neuropeptide Y in the central nervous system

1.63 Neuropeptide Y in the amygdala

vii 1.64 Neuropeptide Y in the prefrontal cortex

1.65 Neuropeptide Y in the hippocampus

1.66 Neuropeptide Y in the locus coeruleus and nucleus of the solitary tract

1.67 Effects of stress on neuropeptide Y expression

1.68 Hypothesis

Chapter 2 The Chronic Variable Stress-Recovery paradigm as a potential model for

posttraumatic stress-like behaviors……………………………………...... 50

2.0 The CVS model

2.1 Physiological measures

2.2 Fear conditioning

2.3 Elevated Plus-maze two conditions

2.4 Social Interaction

2.5 Acoustic Startle

2.6 HPA-axis activation

2.7 Discussion of Results

Chapter 3 Effects of CVS on NPY expression……………………………………………..79

3.0 The effects of CVS on NPY peptide expression in select brain regions

3.1 Immunohistochemical analysis of subregional NPY expression

3.2 Immunohistochemistry for tyrosine hydroxylase

3.3 Discussion of results

Chapter 4 Summary and Conclusions……………………………………………….……..98

4.0 CVS as a model of chronic traumatization

4.1 Integrating CVS induced behavioral changes with NPY dysregulation

viii 4.2 Potential pharmacotherapeutic relevance of NPY for stress associated disorders

Bibliography………………………………………………………………….....103

Appendix 1: Enhanced fear recall and emotional arousal in rats recovering from

chronic variable stress……………………………….……………………….128

ix List of Tables and Figures

Chapter 1

Figure 1: A neurocircuitry model of fear and extinction learning and recall…………………...7

Figure 2: Drawing of the human brain illustrating limbic and brainstem regions regulating

stress, emotion and memory…………………………………………………....….8

Figure 3: NPY in limbic and catecholaminergic structures……………………………...…….42

Table 1: Rodent models of posttraumatic stress disorder…………………………………...….29

Table 2: Physiological role of NPY in the periphery: genetic studies….……………….……...39

Table 3: Physiological role of NPY in the periphery: pharmacological studies………….….…40

Table 4: Physiological role of NPY in the brain: genetic studies…………………………..…..42

Table 5: Physiological role of NPY in the brain: pharmacological studies…………….….…...43

Chapter 2

Figure 1: Representative temporal schematic of the CVS experiments and composition

of stressors…………………………………………………………………………..…..….52

Figure 2: Physiologic measures after CVS………………………………………………..…....54

Figure 3: Sensitization of conditioned fear and fear memory as well as impaired extinction

in CVS animals……………………………………………………………………………..58

Figure 4: Elevated Plus-Maze testing reveals delayed expression of fear-associated arousal

in rats exposed to CVS………………………………………………………………….…..61

Figure 5: Social interaction is not affected by CVS exposure……………………………...... 65

Figure 6: Suppression of startle response in early recovery from CVS…………………….…..69

Figure 7: Rats exposed to CVS exhibit sensitized plasma corticosterone response to a

x novel acute stressor……………………………………………………………….….…..…72

Chapter 3

Figure 1: Post-CVS neuropeptide Y concentrations in forebrain limbic structures at early

and delayed recovery………………………………………………………………..…..…..85

Figure 2: Illustration of the rat brain, sagital view………………………………………….…..86

Figure 3: Reduced amygdalar neuropeptide Y immunoreactivity…………………….…....…..88

Figure 4: NPY immunoreactivity in the medial prefrontal cortex………………………….…..90

Figure 5: NPY immunoreactivty in locus coeruleus and solitary tract…………………….…...91

Figure 6: Tyrosine hydroxylase immunoreactivity…………………………………………..…93

Chapter 4

Figure 1: A model of NPY actions in the development of PTSD pathophysiology……………102

xi List of Acronyms and Abbreviations

5-HT-2c : serotonin receptor subtype 5-HT-2c

α1 : α1 adrenergic receptor

α2: α2 adrenergic receptor

β receptor: β adrenergic receptor

ACC: anterior cingulate

ACTH: adrenocorticotropin hormone

BDNF: brain-derived neurotrophic factor

BLA: basolateral amygdala complex

BNST: bed nucleus of the stria terminalis

BOLD: blood oxygen level dependent

CA1: CA1 region of the hippocampus

CA3: CA3 region of the hippocampus

CBT: Cognitive Behavioral Therapy

CeA: central amygdala

CORT: corticosterone

CRH: corticotropin-releasing hormone

CS: conditioned stimulus

CSF: cerebrospinal fluid

CVS: chronic variable stress dAcc: dorsal anterior cingulate

DBH: dopamine-β-hydroxylase

DEX: dexamethasone

xii DG: dentate gyrus

DMH: dorsal medial hypothalamus

DSM-IV: Diagnostic and Statistical Manual-4th edition

ELISA: enzyme-linked immunoabsorbant assay

EPM: Elevated Plus-Maze

ET: Exposure Therapy fMRI: functional magnetic resonance imaging

GABA: gamma-Aminobutyric acid

HCl: hydrochloric acid

HPA-axis: hypothalamic-pituitary-adrenocortical axis

IL: infralimbic prefrontal cortex

ITC: intercalated cells of the amygdala

LC: locus coeruleus

LTP: long-term potentiation

MeA: medial amygdala mPFC: medial prefrontal cortex mRNA: messenger ribonucleic acid

MRSI: Magnetic resonance spectroscopic imaging

NAA: N-acetylaspartate

NE: norepiniephrine

NET: norepinephrine transporter

NMDA: N-methyl-D-aspartic acid

NPY: neuropeptide Y

xiii NTS: nucleus tractus solitarius or nucleus of the solitary tract

OFC: orbitofrontal cortex

PET: positron emission tomography

PFC: prefrontal cortex

PL: paralimbic prefrontal cortex

PTSD: posttraumatic stress disorder

PVN: paraventricular nucleus of the hypothalamus rCBF: regional cerebral blood flow

RIA radioimmunoassay

SEM: standard error of the mean

SIT: Stress Innoculation Therapy

SNRI: serotonin-norepiniphrine reuptake inhibitor

SPECT: Single-photon emission computed tomography

SPS: single-prolonged stress

SSRI: selective serotonin reuptake inhibitor

TH: tyrosine hydroxylase vmPFC: ventromedial prefrontal cortex

Y1 : NPY1 receptor

Y2: NPY2: receptor

Y3: putative NPY3 receptor

Y4: NPY4 receptor

Y5: NPY5 receptor y6: NPY6 receptor pseudogene

xiv

Posttraumatic stress disorder

1 1.0 Posttraumatic Stress Disorder

Posttraumatic stress disorder (PTSD) is classified as an anxiety disorder and, as the name

implies, is initiated by a traumatizing experience. Research suggests that approximately 25% of trauma

exposed people may eventually develop PTSD (Green BL, 1994). The risk in combat exposed soldiers

may be higher (38%)(Kessler RC, 2000). Additionally, those who suffer chronic or repetitive trauma,

such as combat, severe childhood abuse are more likely to develop chronic, treatment refractory PTSD

(Kessler RC, 2000;Prigerson HG et al., 2001). A longitudinal study of veterans of the 1982 Lebanon

war determined that 20 years after the war, over 26% of soldiers with adaptive responses during

combat had enduring PTSD, while 52% of soldiers who showed obvious distress during combat had

PTSD (Solomon Z and Mikulincer M, 2006).

Posttraumatic stress disorder is associated with adverse changes in life course and interpersonal

instability (Kessler RC, 2000). In a study by Prigerson, et al, men who counted combat as their greatest

trauma had the highest rates of unemployment, of having been fired in the last year, of having been

divorced and of having physically abused women than any other types of trauma (Prigerson HG et al.,

2001).

There is a higher rate of suicide attempts among PTSD sufferers than with any other anxiety

disorder, major depression, bipolar disorder or substance abuse (Nock MK et al., 2009). The suicide

risk in combat-related PTSD is particularly high, perhaps because of access to weapons and knowledge

of how to use them (Panagioti M et al., 2009). Comorbid depression or other mood disorders and/or

substance abuse further increase the risk (Panagioti M et al., 2009). In addition to the individual burden

and risks of PTSD, cost analysis studies put the annual societal cost at over 3 billion dollars (Kessler

RC, 2000).

2 1.1 DSM-IV diagnostic criteria

In addition to an initiating traumatic event, the DSM-IV recognizes a number of symptom

clusters for PTSD (American Psychiatric Association, 2000). The first is persistent re-experiencing of

the event that can manifest as nightmares, intrusive thoughts, flashbacks and intense emotional distress

and cues symbolizing aspects of the event. A second feature of PTSD is avoidance of trauma

reminders and emotional numbing. Avoidance includes suppression of thoughts, feelings and

conversations associated with the trauma and also of activities, places, objects and people that

stimulate memories of the event. Emotional numbing can be experienced as lack of empathy,

dissociation and diminished expectations for the future. A third recognized feature of PTSD is

persistent hyperarousal. These symptoms include insomnia, difficulty in regulating emotion and/or

concentration, hypervigilance and an exaggerated startle response (American Psychiatric Association,

2000). An additional diagnostic criterion is chronicity. Symptoms must persist for more than 3 months

before a PTSD diagnosis can be made. Additionally, symptoms can appear with a delayed onset,

months or years after the initiating event. The final DSM-IV criterion is clinically relevant impairment

of social functioning.

Traumatizing experiences initiating PTSD can include acute trauma such as a violent attack or

accident, or a chronic or repetitive trauma such as extreme childhood abuse and neglect or combat

experience. Symptom severity in PTSD roughly correlates to the degree of trauma (Brinker M et al.,

2007). Combat-related and other chronic traumas can lead to complex and particularly difficult to treat

forms of PTSD (Friedman MJ et al., 2007).

1.2 Treatment options for PTSD

Currently, treatment options for PTSD are suboptimal. As reviewed in Ravindran and Stein, a

variety of pharmacologic agents targeting different systems are currently being employed to treat

3 PTSD (Ravindran LN and Stein MB, 2009). Selective serotonin-reuptake inhibitors (SSRIs) and

Serotonin-Norepinephrine reuptake inhibitors (SNRIs) have yielded mixed results, and the 2007

Committee on the Treatment of PTSD from the Institute of Medicine concluded that there was insufficient evidence to support the efficacy of SSRIs and SNRIs in the treatment of PTSD (Ravindran

LN and Stein MB, 2009). Anticonvusants such as Lamotrigine and Topiramate have also been tested for efficacy in the treatment of PTSD symptoms again with inconclusive results (Ravindran LN and

Stein MB, 2009). Benzodiazepines, although currently in use in PTSD pharmacotherapy, have not been demonstrated to improve core PTSD symptoms (Cates ME et al., 2004;Melman TA et al., 2002).

Agents targeting the noradrenergic system have been promising and will be addressed further in section 1.54.

The common and most effective non-pharmacologic treatment for PTSD is cognitive behavioral therapy (CBT) (Bryant RA et al., 2008). CBT focuses on the retraining of the individual’s belief system, particularly with respect to negative self-appraisal regarding the event and extreme, nonspecific generalizations about the external environment (Cloitre M, 2009). Stress Inoculation

Therapy (SIT) and Exposure Therapy address the traumatic event directly using guided imagery, and eventually in vivo exposure to minimize the emotional impact of event reminders; essentially they are fear extinction paradigms. SIT and ET may be used in combination with CBT (Cloitre M, 2009). In their meta analysis of psychotherapy paradigms for PTSD treatment, Bradley, et al. reported that approximately 50% of patients still meet diagnostic criteria for PTSD following treatment (using either a validated self-report measure, or validated structured interview) and an even greater number failed to meet investigator-defined criteria for symptom improvement (Bradley R et al., 2005).

4

The neurobiology of PTSD and systems regulating fear memory and stress

5 1.3 Neurobiology

Single-photon emission tomography (SPECT), positron emission tomography (PET) and

functional magnetic resonance imaging (fMRI) studies use symptom provocation paradigms in an

attempt to identify brain function disturbances in PTSD. These modalities use blood flow and blood

oxygen level dependent (BOLD) effects to determine relative brain activity. Additional studies using

non-imaging techniques provide further information on system dysregulation in PTSD. Together these

types of studies provide a theoretical foundation for the etiology of PTSD.

One current theory in PTSD research is that PTSD is a disorder of learning and memory. This

model is elegantly described in Elzinga and Bremner 2002 (Elzinga BM and Bremner JD, 2002). The

authors propose that dysregulation of both implicit and explicit memory systems can account for the

majority of the deficits and symptoms seen in PTSD. Explicit or declarative memory is conscious

recollection of facts, events and context, verbal memory, and associations, which is coordinated

through the hippocampus and cortical structures. Implicit memory involves other brain regions

including the amygdala and includes emotional memory and behavioral knowledge without conscious

memory. The authors describe the concerted effort of these two memory systems in this way “When

input (for example a noise) ‘matches’ representations in the emotional memory network (e.g. the noise

of the ambulance), mutual activation automatically spreads among the units, activating verbal,

behavioral, and physiological responses, and declarative and semantic knowledge” (Elzinga BM and

Bremner JD, 2002).

Dysregulation and mis-co-ordination of brain regions responsible for learning and memory functions

may then lead to increased accessibility of emotional memories and inhibited or inadequate regulatory

control from the prefrontal cortex and hippocampus. The trauma memory is “overconsolidated” while

subsequent extinction learning and other regulatory mechanisms are “impaired”. Abnormalities in

6 amygdalar, prefrontal cortical and hippocampal functions are all implicated in the etiology of PTSD.

(see Figure 1).

Figure 1: A neurocircuitry model of fear and extinction learning and recall. The amygdala is critical in acquisition of both fear and extinction learning. The infralimbic mPFC integrates sensory information with contextual information from the hippocampus to determine extinction retrieval and inhibitory status on amygdalar output. Regulatory control is also exerted by the brainstem noradrenergic system CS=conditioned stimulus

Adapted from Quirk and Mueller, Neuropsychopharmacology (2008) 33,56-72 with modifications

7 Contributions of limbic forebrain circuits and stress response systems in the pathophysiology of PTSD

Responses to stress involve the coordinated efforts of mutiple brain regions and systems. Figure

2 is an illustration of the human brain with key regions of the stress response systems identified

(Figure 2). The potential role of regions labeled in red in PTSD pathophysiology will be described in further detail below.

Figure 2: Drawing of the human brain illustrating limbic and brainstem regions regulating emotion memory and stress responses. Regions highlighted in red are believed to contribute to stress-related psychopathology, including PTSD.

8

1.31 The amygdala

The amygdala is a small almond shaped structure situated bilaterally in the medial temporal

lobe. The amygdala has been implicated in a broad range of functions related to emotional processing,

memory, attention, and both aversive and appetitive conditioning (Kentridge RW et al., 1991;LeDoux

JE, 2008;McDonald RJ et al., 2007;McGaugh JL et al., 1996;Wilensky AE et al., 2006). The

amygdala is subdivided into a number of nuclei and subnuclei based on histological features,

biochemical signature and/or afferent and efferent connections (LeDoux JE, 2008;Pitkanen A et al.,

1997). The lateral-basolateral complex of the amygdala consists of multiple smaller subnuclei critical

to the acquisition, consolidation, recall and expression of emotional/arousing memories and innate

behavioral responses (Doyere V et al., 2007;M Fendt and MS Fanselow, 1999;Pitkanen A et al., 1997).

Hereafter the lateral-basolateral complex will be referred to as the basolateral amygdala (BLA) or

basolateral complex. The basolateral complex is the major input region for the amygdala, with efferent

connections from the thalamus and sensory cortices, the hippocampus and the medial prefrontal cortex

(mPFC) (LeDoux JE, 2008). The BLA in turn projects back to the mPFC and to other nuclei within the

amygdala, primarily the central nucleus (LeDoux JE, 2008;Pitkanen A et al., 1997).

The medial amygdalar nucleus receives sensory input as well, primarily olfactory input directly

from the olfactory bulb (LeDoux JE, 2008). Additionally, the medial amygdala receives inputs from

the infralimbic prefrontal cortex (McDonald AJ et al., 1999). The medial amygdala has reciprocal

connection to the BLA, input to the central amygdala and projections to the periaqueductal grey

(Eberhardt JA et al., 1985). The medial amygdala has been implicated in the emotional processing of

olfactory cues, including predator scent and pheromones, and in social, sexual and defensive behaviors

(Takahashi LK et al., 2007).

9 The central nucleus of the amygdala is the main external relay point for the amygdalar complex. The central amygdala receives input from the other amygdalar nuclei, the medial prefrontal cortex and brainstem nuclei including the locus coeruleus (LC) and nucleus tractus solitaris (NTS)

(McDonald AJ et al., 1999;Riche D et al., 1990). The central nucleus projects back to multiple brainstem regions modulating sympathetic nervous system function, neurotransmitters and catecholamines, and response of the hypothalamic-pituitary-adrenocortical (HPA) axis (Herman JP et al., 2003).

In animal studies, the amygdala, particularly the basolateral complex, has been shown to be critical to the acquisition and expression of fear memory. Pavlovian fear conditioning induces long- term potentiation (LTP), an index of learning and memory, in the amygdala (Rogan MT et al., 1997).

Musimol inactivation of the BLA prior to fear conditioning prevents the formation of fear memory

(Wilensky AE et al., 1999). Retrieval of fear memories, and reconsolidation of retrieved fear memory has also been shown to activate LTP in the basolateral complex (Doyere V et al., 2007).

Consolidation and reconsolidation of fear can be enhanced or impaired respectively through the agonism or antagonism of the NMDA receptor in the BLA (Lee JLC et al., 2006). There is also evidence that the BLA is part of the circuitry mediating extinction learning via NMDA signaling

(Sotres-Bayon F et al., 2007). Additional studies have demonstrated the central nucleus is a site of fear memory reconstruction. Musimol inactivation of the central nucleus prior to conditioning prevents fear learning, while blocking protein synthesis prevents consolidation of fear memory indicating that the basolateral amygdala of itself is not sufficient for the acquisition and maintenance of emotional memory (Wilensky AE et al., 1999).

The medial amygdala has been less extensively studied than either the central or basolateral nuclei with respect to fear conditioning. While there is little evidence to support a role for the medial

10 amygdala in the consolidation of conditioned or unconditioned fear, that medial amygdala was shown to be important for recall of both conditioned and unconditioned fear (Takahashi LK et al., 2007) and in translating fear recall into defensive behaviors (Herdade KCP et al., 2006).

Fear conditioning studies in humans using imaging technology have confirmed nearly identical pathways for fear learning and memory as those worked out in animals (Milad MR et al., 2006) and demonstrated activation of the amygdala during fear learning and recall (Fischer H et al., 2000). This validates animal models of fear conditioning and extinction in mechanistic and pharmacological intervention studies.

Although imaging techniques do not have the sensitivity to identify specific subnuclei, the most consistent finding in PTSD imaging studies is hyperactivity of the amygdala following an emotionally challenging stimulus. These findings are consistent for both visual and auditory stimuli including combat footage and sounds (Bremner JD et al., 1999b;Shin LM et al., 1997), trauma-relevant narratives (Rauch SL et al., 1996), and negative but not implicitly trauma related images (Rauch SL et al., 2000). The degree of amygdalar activation was predictive of poor response to CBT, although the sample size was small (Bryant RA et al., 2008). There is not, however complete agreement between studies. Different groups report activation of the right amygdala (Pissiota A et al., 2002;Rauch SL et al., 1996;Schiller D et al., 2008;Shin LM et al., 1997), left amygdala (Liberzon I et al., 1999c;Williams

L et al., 2006), or bilaterally (Shin LM et al., 1999). One group reported decreased activity (Britton JC et al., 2005). The diversity in results may be a reflection of the imaging modalities, study populations or sample size.

The essential role of the amygdala in the etiology of PTSD is supported by a study that recruited veterans from the Vietnam Head Injury Study (Koenigs M et al., 2008). Participants were divided into groups based on focal injury to the ventromedial prefrontal cortex, amygdala, other brain

11 regions, or no injury based on CT scan analysis. Of 15 participants with amygdalar damage

(unilateral), 0% developed PTSD (Koenigs M et al., 2008). Due to the location of the amygdala within

the brain, participants with amygdalar damage had additional temporal lobe damage. Participants with

hippocampal damage without amygdalar damage did not differ significantly from non-injured controls

in the incidence of PTSD (44% vs 48% respectively) nor did non-PFC-non-amygdala brain injuries as

a whole (40% vs 48%).

Data from both animal and human studies indicate that the amygdala is required for the

acquisition and maintenance of fearful and emotional memory. The impact of these memories can be

modified by extra-amygdalar processes such as the acquisition of extinction learning. In extinction,

aversive stimuli are dissociated from environmental and sensory cues through repetitive presentation of

those cues without negative consequence. Extinction learning and memory does not replace or

overwrite fearful memories, but involves the creation of new memories (Delgado MR et al., 2006). In

both humans and rodents, the medial prefrontal cortex (mPFC), is critical in the acquisition and

expression of extinction learning (Quirk GJ et al., 2006;Quirk GJ and Mueller D, 2008). Impairment in

extinction learning is believed to be an additional component of refractory PTSD.

1.32 The prefrontal cortex

There is strong evidence from humans and non-human primates that the prefrontal and frontal

cortices are centers of higher order cognitive processing and executive function (Barbas H, 2000). The

prefrontal cortex is made up of a number of distinct regions with individual roles in cognitive,

emotional and mneumonic processes (Barbas H, 2000). The rodent prefrontal cortex shares a number

of anatomical, electrophysiological characteristics with regions of the primate dorsolateral and medial

prefrontal cortices, suggesting specialization of these regions in the course of primate evolution

12 (Brown VJ and Bowman EM, 2002;Seamans JK et al., 2008). The rat prefrontal cortex shares the most similarity with the primate anterior cingulate (ACC), with the rat anterior cingulate, prelimbic cortex and infralimbic cortex corresponding to areas 24, 32 and 25 of the primate ACC respectively (Seamans

JK et al., 2008). Although not definitive, the data suggests that the rat prefrontal cortex is involved in response control and flexibility, attention and anticipation (Seamans JK et al., 2008), functions also attributed to primate PFC. Despite the lack of direct anatomical homology, there are functional correlates between primate and rat prefrontal cortices that support rat as a valid research model into

PFC function (Brown VJ and Bowman EM, 2002)

Connections between the prefrontal cortex and amygdala are reciprocal, with a large number of projections between the rat infralimbic (primate area 25) and both the central and medial amygdalar nuclei (McDonald AJ et al., 1999). The rat prelimbic PFC (primate area 32) sends and receives projections primarily to the basal nucleus of the BLA (McDonald AJ et al., 1996). Additional evidence supports projections from a discreet area of the rat rostral anterior cingulate to the basolateral complex

(Bissiere S et al., 2008).

In primates there are also abundant projections from areas 24, 25 and 32 to the amygdala, primarily the basolateral complex (Ghashghaei HT et al., 2007). Outputs in these regions out number input connections from the amygdala (Ghashghaei HT et al., 2007). Additionally in primates there are extensive connections between the orbitoprefrontal cortex (OFC) and amygdalar nuclei (Ghashghaei

HT et al., 2007). The OFC is known to help calibrate emotional responses while dysfunction of the

OFC is associated with impulsive and aggressive behavior. While there is some evidence suggestive of

OFC dysfunction in PTSD patients (Dileo JF et al., 2008;Rauch SL et al., 1996;Shin LM et al., 1999), human imaging studies to date have focused primarily on the medial prefrontal cortex.

13 Data from animal studies overwhelmingly supports a critical role of the PFC in extinction consolidation and recall. In rats, lesioning the mPFC does not impair short-term extinction, but prevents recall of extinction learning 24 hours later (LeBron K et al., 2004;Quirk GJ et al., 2000).

Infusion of NMDA receptor antagonists (Burgos-Robles A et al., 2007) or protein synthesis inhibitors

(Santini E et al., 2004) into the mPFC also prevent consolidation of extinction learning. Conversely, stimulation of the mPFC can strengthen extinction learning while stimulation of the BLA reduces spontaneous firing of neurons in the mPFC (Herry C and Garcia R, 2002;Milad MR and Quirk GJ,

2002). Electrophysiology studies in both slice models (Schroeder BW and Shinnick-Gallagher P, 2004) and in vivo recordings (Maroun M and Richter-Levin G, 2003) found that high frequency stimulation of the basolateral amygdala (or acute stress) suppresses LTP in the amygdalo-cortical pathway.

Correlations between increased activity in the infralimbic prefrontal cortex (IL) and enhanced extinction learning suggests an inhibitory circuitry between the IL and amygdala (Milad MR and Quirk

GJ, 2002). Additionally, there is reduced responsiveness in central amygdala output neurons following electrical stimulation of the IL (Quirk GJ et al., 2003), possibly via inhibitory activation of the intercalated cells (ITC) (Stephen Maren and Gregory J Quirk, 2004). In non-human primates, projections from anterior cingulate area 32 to the amygdala are surrounded by calbindin and parvalbumin positive inhibitory interneurons (Ghashghaei HT et al., 2007), further supporting inhibitory mechanisms for PFC regulation of the amygdala.

Inhibition of amygdalar fear response in rats appears to be selective for the infralimbic region as electrical stimulation of the infralimbic facilitated extinction to a conditioned stimulus while the extinction profile between unstimulated animals and animals receiving stimulation of the prelimbic prefrontal cortex or anterior cingulate did not differ significantly (Vidal-Gonzales I et al., 2006).

14 Fear conditioning and extinction paradigms in human imaging studies have found similar involvement of the ventromedial PFC (vmPFC) in extinction recall (Phelps EA et al., 2004). Milad et al, found that the degree of extinction recall correlated to thickness of the vmPFC (Milad MR et al.,

2005). A recent study examining acquisition and extinction of context rather than a conditioned stimulus found the dorsal anterior cingulate (dACC, area 32) to be activated during extinction training

(Lang S et al., 2009). In the same study, connectivity analysis linked the dAcc with area 24 of the anterior cingulate and with the right amygdala (Lang S et al., 2009). A reversal paradigm, requiring simultaneous extinction to a cue previously associated with a shock and new conditioning to a previously unpaired cue, determined an exclusive role of the vmPFC in discriminating the extinguished cues from cues not previously associated with shock (Schiller D et al., 2008).

Human imaging provocation studies generally indicate a decrease in regional cerebral blood flow (rCBF) and BOLD effects in the mPFC of PTSD patients (Bremner JD et al., 1999b;Britton JC et al., 2005;Liberzon I et al., 1999b;Rauch SL et al., 2000;Shin LM et al., 1997;Shin LM et al., 1999;Shin

LM et al., 2004a;Williams L et al., 2006). However, activation patterns in the prefrontal cortex are particularly sensitive to the provocation paradigm. In healthy subjects, mental visualization of negative images activated an entirely different set of loci than viewed images (Kosslyn SM et al.,

1996). In PTSD patients and controls, mental visualization of traumatic events increased rCBF in the anterior cingulate, while viewing images of trauma scenes decreased rCBF in the same region only in the PTSD subjects (Shin LM et al., 1997).

Shin found diminished activation following script-driven imagery in the anterior cingulate in both combat and sexual abuse-related posttraumatic stress disorder (Shin LM et al., 1999;Shin LM et al., 2001). The same group later found decreased rCBF in medial prefrontal areas concurrent with increased activation in amygdala (Shin LM et al., 2004a). Additionally, Bremner et al. found increases

15 in rCBF in PFC areas 9,10 and 31, but deactivation in areas 25 and 32 following script-driven provocation (Bremner JD et al., 1999a).

Corroborating provocation studies, Magnetic Resonance Spectroscopic Imaging (MRSI) analysis found reductions in N-acetylaspartate (NAA), a putative marker of neuronal integrity, in anterior cingulate of PTSD patients. In a study using identical twins discordant for combat-related

PTSD, Kasai et al. found reduced gray matter density in the anterior cingulate in the PTSD twin strongly suggesting that deficits in the prefrontal cortex are a symptom of PTSD rather than a pre- existing risk factor (Kasai K et al., 2008;Schuff N et al., 2008). There are several recent and comprehensive reviews of the neuroimaging literature (Francati V et al., 2007;Liberzon I and Sripada

CS, 2008;Shin LM and Liberzon I, 2009).

One current hypothesis in PTSD research is that deactivation of the prefrontal cortex with concurrent increased activity in the amygdala in PTSD imaging studies depicts a failure of top-down regulatory control by the PFC on amygdalar activity. Two studies cast some doubt on that model.

Koenigs, et al., reported that of 40 participants recruited from the Vietnam Head injury Study with damage to the ventromedial PFC (unilateral or bilateral) only 18% had developed PTSD compared to almost 50% in non brain-damaged controls and 40% in non-PFC/non-amygdala brain-damaged controls (Koenigs M et al., 2008). The authors conceded that the effects of vmPFC damage likely depend on the subfields affected and further hypothesized that an inability for self-reflection may be protective in the development of PTSD (Koenigs M et al., 2008). This study does however suggest that diminished PFC regulatory control is not the primary contributing factor to the development of PTSD.

A study by Bryant, et al reported that, as anticipated higher pre-treatment activation in the amygdala was associated with non-responsiveness to treatment, but contrary to expectations, higher pre-treatment activation in the ventral anterior cingulate was also associated with treatment non-

16 responsiveness (Bryant RA et al., 2008). This may again be a reflection of specialization between the

subfields of the PFC, the provocation paradigm, ‘bottom-up’ activation from the brainstem, or a

combination of the above (Bryant RA et al., 2008). While the results of the two studies do not support

inadequate regulation of amygdala function by the PFC as the primary deficit in PTSD, they are

consistent with an ‘amygdalocentric’ model whereby hyperactivity of the amygdala overwhelms

regulatory networks. Other studies strongly indicate reduced regulatory capacity of the PFC as a

contributing factor to PTSD pathology.

1.33 The hippocampus

The hippocampus is another medial temporal lobe structure and has abundant reciprocal

connections to the amygdala and prefrontal cortex. The hippocampus is critically involved in processes

of learning and memory. Some aspects of declarative memory cannot be described in animal models

(e.g. verbal recall, conscious recall and perceived meaning), which can make generalizations from

animal studies to humans problematic (Elzinga BM and Bremner JD, 2002). However, some aspects of

hippocampal function such as spatial learning and recall and contextual association can be tested in

animal models and are consistent between rodent, non-human primate and human (Squire LR, 1992).

Lesion studies in rats have delineated the contributions of three subunits of the hippocampus,

the CA1, CA3 and dentate gyrus (DG) to the acquisition and retrieval of contextual associations (Lee I

and Kesner RP, 2004). In a fear conditioning paradigm, animals with lesions to any of these three

subregions showed delayed acquisition of fear, requiring two (CA3) or three (CA1 or DG)

conditioning sessions for equivalent freezing behavior between lesioned and control animals (Lee I and

Kesner RP, 2004). When tested for recall of the shock context, animals with lesions to the CA1 or DG

froze significantly less than either control of CA3-lesioned animals (Lee I and Kesner RP, 2004).

Lesions to any of the three regions tested did not affect freezing to an unconditioned stimulus (Lee I

17 and Kesner RP, 2004). Overall, these data indicate that the CA1, CA3 and DG are active during acquisition of contextual associations, while the CA1 and DG are required for later retrieval of that information (Lee I and Kesner RP, 2004).

Both acute and chronic stress are known to impair later hippocampus-dependent learning and memory in humans (Kirschbaum C et al., 1996) and in rats (Diamond D et al., 1999;Garcia R et al.,

2007;Park CR et al., 2008). In rats, stress exposures inhibit induction of LTP both in vivo (Garcia R et al., 2008) and in ex vivo slice models (Collins DR, 2009). Kozlovsky, et al also report reduced activation of immediate-early genes following stress, indicative of reduced neuronal activity in the

CA1 (Kozlovsky N et al., 2008). Impairment of hippocampal function is attributed to prolonged exposure of hippocampal neurons to elevated levels of the adrenal stress hormone corticosterone

(rodents) or cortisol (human) (Kirschbaum C et al., 1996;Magarinos AM and McEwen BS, 1995).

There is evidence in rodents as well that acute stress impairs hippocampal neurogenesis (Kikuchi A et al., 2008).

A large number of imaging and provocation studies in human subjects with PTSD have looked at hippocampal size and activation patterns. Different groups report both increased (Shin LM et al.,

2004a;Shin LM et al., 2004b;Thomases K et al., 2009) and decreased (Britton JC et al., 2005;Moores

KA et al., 2008) activation in the hippocampus. The results of volumetric analyses are inconsistent as well. Decreased (Bremner JD et al., 1995;Bremner JD et al., 2003b;Shin LM et al., 2004b) and unchanged (Golier JA et al., 2005;Schuff N et al., 2008) hippocampus size are reported. Volumetric analysis may be complicated by high incidence of co-morbid depression and addiction associated with

PTSD, both of which are reported in association with smaller hippocampi (reviewed in (Geuze E et al.,

2005)). Overall, inconsistencies in these studies may be attributable to differences in provocation paradigms, subject populations, and statistical analysis (Shin LM and Liberzon I, 2009).

18 Nonetheless, there is evidence of hippocampal dysfunction in PTSD. A number of MRSI

studies report reduced N-acetylaspartate in the hippocampi of PTSD patients, indicative of poor

neuronal health (Schuff N et al., 2008;Shin LM and Liberzon I, 2009). In some (Moores KA et al.,

2008) but not all (Bremner JD et al., 1995;Shin LM et al., 2004b) studies, PTSD patients perform

worse on verbal recognition tasks. Imaging studies examining activation patterns during non-

emotionally loaded memory tasks suggest alterations in tasks requiring hippocampal-cortical

connectivity (Bremner JD et al., 2003b;Moores KA et al., 2008;Thomases K et al., 2009). However,

twin studies suggest that both smaller hippocampus size and poorer performance on hippocampal-

dependent learning and memory tasks may be pre-existing vulnerability factors rather than symptoms

of posttraumatic stress disorder (Gilbertson MW et al., 2007;Gilbertson MW et al., 2002).

1.34 Noradrenergic system

The locus coeruleus (LC) is a small, well-delineated nucleus of the pontine brainstem. The

majority or norepinephrine (NE) containing neurons are located within the LC and it is the exclusive

source of norepinephrine in hippocampus and neocortex and primary source in the amygdala (Berridge

CW and Waterhouse BD, 2003). The LC receives afferent connections from central amygdala, lateral

hypothalamus, bed nucleus of the stria terminalis (BNST) and cortical structures (Berridge CW and

Waterhouse BD, 2003). The prefrontal cortex has strong projections to the locus coeruleus and is a

primary excitatory input (Jodo E et al., 1998).

Three receptor classes: α 1 (subtypes α1a, α1b,α 1d), α2 (subtypes α2A-D) and β (subtypes β1-3)

mediate the diverse effects of NE. The α1 and β receptors work in combination to mediate wakefulness

and attentional processes. In the prefrontal cortex, activation of the α1 produces deficits in response

inhibition and impairs performance on working memory tasks, while activation of the α2 receptor

19 improves concentration and cognitive function (Elzinga BM and Bremner JD, 2002). The α1 receptor has a lower affinity for NE than the α2 and may mediate suppression of cortical function when NE concentrations are elevated (Arnsten AFT, 1998). Expression of the various receptor subtypes varies within and across brain regions and helps direct cellular response to norepinephrine with the target region (Berridge CW and Waterhouse BD, 2003). Cellular responses to norepinephrine are biphasic, with high and low NE concentrations inhibiting neuron firing while median concentrations stimulate neuronal activity and facilitated neuronal response to other transmitters (Berridge CW and Waterhouse

BD, 2003).

Norepinephrine is released following acute stress in hippocampus, amygdala and medial prefrontal cortex (Miyashita T and Williams CL, 2004;Nisenbaum LK et al., 1991;Smagin GN et al.,

1994;Williams CL et al., 2000). Corticotropin-releasing hormone (CRH) stimulates LC discharge in response to stress (Valentino RJ et al., 1988;Valentino RJ et al., 1993). NE stimulates release of CRH from the paraventricular nucleus of the hypothalamus (Plotsky PM et al., 1989). Chronic stress depresses peak LC discharge, but reduces the threshold at which CRH initiates NE release (Curtis AL et al., 1995). NE release in response to environmental stressors occurs rapidly, while neuroendocrine stress response initiates more slowly but lasts longer (O'Donnell T et al., 2004). One can envision a feed-forward mechanism in which noradrenergic and neuroendocrine dysregulation reinforce and maintain hyperarousal in pathological anxiety and arousal. In fact, both CRH and NE have been shown to be elevated in the cerebrospinal fluid of combat-related PTSD patients (Geracioti TD et al.,

2001;Geracioti TD et al., 2008).

Retrograde tract-tracing from CRH immunoreactive fibers in the LC identify the central amygdala as an abundant source of LC CRH (Van Bockstaele EJ et al., 1998). The reciprocal connections between the LC, prefrontal cortex and amygdala suggest the locus coeruleus as a site for

20 the integration of behavioral, noradrenergic and neuroendocrine response to stress (Morilak DA et al.,

2005).

The nucleus tractus solitarius (NTS) also exerts effects on norepinephrine release in limbic structures and on memory retention (Miyashita T and Williams CL, 2004;Williams CL et al., 2000).

The NTS is situated in the caudal medulla and also has reciprocal connections with the amygdala

(Williams CL et al., 2000). The NTS is the relay point for CNS response to peripheral stimuli via ascending vagal nerve projections (Williams CL et al., 2000). The NTS is a major excitatory input for the HPA-axis, with direct noradrenergic and glutamatergic connections to the paraventricular nucleus of the hypothalamus (Herman JP et al., 2003). In turn, noradrenergic activation of the α1 receptor in the

PVN stimulates both CRH and ACTH release (Plotsky PM et al., 1989)

In the rat NTS, stimulation of the β- adrenergic receptor by clenbuterol or the peripheral hormone epinephrine induce norepinephrine release in the amygdala and hippocampus (via NTS stimulation of the locus coeruleus), while temporary inactivation of the NTS using lidocane abolishes the effects of epinephrine (Miyashita T and Williams CL, 2004;Williams CL et al., 2000).

Furthermore, facilitated release of amygdalar NE by clenbuterol enhanced retention of fear memory in a Y-maze conflict task (Williams CL et al., 2000).

Human imaging studies corroborate the findings of animal studies. In healthy subjects, inhibition of norepinephrine re-uptake increases rCBF in amygdala following fearful, but not neutral or happy faces over placebo controls (Onur OA et al., 2009). Yohimbine, an α2 adrenergic receptor antagonist known to be anxiogenic in both animals and humans initiates an exaggerated response in

PTSD patients over both trauma-exposed and healthy individuals (Morgan III CA et al.,

1995;Southwick SM et al., 1999;Southwick SM et al., 1993). George Koob sums up the potential role of the noradrenergic system in a recent review “The hypothesis outlined here is that brain stress

21 systems respond rapidly to to anticipated challenges to homeostasis (excessive drug taking) but are

slow to habituate or do not readily shut off once engaged. Thus the very physiological mechanism that

allows a rapid and sustained response to environmental challenge becomes the engine of pathology if

adequate time or resources are not available to shut off the response. The interaction between CRF and

norepinephrine in the brainstem and basal forebrain, with contributions from other brain stress

systems, could lead to the chronic negative emotional-like states . . . “ (Koob GF, 2009).

Although he is referring to the phenomenom of compulsive drug seeking, he could easily be

describing the maintenance of PTSD symptoms; this is not coincidental as the “reward” circuitry

attributed to the development and maintenance of drug addiction is nearly identical to the circuitry of

emotional fear and memory.

Not surprisingly, the noradrenergic system has been a target of pharmacological treatments in

PTSD. The data on α2 receptor agonists is conflicting. In placebo-controlled trials, guanfacine was

found to be ineffective in the treatment of PTSD (Davis LL et al., 2008;Neylan TC et al., 2006).

Clonidine, another α2 agonist was reported to improve hyperarousal symptoms, also in placebo-

controlled trials. The selective α1 adrenergic receptor antagonist, prazosin improved sleep and

hyperarousal symptoms in combat veterans with PTSD in multiple studies (Raskind MA et al.,

2002;Raskind MA et al., 2003;Raskind MA et al., 2007;Thompson CE et al., 2008). Systemic β-

blockers such as propanolol disrupted both consolidation and reconsolidation of fear memories in rats

and showed promise as a prophylactic treatment for PTSD (Debiec J and LeDoux JE, 2006). However

randomized placebo-controlled studies failed to demonstrate efficacy in human subjects (Stein MB et

al., 2007).

1.35 Hypothalamic-Pituitary-Adrenal Axis

22 The hypothalamo-pituitary-adrenal axis (HPA-axis) is comprised of the paraventricular nucleus of the hypothalamus (PVN), the anterior pituitary and the adrenal gland. The HPA-axis is one of the systems mediating physiologic adjustments to changes in the external or internal environment. The

PVN secretes corticotropin releasing hormone (CRH) and vasopressin that stimulates adrenocorticotropic hormone (ACTH) release from the anterior pituitary. ACTH in turn stimulates release of glucocorticoids from the adrenal glands. Tonic release of glucocorticoids fluctuates over the daily circadian cycle, peaking at the onset of wakefulness. In response to stress, a surge of ACTH is released driving an increase in circulating glucocorticoids. Elevated glucocorticoids act on a number of systems, diverting resources to address the immediate challenge. Elevated glucocorticoids also feed back on the hypothalamus, inhibiting CRH and ACTH release terminating the stress response.

The HPA-axis responds to both direct physiologic threats to homeostasis, such as hemorrhage, infection, hypoglycemia, hypothermia etc. and to anticipatory threats such as novel situations, social challenge and contextual or conditioned stimuli. Anticipatory and psychologic stressors are potent activators of the HPA-axis and autonomic nervous system (McEwen BS, 2002)

The PVN receives direct connections primarily from regions regulating homeostatic functions including the nucleus of the solitary tract (NTS), bed nucleus of the stria terminalis (BNST), and the dorsal medial hypothalamus (DMH), arcuate nucleus and other hypothalamic nuclei (reviewed in

(Herman JP et al., 2003)). Activation of the HPA-axis by anxiogenic stressors involves contributions from the medial and basolateral amygdalar nuclei, periaqueductal grey and the infralimbic medial prefrontal cortex (Herman JP et al., 2003;Masini CV et al., 2009;Radley JJ et al., 2008;Ulrich-Lai YM and Herman JP, 2009). In contrast, the prelimbic mPFC and hippocampus appears to inhibit HPA-axis response to psychogenic stress (Radley JJ et al., 2008).

23 Glucocorticoids enhance memory formation and impair retrieval, depending on the time of administration (reviewed in (Roozendaal B et al., 2008))(Cohen H et al., 2008;Kirschbaum C et al.,

1996;Roozendaal B et al., 2006;Thompson BL et al., 2004). Strong encoding of emotionally charged memories is due in part to the effects of glucocorticoids. These effects on memory actually require NE and glucocorticoid signaling in conjunction, as metyrapone, a corticosterone-synthesis inhibitor, and β- adrenergic receptor antagonists ablate memory enhancement (Roozendaal B et al., 2006). Like norepinephrine, the effects of glucocorticoids on memory enhancement are dose dependent, with maximum effect occurring with a moderate range of glucocorticoid availability (Roozendaal B et al.,

1999). In addition to modulation of memory consolidation, noradrenergic-glucocorticoid interactions affect emotional reactivity, increasing arousal to negative stimuli (Kukolja J et al., 2008;van Stegeren

AH et al., 2007;van Stegeren AH et al., 2008).

Chronic stress affects is known to sensitize the HPA-axis response to novel stressors (Dal-Zotto

S et al., 2000;Servatius RJ et al., 1994;Ulrich-Lai YM and Herman JP, 2009;Weinberg DH et al.,

2009). At the same time, HPA-response to familiar stressors is suppressed (Dal-Zotto S et al.,

2000;Weinberg DH et al., 2009). The same phenomenon occurs following severe acute stressors

(Adamec RE and Shallow T, 1993;Armario A et al., 2004;Belda X et al., 2008). Other groups report suppressed corticosterone response to chronic variable stress, immobilization stress and single prolonged stress (Adamec RE and Shallow T, 1993;Cohen H et al., 2007b;Kohda K et al.,

2007;Ostrander MM et al., 2006). One report suggests that this may be due to enhanced feedback inhibition of the HPA-axis (Kohda K et al., 2007).

Initial reports in neuroendocrine studies on PTSD patients reported low basal cortisol (Yehuda

R et al., 1990). Follow up studies are inconsistent (reviewed in (de Kloet CS et al., 2006)). Low (Olff

M et al., 2006;Yehuda R et al., 1996), high (Baker DG et al., 2005;Liberzon I et al., 1999), or no

24 differences (Baker DG et al., 1999;Metzger LJ et al., 2008) in basal cortisol levels are reported. Studies vary widely in methodology and study populations, which may account for some of the discrepancy.

Basal cortisol may be affected in a subset of PTSD patients, or low circulating cortisol may be a pre- existing susceptibility factor to the effects of severe stress. At this time, it is not clear whether there are irregularities in basal cortisol rhythms in PTSD.

There are consistent reports of enhanced feedback inhibition of the HPA-axis in PTSD.

Suppression of the HPA-axis is enhanced relative to traumatized and healthy controls following low doses of dexamethasone, a glucocorticoid receptor agonist (Yehuda R et al., 1995;Yehuda R et al.,

2003). However, more recent studies using a combined dexamethasone-CRH test, a more refined test of HPA-axis alterations, found no differences between patients with chronic PTSD and controls in either ACTH or cortisol concentration curves (CS de Kloet et al., 2008;Muhtz C et al., 2008). Despite inconsistencies in peripheral measures, elevated cortisol, CRH and norepinephrine are reported in the cerebrospinal fluid of PTSD patients (Baker DG et al., 2005;Geracioti TD et al., 2001). This is consistent with persistently active noradrenergic and HPA-axis signaling with consequent hyperactivity in the amygdala and enhanced sympathetic reactivity.

25

Rodent models of PTSD

26 1.4 Rodent models of PTSD

Animal models may show the expression of behaviors with similarity to certain

pathophysiological outcomes. Models are useful for conducting detailed mechanistic studies and the

identification of potential therapeutic targets. However, there are certain criteria that need to be met.

Animal models should share phenotypic similarity with the human disorder (face validity)(Yehuda R

and Antelman SM, 1993). Additionally, animal models should share a theoretical framework for the

development and maintenance of that phenotype (construct validity) (Yehuda R and Antelman SM,

1993). Finally an animal model should have some predictive value in the testing of pharmacological

interventions (predictive validity) (Yehuda R and Antelman SM, 1993). Animal models of PTSD

should ideally show biological and behavioral changes that persist or grow stronger over time,

gradation of PTSD-like alterations that depend on the intensity of the stressor, and individual

variability in the long-term effects of stress reflecting previous experience and genetic variability

(Yehuda R and Antelman SM, 1993). According to Yehuda and Antelman (1993), the critical

behavioral characteristic is a clear conditioned response to trauma reminders, and sensitization to novel

stressors (Yehuda R and Antelman SM, 1993). Models relevant to PTSD should also reflect some of

the somatic disturbances associated with PTSD such as HPA axis dysregulation, sleep disturbances and

sympathetic reactivity.

Acute stress models predominate in the PTSD field. Table 1 summarizes current animal models

of PTSD and the relevant findings. In shock models, animals are given foot-shocks or tail-shocks as

the initiating traumatic event. The SPS model involves three intense stressors given back to back: 2

hours of restraint, a 20 minute swim and then ether anesthesia to unconsciousness. Predator exposure

paradigms expose the rodent subjects to either cat scent (Cohen H et al., 2007a), or to the presence of

a cat while protected (Zoladz PR et al., 2008) or unprotected (Adamec RE et al., 1999). Other animal

27 models include immobilization (Hegde P et al., 2008), submergence underwater (Richter-Levin G,

1998) and social defeat (Pulliam JVK et al., 2009). Sub-chronic (2-4 day stress exposure) and chronic

(5+ days stress exposure) models of PTSD have primarily extended acute models into multiple presentations of shock or predator exposure. None of these models reflect the multiple single-episode traumas thought to instrumental in the development of the complex and entrenched PTSD symptoms associated with combat trauma and other chronic traumas (Kaysen D et al., 2009)

Studies in animal models of PTSD have begun to examine neuropeptide and neurotransmitter systems with implications for treatment of PTSD. Pharmacological blockade of cholecystokinin receptors before or after stress exposure produced lasting reductions in anxiety-like behavior (Adamec

RE et al., 1997). In a stress-restress model of PTSD, dopamine in the prefrontal cortex was depleted at least 7 days after re-stress (Adamec RE et al., 1997;Harvey BH et al., 2006). Changes in serotonin receptor subtype expression were identified after a recovery period from single-prolonged-stress

(Harada K et al., 2008). Pharmacological investigation implicated the 5-HT2c receptor subtype as mediating the behavioral responses to SPS (Harada K et al., 2008).

Selective depletion of brain-derived neurotrophic factor (BDNF) in the hippocampus is reported in both shock and predator-scent models of PTSD (Kozlovsky N et al., 2007;Rasmusson AM et al., 2002). More recently, galanin was determined to be up-regulated in hippocampus of more stress- resistent rats but downregulated in the hippocampus and prefrontal cortex of more anxious rats

(Kozlovsky N et al., 2009). This study went even further and showed that the galanin receptor agonist galnon could reduce anxiety behavior in rats more severely affected by predator scent stress

(Kozlovsky N et al., 2009).

28

TABLE 1: RODENT MODELS OF POSTTRAUMATIC STRESS DISORDER

Contd ↑ = increase; ↓= decrease; NC=no change

29

Contd

30 Contd

31

32

Stress resilience and neuropeptide Y

33 1.5 Resilience and Resistance

Virtually everyone who experiences a severe traumatic event develops PTSD-like symptoms in

the time period immediately following the event. For the majority, those symptoms fade and PTSD

does not develop. In this case, resilience can be defined as the ability to persevere during an acutely

stressful or traumatic experience, re-adjust and return to normal functioning. While a number of

psychological characteristics are identified as resilience factors (reviewed in (Haglund MEM et al.,

2007;Yehuda R et al., 2006b), identification of biological stress-regulatory systems conferring

resistance to and resilience from the effects of traumatic stress has become an increasing focus of

researchers (Pitman RK et al., 2006). A number of features make neuropeptide Y (NPY) an attractive

candidate as a factor influencing trauma resilience (Yehuda R et al., 2006b). First, NPY is abundantly

expressed in brain regions pertinent to the development and maintenance of PTSD symptoms. Also,

NPY is known to have anxiolytic properties (reviewed in Kask, et al)(Allen YS et al., 1983;Kask A et

al., 2002). Furthermore, NPY negatively regulates excitatory neurotransmitter release (Sorensen AT et

al., 2008;Whittaker E et al., 1999) and antagonizes the actions of the stress response hormone,

corticotropin-releasing hormone (CRH) (KashTL and Winder DG, 2006;Sajdyk TJ et al., 2006).

Genetically low levels of NPY production are associated with increased emotionality in both humans

(Zhou A et al., 2008) and rodents (Karl T et al., 2008;Sudakov SK et al., 2001). On the other hand

high NPY expression promotes stress resilience in rodents (Thorsell A et al., 2000).

Reports regarding circulating NPY levels in PTSD patients are conflicting. A study by Morgan

et al., reported that trauma exposure suppressed baseline NPY levels, but reduced NPY did not

correlate with PTSD (Morgan, III et al., 2003). A subsequent study found that lower plasma NPY was

associated with PTSD, and that treatment increased NPY levels (Yehuda R et al., 2006a). Importantly,

the increase in circulating NPY positively correlated to the extent of symptom improvement (Yehuda

34 R et al., 2006a). A study in active military personnel found that elite Special Forces soldiers mounted a higher plasma NPY response under stress (Morgan III CA et al., 2000). Additionally, while non-

Special Forces soldiers had plasma NPY levels below baseline 24 hours after stress, Special Forces soldiers had returned to baseline levels (Morgan III CA et al., 2000). Finally, combat veteran PTSD patients show depletion of NPY in cerebral spinal fluid, corroborating the accumulating data implicating NPY in stress-protection and resilience (Sah R et al., 2009).

While promising, research into the stress protective effects of NPY in PTSD is circumstantial and requires detailed investigation. Surprisingly, only a single study has looked at the effects of trauma on NPY in an animal model of PTSD (Cui H et al., 2008). The authors found an increase in NPY expression in the basolateral complex of the amygdala, but a sharp decrease in the central nucleus (Cui

H et al., 2008). The authors were unable to explain the apparent contradiction to previous reports on the effects of acute stress in the basolateral amygdala and the anxiolytic properties of NPY (Thorsell A et al., 1998), but speculated that the increase in NPY content was related to the neuronal hypertrophy in the BLA also reported (Cui H et al., 2008).

Identification and validation of potential PTSD-relevant targets requires a robust and reliable animal model of PTSD-like sequelae. Of particular importance is the development of models pertinent to the complex and entrenched PTSD symptoms seen in survivors of sustained and repetitive trauma such as refugees, survivors of severe abuse and torture, and combat veterans. Kaysen, et al. define chronic traumatization as “repeated exposures to stressors with the same overall context over time”

(Kaysen D et al., 2009). Studies in this dissertation were designed with the dual objectives of a) developing an animal model pertinent to chronic trauma, and b) investigating potential relevance of

NPY as a resiliency peptide in this model.

35

Neuropeptide Y: physiological functions, roles in behavioral regulation and potential

in stress-resilience

36

1.6 Neuropeptide Y

Neuropeptide Y is a 36 amino acid peptide identified in the early 1980s due to its homology

with the enteric peptides pancreatic polypeptide and peptide YY (Tatemoto K et al., 1982). NPY is

widely expressed in the central nervous system and in sympathetic ganglia (Allen YS et al.,

1983;Lundberg NM et al., 1982). NPY expression is high in a number of hypothalamic nuclei, the

amygdala, cortex, hippocampus, nucleus accumbens, the dorsal and ventral periaqueductal grey and

the dorsal raphe nucleus (Allen YS et al., 1983;Yamazoe M et al., 1985). NPY is also expressed across

a number of catecholaminergic brainstem nuclei: A1-A3 noradrenergic cell groups, the locus

coeruleus, and the C1 and C2 adrenergic groups, (Wahlestedt C et al., 1989;Yamazoe M et al., 1985).

1.61 NPY in the periphery

Peripherally, NPY is expressed in sympathetic nerves, adrenal gland nerves fibers and adrenal

chromaffin cells (Kuramoto H et al., 1986;Lundberg JM et al., 1983). NPY is released from

sympathetic nerve terminals and adrenals in response to intense stressors activating the sympathetic

nervous system including hemorrhage, exercise, hypoxia and intense anxiogenic stress (Kaijser L et

al., 1990;Morris MJ et al., 1986;Qureshi N et al., 1998;Zukowska-Grojec Z and Vaz AC, 1988).

Circulating NPY is believed to derive from sympathetic neurons co-expressing norepinephrine rather

than adrenal pools, as adrenalectomy does not affect either baseline or stimulated levels of plasma

NPY (Bernet F et al., 1998). The effects of NPY are mediated through at least four G protein-coupled

receptors: Y1, Y2, Y4, and Y5 (Blomqvist A and Herzog H, 1997;Dumont Y et al., 1998;Wahlestedt C

et al., 1986). The existence of a putative Y3 receptor is suggested by receptor binding profiles, but has

yet to be cloned (Wahlestedt C et al., 1992). The gene for a Y6 receptor encodes a full-length peptide

in mice, but a non-functional truncated peptide in primates (Starback P et al., 2000;Weinberg DH et

al., 1996). An excellent review by Pedrazzini, Pralong and Grouzmann details the functional

characterization and downstream effectors of the NPY receptors (Pedrazzini T et al., 2003).

37 There are extensive studies on the physiological role of NPY using in vitro and in vivo manipulation of NPY signaling and in genetically modified animals. Tables 2 and 3 outline the genetic and pharmacological studies of NPY in the periphery. NPY is released into the peripheral circulation following activation of the sympathetic nervous system (Lundberg NM et al., 1982;Morris MJ et al.,

1986). NPY has potent vasoconstrictive properties (Lundberg JM and Modin A, 1995;Zukowska-

Grojec Z et al., 1996) NPY at lower doses also potentiates the actions of other vasoconstrictors including norepinephrine (Edvinsson L et al., 1984). NPY is not required for the homeostatic maintenance of resting blood pressure (Pedrazzini T et al., 1998). Under acute physiologic stress such as septic or hemorrhagic shock, NPY functions both directly and synergistically with other vasoconstrictors to help maintain blood pressure (Qureshi N et al., 1998). NPY mediates further cardiac actions through negative regulation of neurotransmitter release (Smith-White MA et al., 2002).

In adrenal glands, NPY is found in catecholaminergic nerve terminals and is synthesized endogenously in the adrenals by medullary chromaffin cells. NPY release in the adrenals has been found to stimulate catecholamine release and adrenal hormone secretion (Hexum TD and Russett LR,

1989;Lundberg JM et al., 1986). Paradoxically, in an in vitro study using primary chromaffin cultures from Y1 receptor knockout mice and transfected cells, NPY was also found to exert tonic inhibitory control of catecholamine synthesis by regulating tyrosine hydroxylase transcription (Cavadas C et al.,

2006).

38 TABLE 2: PHYSIOLOGICAL ROLE OF NPY IN THE PERIPHERY: GENETIC STUDIES

++ = overexpresses NPY; – = knock out ; ↑ = increase ; ↓ = decrease

39 TABLE 3: PHYSIOLOGICAL ROLE OF NPY IN THE PERIPHERY: PHARMACOLOGICAL STUDIES

↑ = increase ; ↓ = decrease

40 1.62 NPY in the central nervous system

NPY is widely expressed in the central nervous system (CNS) and participates in a broad

spectrum of central activities including energy homeostasis, learning and memory, and regulation and

expression of anxiety and anxiety-related behaviors. Tables 4 and 5 outline the genetic and

pharmacological studies on NPY functions in the central nervous system.

NPY is well studied with respect to energy homeostasis. Administered

intracerebroventricularly or directly into the hypothalamus, NPY strongly induces feeding (Stanley BG

et al., 1985) and chronic administration induces obesity (Zarjevski N et al., 1993). However focal

administration of NPY into the amygdala (Heilig et al., 1993;Primeaux SD et al., 2005;Sajdyk TJ et al.,

2008) or hippocampus (Sorensen AT et al., 2008) do not induce feeding or weight gain. Energy

balance is regulated by complex interactions of a large number of peptides and hormones and multiple

regulatory loops. NPY knockout animals grow and develop normally (Erickson JC et al., 1996) and

NPY transgenics do not develop an obese phenotype (Thiele TE et al., 1998), indicating compensatory

mechanisms for body weight regulation. Schwartz et al, (2000) provide and excellent review of the

central nervous system pathways controlling food intake including NPY (Schwartz MW et al., 2000).

NPY is found in a number of regions controlling the acquisition and expression of fear and anxiety.

Figure 2, from a review by Kask (2002), is a simplified schematic of the principal regions in the

circuitry of fear and anxiety (Kask A et al., 2002). Areas in which NPY has been shown to regulate

anxiety and anxiety behaviors are shaded.

41

Figure 3: Neuropeptide Y in limbic and catecholaminergic sructures has regulatory action on cognitive, behavioral, neuroendocrine and sympathetic response to stress. Abbreviations: HCF=hippocampal formation, dPAG= dorsal periaqueductal gray, vlPAG=ventrolateral periaqueductal gray, LC=locus coerulues, Pb= parabrachial nucleus

42

TABLE 4: PHYSIOLOGICAL ROLE OF NPY IN THE BRAIN: GENETIC STUDIES

++ = overexpresses NPY; – = knocked out ; ↑ = increase ; ↓ = decrease

43

TABLE 5: PHYSIOLOGICAL ROLE OF NPY IN THE BRAIN: PHARMACOLOGICAL STUDIES

↑ = increase ; ↓ = decrease

44

1.63 NPY in the amygdala

Due to its’ anxiolytic properties, there are numerous studies on NPY function in the amygdala

(reviewed in (Heilig M, 2004)). Infusion of NPY into the amygdala decreases anxiety-like behaviors

while antagonists to the NPY-Y1 receptor block the effects of exogenous NPY (Heilig et al.,

1993;Kask A et al., 2002;Primeaux SD et al., 2005;Sajdyk TJ et al., 2004;Sajdyk TJ et al., 2006;Sajdyk

TJ et al., 2008). The behavioral effects of NPY appear to be mediated primarily through the basolateral

and central amygdala, as NPY injected into the medial amygdala has no effect on aggression (Katoaka

Y et al., 1987) or arousal (Gutman AR et al., 2008) behaviors. NPY injected in the basolateral

amygdala before extinction training, enhanced extinction learning for both context and cue (Gutman

AR et al., 2008). Loss of NPY signaling in the amygdala may therefore be highly relevant to the

extinction deficits seen in PTSD.

NPY signaling may be particularly important in the central nucleus of the amygdala.

Corticotropin-releasing hormone from the central amygdala is a major excitatory input into the locus

coeruleus (Van Bockstaele EJ et al., 1998). NPY antagonizes CRH receptor signaling (Kash TL and

Winder DG, 2006). The interactions between these two peptides are important for calibrating

emotional state (Heilig M et al., 1994;Kask A et al., 2002;Sajdyk TJ et al., 2006) Corticotropin-

releasing hormone from the central amygdala is a major excitatory input into the locus coeruleus (Van

Bockstaele EJ et al., 1998). Loss of NPY inhibitory regulation of CRH could directly increase both

amygdalar excitability and tonic noradrenergic signaling (Curtis AL et al., 1995).

1.64 NPY in the prefrontal cortex

The role of neuropeptide Y in the prefrontal cortex has yet to be elucidated. NPY expressing

interneurons within the prefrontal cortex are almost exclusively GABA-ergic (Karagiannis A et al.,

2009), indicating negative regulatory actions on excitatory neurotransmission. Electrophysiological

45 data indicates that increases in NPY decrease excitability in rat neocortex (Bacci A et al., 2002). Loss

of NPY in the prefrontal cortex would be expected to release inhibition of PFC excitation. Increased

activity in the PFC would suppress amygdalar activity via inhibitory projections from the infralimbc

cortex (Milad MR and Quirk GJ, 2002;Quirk GJ et al., 2003). Increased activity would also suppress

HPA-axis activity via inhibitory projections from the prelimbic cortex to the bed nucleus of the stria

terminalis and from there to the paraventricular nucleus (Radley JJ et al., 2009).

1.65 NPY in the hippocampus

Hippocampal NPY also has anxiolytic effects, as well as effects on learning and memory.

Transgenic rats over-expressing NPY in the hippocampus are resistant to anxiogenic stress (Thorsell A

et al., 2000). Intra-hippocampal injection of NPY reversed the behavioral effects of 3 weeks of chronic

mild stress (Luo DD et al., 2008), indicating that the anxious and depressed-like behavior exhibited

following CMS are due, in part, to loss of NPY signaling.

NPY inhibits release of the excitatory transmitter glutamate from hippocampal neurons both in

in vitro slice models (Whittaker E et al., 1999) and in vivo (Sorensen AT et al., 2008). Inhibition of

glutamate signaling attenuates long-term potentiation, a necessary component of learning (Sorensen

AT et al., 2008;Whittaker E et al., 1999). Predictably, NPY transgenic rats show mild impairments in

spatial learning (Thorsell A et al., 2000).

1.66 NPY in the locus coeruleus and nucleus of the solitary tract

Neuropeptide Y co-localizes with noradrenergic cells in both the locus coeruleus and nucleus

of the solitary tract (Everitt BJ et al., 1984). Neuopeptide Y injected into the area of the locus

coeruleus has anxiolytic effects (Kask A et al., 1998). While this may be an indirect effect of NPY on

the NE system through CRH signaling, the mechanism is not yet known. NPY in the NTS contributes

to cardiovascular and metabolic regulation (Diaz-Cabiale Z et al., 2007;Schwartz MW et al., 2000).

46 More recently, a mouse over-expressing neuropeptide Y behind the dopamine-β-hydroxylase

(DBH) promoter was described (Ruohonen ST et al., 2009;Ruohonen ST et al., 2008). DBH catalyzes

the conversion of dopamine to norepinephrine and is expressed exclusively in noradrenergic and

adrenergic cells. Ruohonen et al, reported increased NPY in adrenal glands and brainstem of transgenic

mice, with no difference in NPY content between hypothalami of transgenic and wildtype mice

(Ruohonen ST et al., 2008). Peak plasma corticosterone following restraint or cold-stress did not differ

between transgenic and wild-type animals (Ruohonen ST et al., 2009) Male transgenic animals were

reported to have a moderate reduction in anxiety-like behavior in the elevated plus maze and light-dark

box; no behavioral data was reported for female animals (Ruohonen ST et al., 2009). As both NTS

and LC excitation facilitate memory consolidation is will be interesting when a more detailed

behavioral phenotype is reported for these animals (Lemon N et al., 2009;Miyashita T and Williams

CL, 2002).

1.67 Effects of stress on neuropeptide Y expression

The effects of stress on NPY expression are complex. Following acute stress, there is a

transient depletion of NPY in the amygdala, presumably due to release of intracellular NPY into

circulation (Thorsell A et al., 1998). The effects of chronic stress on NPY expression are more

complex. Chronic homotypic stress is reported to increase NPY levels (Makino S et al., 2000;Thorsell

A et al., 1999;Thorsell A et al., 2006). This may be part of the process of stressor habituation or may

parallel behavioral adaptations to stress and development of coping mechanisms. On the other hand,

chronic heterotypic stress decreases NPY-imunoreactivity in multiple brain regions (Kim H et al.,

2003;Sergeyev V et al., 2005). The maternal separation model of early life stress is reported to

decrease NPY levels into adulthood (Jimenez-Vasquez PA et al., 2001;Park HJ et al., 2005).

47 Repeat exposure, chronicity and unpredictability are key features of combat stress and likely

contribute to the elevated risk for severe refractory PTSD in combat experienced veterans (Kaysen D

et al., 2009;Prigerson HG et al., 2001). Chronic variable stress (CVS) is a heterotypic chronic stress

protocol used in rodents to test the effects of sustained, non-habituating stress. The chronic variable

stress model incorporates multiple, discrete, and unpredictable events within the traumatization period

to simulate chronic trauma as described by Kaysen (Kaysen D et al., 2009). Stressors such as cold

swim and hypoxia represent an event involving actual threat of serious injury or threat to physical

integrity”: a criterion for a traumatic event. The CVS model of multiple single-unit traumas has face

validity in modeling the primary characteristics of combat stress associated with the development of

PTSD pathophysiology.

1.68 Hypothesis

The purpose of these studies is to understand the pathological consequences of long-term stress

using chronic variable stress to induce physiological and behavioral changes consistent with traumatic

experience. The effects of chronic stress may include enhancement of fear memory consolidation and

impaired extinction, social withdrawal or aggression, and signs of arousal and sensitized sympathetic

response.

There is considerable evidence suggesting that neuropeptide Y is a stress resilience/resistance

factor. NPY is expressed in all PTSD-relevant brain regions, low endogenous NPY expression is

associated with higher trait anxiety and amygdalar activity while increased NPY expression promotes

stress resilience in rats (Thorsell A et al., 2000;Zhou Z et al., 2008). However, these are correlative

studies and fail to directly address the effects of traumatic stress on the NPY system. Therefore,

additional studies in this dissertation tested whether NPY is dysregulated in PTSD relevant brain

48 regions, particularly the amygdala, prefrontal cortex, hippocampus and noradrenergic brainstem nuclei following chronic variable stress and recovery. Finally, immunohistochemistry for tyrosine hydoxylase, the rate-limiting enzyme in catecholamine production was performed to evaluate potential dysregulaion of chatechoaminergic systems mediating arousal.

49

The Chronic Variable Stress-Recovery paradigm as a potential model for posttraumatic

stress-like behaviors

50 Emergent and persistent behavioral changes in the chronic variable stress-recovery model

Emergence of PTSD-like behaviors was examined in a rat model of chronic variable stress. The

CVS model is based on deficits arising after repeated and unpredictable exposure to variable stressors and has face validity to repetitive trauma exposure such as that found in combat operations. In previous work, there was a delayed emergence of neuroendocrine deficits in rats during recovery from CVS.

The CVS-recovery model was not investigated for the emergence of behaviors pertinent to PTSD-like pathophysiology. The current study tested whether CVS exposure invokes expression of potentiated fear memories, social or generalized anxiety, dysregulation of HPA function and enhanced sympathetic reactivity.

2.0 The CVS model

The animals used in these experiments were male Long-Evans rats (Harlan, Indianapolis, IN).

Subjects were randomly assigned to weight matched control and chronic stress groups. The chronic variable stress procedure was a modification of that described in Ostrander, et al (Ostrander MM et al,

2006). Experimental animals underwent 2 stressors a day, morning and afternoon, for 7 days.

Morning and afternoon stressors were administered between 0900 - 1100 h and 1400 - 1600 h, respectively. Overnight stressors began immediately after cessation of afternoon stressors and terminated at the initiation of the next day’s morning stressor.

Stressors were administered in a randomized order but the composition of stressors was identical across experiments. Stressors included i) cold swim (10m, 16-18°C), ii) warm swim (20m,

30-32°C), iii) hypoxia (30m, 8%O2), iv) 1h shaker (100rpm), v) 1h cold room and vi) 1h restraint.

Cold and warm swim consisted of trials of 5-8 animals per trial. For hypoxia, all animals were tested at the same time in hypoxia chambers divided in the center, with 5-8 animals on a side. During the shaker stress, animals were grouped 5-8 per cage in neutral cages. For the cold room animals were paired in

51 cages without bedding. For the restraint stressor, the animals’ home cages were moved out of the housing room and the animals were placed in restrainers and returned to their home cage. Figure 1 shows the distribution of stressors and temporal layout of the CVS paradigm. All stressors were administered twice during the 7 days; cold room and restraint stress were administered thrice.

Figure 1: representative temporal schematic of the CVS experiments and composition of stressors

52 2.1 Physiologic Measures

Indices of chronic stress in rodents are failure to gain weight, enlargement of adrenal glands

and thymic involution (Konkle ATM et al., 2003;Ostrander MM et al., 2006;Tache Y et al., 1978). To

evaluate the efficacy of the CVS paradigm in our hands, animals were weighed prior to CVS and again

on the day of behavioral testing. At sacrifice, adrenal glands and thymus were dissected out, cleaned of

fat and other residual tissue and weighed. Adrenal and thymus data are reported as adjusted

(normalized to body weight) data. Physiological measures were analyzed by two-way ANOVA with

Stress (Control, CVS) and Recovery Time (Early, Delayed) as between-subject factors.

Chronically stressed animals showed consistent and significant failure to gain weight both at

the early recovery and later recovery timepoints (Figure 2A). Adrenal and thymus weights were

normalized per 100mg body weight to control for initial size differences between animals. At early

recovery (16 hr), CVS animals showed both adrenal hypertrophy and thymic atrophy indicative of

chronic hypothalamo-pituitary-adrenal axis activation (Figure 2B). At the delayed recovery point (7d)

adrenal and thymus size recovered to control levels.

53 A

B

Figure 2: A. CVS animals gain less weight than controls and fail to regain body weight after seven days recovery. B. Adrenal hypertrophy and thymic involution is observed 1 day after CVS, but recovers to control levels after seven days recovery. *=p<.05

54 2.2 Fear Conditioning

The dysregulated memory model of posttraumatic stress disorder suggests that over-

consolidation of emotional/fearful memory and inadequate consolidation of extinction learning and

extinction memory underlie the symptomology of PTSD (Elzinga BM and Bremner JD, 2002).

This model is supported by imaging studies showing hyperactivity in the amygdala and altered

patterns of activity in the prefrontal cortex. Additional support for this model comes from Milad, et

al., who found deficits in extinction recall in PTSD patients (Milad MR et al., 2008). Furthermore,

these deficits were determined to be symptomatic of PTSD, as neither identical co-twins to the

PTSD patients nor combat-exposed non-PTSD controls exhibited the same defect (Milad MR et al.,

2008). To assess the impact of chronic variable stress on fear memory related behaviors, animals

were tested using a fear conditioning paradigm. This experiment tested three phases of training:

fear conditioning, extinction and post extinction recall of fear memory. All of these phases were

conducted in the same context. In the recall phase, a single shock was administered to investigate

whether re-exposure to mild trauma reminder post-extinction can produce sensitized fear responses

and recall.

Methods

Twenty male Long-Evans rats were weighed and randomly assigned to CVS and control

groups. The CVS procedure was described above. Fear memory behaviors were examined after a

seven-day recovery period. The rationale for this was that behavior with delayed emergence was more

pertinent to PTSD-like phenomenom. Animals underwent a contextual conditioning paradigm to

investigate three phases of training: fear conditioning, extinction and post extinction recall of fear

memory. All of these phases were conducted in the same context. Exposure to a post extinction re-

shock was chosen to investigate whether re-exposure to mild trauma reminder post extinction can

55 produce sensitized fear responses and recall. A schematic of the conditioning procedure is shown in

figure 3A.

On day 1 each animal was placed in the Gemini Shock apparatus (San Diego Instruments) and

acclimated to the chamber for 3 minutes. Acclimation was followed by 3 shocks of 1mA intensity, 1s

duration administered 1 minute apart. Animals were recorded for post shock freezing for 3 minutes.

The animals were placed in the chamber the next five days and recorded for 5 minutes without shocks

to measure fear conditioning and extinction. Seven days after the initial shock training, the animals

were placed in the same context and after 1 minute received a single, 1mA, 1s shock. Behavior was

recorded for 3 minutes post shock. Sensitized fear (post shock freezing after initial shocks),

conditioned fear and extinction (freezing on exposure to context with no shock) and fear memory

recall (post shock freezing after mild trauma reminder shock) were scored. Freezing was defined as

the absence of all movement except that necessary for respiration (Fanselow MS, 1980).

Results Freezing to the shock context was measured for 4 consecutive days following the initial

conditioning (Fig 3B). A significant main effect of stress [F (1,30) = 5.909; p<0.05] and time [F (3,30)

= 26.76; p<0.05] was present; no stress x time interaction was noted. Post -hoc tests revealed no

significant difference between control and CVS groups at specific days. Significantly elevated freezing

in CVS animals was observed on test day 4 (p<0.05 using unpaired t- test).

In the test of fear recall, rats with CVS experience exhibited significant potentiation of freezing

following a mild reminder shock administered in the same context following extinction (Fig 3C). Two

way analysis of variance revealed a significant main effect of stress [stress, F(1,22) = 5.924; p<0.05].

However, no significance effect of time or an interaction between stress and time were observed. Post

hoc analysis revealed significantly increased freezing in the CVS group after the reminder shock (t=

56 2.756; p<0.05). No significant difference between CVS and control groups was observed following the initial conditioning shock.

57 A

B

C

D

Figure 3: Sensitization of conditioned fear and fear memory recall as well as impaired extinction in CVS animals. (A) Fear conditioning, extinction and reinstatement training testing schedule. 7 days post CVS, rats were administered three shocks for conditioning, then retest and extinction (Test 1-Test 4). To assess fear memory recall, a single reminder shock was administered 48 hr after the last extinction test. (B) Percent freezing ± SEM per minute in groups of control and CVS rats exposed to context 24 hr after conditioning. (C) Extinction of conditioned fear in groups of control and CVS rats between Test 1 to Test 4. Mean percent freezing ± SEM over five minutes was measured. (D) Mean post shock percent freezing ± SEM following initial conditioning shocks and reminder shock in control and CVS rats. * p<0.05 versus control responses (n= 6-7 per group)

58 2.3 Elevated Plus Maze, two conditions

The EPM test was conducted based on previously reported procedures (Pellow S et al., 1985).

To study the expression of anxiety and innate fear, we assessed behavior on the EPM under two

conditions, a “low threat” minimal light setting and an “aversive” bright light setting. CVS and

controls were tested on the EPM apparatus at early (16 hr) or delayed (7 d) recovery time points after

chronic variable stress. The apparatus was comprised of a PVC maze with two open (40cm x 10cm)

and two enclosed (40cm x 10cm x 20cm) plexiglass arms. The arms radiate from a 10cm central

square. The entire apparatus is elevated 60cm off the floor. For testing, each animal was placed on the

center square of the maze facing the same open arm. The low light condition consisted of a red light

facing away from the maze. In the bright light condition, a bright lamp was positioned over the maze in

addition to the room lights.

Behavior was recorded from an overhead ceiling camera for 5 minutes. Video files were

captured and saved for later scoring. A number of behavioral measures were analyzed, including

standard measures of exploration and anxiety-like behavior as well as additional measures of fear and

arousal. Two experimenters blinded to the treatment group manually scored arm time (open and

closed) arm entries (open and closed), rearing, grooming, freezing, stretch-attend posture (SAP)/risk

assessment, and head dips. The Topscan program from CleverSys (CleverSys Inc. Reston, VA) was

used for certain endpoints such as locomotor activity.

Results

There were striking differences in behavior between the two testing conditions. Under aversive

bright light conditions there was a delayed emergence of an unusual constellation of behaviors

representing fearful arousal in CVS animals. An increase in open arm time was observed which

trended towards statistical significance (p= 0.08). This was accompanied by a significant increase in

59 freezing (p=0.049), motor activity (p=0.023) and a strong trend toward increased rearing (p=0.055) as determined by the Wilcoxon non parametric statistical analysis. Grooming was significantly increased in CVS animals at early recovery (p=0.004), however, open arm time was not significantly different between CVS and control animals at the later timepoint. In contrast to bright light conditions, testing on the EPM under low light produced increased open time times at early recovery (p<0.05), accompanied by a significant increase in grooming. Other parameters (motor activity and rearing) were not significantly different between CVS and control cohorts (data not shown). Animals did not show any freezing responses under low light conditions.

60 A

B

C

D

Figure 4: Elevated Plus-Maze testing reveals delayed expression of fear associated arousal in rats exposed to CVS. (A) EPM testing schedule schematic. Testing at early and delayed recovery was conducted under bright light or low light conditions using separate cohorts of CVS and control animals for each condition. (B) Open arm time in CVS and Control groups tested under bright (left) and low (right) light conditions. Data as shown as mean ± SEM represented as percentage of control. (C) Panel shows motor activity, freezing, grooming and rearing outcomes measured for bright light conditions in CVS and control rats at early and delayed recovery. Data are represented as mean ± SEM values * p<0.05 versus control; # trends for significance p=0.08 (open arm time) and p=0.053 (rearing) n = 10-12 per group.

61 2.4 Social Interaction

While not is a defining characteristic, posttraumatic stress disorder is associated with social

instability (Kessler RC, 2000) and diminished social functioning (Solomon Z and Mikulincer M, 2007

American Psychiatric Association, 2000). Previous studies investigating the effects of stress on social

interaction are equivocal. Social interaction following footshock or an emotional stressor (being

present during the footshocks without receiving any) was unchanged (Pijlman FTA and van Ree JM,

2002), while other studies reported reduced social interaction following a single exposure to predator

(Adamec R et al., 2007) or footshock (Louvart H et al., 2005;Siegmund A and Wotjak CT, 2007).

Methods

The Social Interaction test procedure was based on previous studies (Adamec RE et al.,

2007;Gehlert DR et al., 2005) with modifications. Seventy-five male Long-Evans rats were used for

this study. CVS and handled controls were tested at early and delayed timepoints (n=12-13/group). An

additional cohort of neutral “interactor” animals provided the social experience to CVS and control

animals. To acclimate the animals to testing procedures, the animals were brought to the testing room

and briefly handled daily during the CVS and recovery period prior to testing. Animals were

habituated to the testing arena during that time. The testing arena was a standard rat shoebox cage

without bedding. On the day prior to testing, animals were weighed and weight matched to an

interactor to within 5% of body weight. For identification, interactor animals were marked on their

white sides with a non-toxic black marker.

For the test, experimental animals were placed into the arena and the interactor animal

introduced immediately thereafter. Test cages were videotaped for 10 minutes. Two observers blinded

to the experimental conditions scored the interactions. Videos were scored for total interaction, active

62 interaction, grooming, freezing, rearing and fighting. Active interaction was defined as active engagement of the experimental animal with the conspecific, sniffing, following, climbing over or fighting.

Social interaction data did not meet the criteria for Gaussian distribution and were analyzed by non- parametric Wilcoxon Rank Sum test for treatment group differences. Statistical significance was taken as p<0.05.

Results

Exposure of CVS animals to a novel conspecific produced no significant effect on social interaction at either the early or delayed recovery times (Fig 4A). Time spent grooming and time spent fighting also did not differ between CVS and control groups (Figure 4 B and C). A significant increase in rearing frequency was found in the CVS group after seven-day recovery.

63 A

B

Figure 5: Social interaction is not affected by CVS exposure. (A) Social Interaction testing schedule schematic. (B) Panel shows active interaction, rearing, grooming and duration of fights in CVS and control rats at early and delayed recovery. Data are represented as mean ± SEM values; n = 12 animals/group, * p<0.05 versus control

64 2.5 Acoustic Startle

Exaggerated startle response is one of the most recognized features of PTSD and a diagnostic

criterion within the hyperarousal category. The cluster of symptoms including exaggerated startle,

insomnia, and hyperarousal are indicative of hyperactivity of the sympathetic nervous system. Heart

rate, blood pressure, skin conductance and eye-blink startle response have all been used in human

subjects to reflect sympathetic functioning (Orr SP et al., 1995;Orr SP et al., 2003;Orr SP et al., 1997).

Startle is involuntary muscular contractions and an increase in heart rate to unexpected tactile,

visual or acoustic stimuli. Startle is a defensive behavior and may reflect preparation for a fight or

flight response (Koch M, 1999). Identical stimuli generate a startle response in rats and humans (Koch

M, 1999). Magnitude of the startle response can be manipulated bidirectionally and varies between

individuals, suggesting contributing genetic, epigenetic or environmental factors (Koch M, 1999).

In both animals and humans acoustic startle can be potentiated by stress and by conditioned or

unconditioned aversive events (Koch M, 1999). Enhancement of acoustic startle in PTSD patients has

been demonstrated (Orr SP et al., 1995;Jovanovic T et al., 2009; Shaylev AY et al., 2000).

Additionally, two studies indicate that the enhanced sympathetic reactivity is a result of trauma

exposure and the development of PTSD rather than a preexisting feature of those who go on to develop

PTSD (Orr SP et al., 2003;Shalev AY et al., 2000).

In the first study by Orr, et al. heart rate, skin conductance and eye-blink startle in response to

sudden loud noise was measured in identical twins discordant for combat exposure-related PTSD (Orr

SP et al., 2003). Heart rate response was elevated in the twin with PTSD over the twin who had not

developed PTSD and remained significant even after adjustment for potentially confounding factors

(for example: age, medication use, affective disorders or non-PTSD anxiety disorders)

65 In a study by Shalev, et al., patients arriving at emergency rooms after psychologically traumatic events were recruited and tested for acoustic startle at 1 week, 1 month and 4 months following the event (Shalev AY et al., 2000). At 1 week, there were no differences in startle responses between study volunteers. At 1 month and 4 months, those who developed PTSD showed a significant increase in heart rate response compared to the non-PTSD participants. Both of these studies indicate that increases in startle are an acquired symptom of PTSD (Shalev AY et al., 2000).

A number of acute stress models of PTSD including predator stress models (Adamec RE et al.,

2003;Adamec RE et al., 1997;Cohen H et al., 2008), footshock models (Garrick T et al., 2001;Pynoos

RS et al., 1996) and single-prolonged stress (Khan S and Liberzon I, 2004;Kohda K et al., 2007) have demonstrated enhanced acoustic startle. Enhanced acoustic startle has been reported after both short (1 day) and longer (as long as 30 days) recovery periods (Adamec RE et al., 1997;Cohen H et al.,

2007a;Khan S and Liberzon I, 2004;Richter-Levin G, 1998). Impaired habituation to the startle stimulus has been seen in some (Adamec RE et al., 1997;Cohen H et al., 2008), but not all (Adamec

RE et al., 2006;Garrick T et al., 2001;Pulliam JVK et al., 2009) studies. Based on numerous previous studies in both human and rodent using startle as an outcome measure for hyperarousal, a rodent acoustic startle test was used to test sympathetic reactivity in the chronic variable stress-recovery model.

Methods

Forty-eight 250-275g male Long-Evans rats were used in this experiment. The animals were divided into 4 weight matched cohorts (n=12 for each): CVS animals tested either 1 day or 7 days following completion of CVS, and equivalent control groups. The CVS protocol described above was used.

66 Startle response to an unexpected acoustic stimulus was measured using the SR-LAB startle response system (San Diego Instruments, San Diego, CA). The apparatus included ventilated, soundproof chambers measuring approximately 52cm x 52cm 76cm and contained an enclosure of approximately 12.5cm diameter to keep the animal over the sensor. The enclosure was of sufficient size to restrict but not restrain the animal and allow it to turn around. The chambers were calibrated prior to testing. To further minimize any effect of measurement differences between the chambers, an equal number of control and CVS animals were tested in each chamber. The chambers and enclosures were cleaned between animals with 0.1% acetic acid.

The test consisted of 30 trials. Background noise in the chamber was maintained at 68 dB. The acoustic stimulus was a 40ms, 108 dB burst of white noise emitted at intervals determined semi- randomly by computer. Inter-stimulus intervals were between 3 and 30 seconds with a minimum step between interval lengths of 3s. Startle measurements were taken at 1ms intervals for 200ms following the startle stimulus using National Instruments data acquisition software. Startle amplitude is reported in millivolts (mV).

Data is represented as mean +/- SEM of peak startle across all 30 trials and for 5 blocks of 6 trials, to evaluate short-term habituation to the startle stimulus. To address effects of body weight variability on startle measurements values were normalized and expressed as mV/g body weight. Data was analyzed by. Two-way ANOVA with stress regimen (control or CVS) and recovery time (16h or

7d) as between subject factors.

Results

As mentioned previously, the chronic stress paradigm results in a failure of the stressed animals to gain weight and significant weight differences between the CVS animals and controls at both 16 hours and 7 days following the completion of CVS. The sensor for the startle chamber is a

67 piezoelectric accelerometer that converts applied for into electrical signals. Potentially, differences in body weight could affect measurements. Startle magnitude was normalized to body weight to minimize the weight confounds. Both normalized and unnormalized startle data showed statistically significant differences between stressed and unstressed animals.

The magnitude of the mean peak startle across the 30 trials was significantly lower in CVS animals compared to unstressed controls at the 16h early recovery time-point (figure 6). After 7 days recovery, the mean peak startle did not differ between the 2 groups (figure 6). When the data was analyzed in 5 blocks of 6 tones for short-term habituation to the stimulus, startle response in the 16 hour CVS group did not differ significantly between the first block and the last, whereas startle response in the control group was significantly greater in the first block than in the last block.

Although these data suggest an initial failure to habituate to the tone after chronic stress, the low initial startle makes the interpretation less clear. At the 7-day delayed recovery time-point, both CVS and control animals showed equivalent short-term habituation to the stimulus (Figure 6). In any event, a delayed, or prolonged enhancement in startle response induced by CVS-exposure was not evident in either the magnitude of startle of rate of habituation to the stimulus in this study.

68

A

B

Figure 6: Suppression of startle response in early recovery from CVS A: A diagram of the experimental design showing the testing days in relation to the termination of CVS.B: Mean peak startle across 30 trials at early and delayed recovery time-points. CVS animals showed a blunted startle response relative to control animals (p<.05) at early recovery. C: Mean peak startle across 5 blocks of 6 trials at early and delayed recovery time-points. The blunted response to the acoustic stimulus is due to a suppressed response in the initial block of 6 trials. CVS animals do not show further habituation across the 30 trials. Testing after a 7 day recovery showed equivalent startle amplitude across all 5 blocks of 6 trials, and equivalent short-term extinction to the stimulus. *=p<.05 n=12 per group

69

2.6 Hypothalamic-pituitary-adrenal (HPA) axis response to acute stress

Many (Yehuda R et al., 1990;Yehuda R et al., 1996), but not all (Metzger LJ et al., 2008)

studies of HPA-axis function in PTSD report low basal cortisol and hyper-suppression of the HPA

response to pharmacological challenge (Liberzon I et al., 1999a;Yehuda R et al., 2003). However, an

increase in salivary cortisol is reported in anticipation of and during stressful events in PTSD patients

over controls (Elzinga B et al., 2003;Liberzon I et al., 1999a). Elevated levels of cortisol are reported

in the cerebrospinal fluid of PTSD patients as well (Baker DG et al., 2005).

Previous studies in animal models of PTSD report both sensitized and blunted (Adamec RE and

Shallow T, 1993;Cohen H et al., 2007b) HPA-axis response to acute challenge. To investigate whether

the sensitivity of neuroendocrine responses are altered by CVS trauma, plasma corticosterone

following an acute stressor was analyzed at early and delayed recovery timepoints.

Methods

Following the acoustic startle testing animals were returned to their home cage and moved to a

separate room where peripheral blood was collected in unrestrained animals at 30 minutes, 60 minutes

and 120 minutes from the first acoustic stimulus. These timepoints were selected to measure peak or

near peak levels of corticosterone (30 minutes) as well as, the termination of the response by feedback

inhibition. Plasma corticosterone was measured using the ImmuChem Double Antibody Corticosterone

125I RIA kit (MP Biomedicals, Orangeburg, NY) according to the manufacturer’s protocol.

Corticosterone concentration was calculated using AssayZap software (Biosoft, Cambridge, UK).

Time course of corticosterone responses were analyzed by repeated measures two-way ANOVA with

stress as between subject factor and time as a within-subject factor. Statistical significance was taken

as p<0.05

70 Results

Following the novel acoustic stimulus, peak corticosterone levels were significantly elevated in the CVS animals over controls at both the initial 16 hour timepoint and after 7 day recovery. This agrees with previous reports of facilitated HPA-axis response to a heterotypic stressor after chronic stress (Bhatanger S and Dallman M, 1998;Dal Zotto S et al. 2000,Choi DC et al., 2008). HPA-axis sensitization persisted into recovery, also in agreement with previous work (Servatius RJ et al.

1994;Weinberg DH et al., 2009). Feedback inhibition of the HPA-axis did not differ between stressed and unstressed animals at either timepoint indicating normal termination of the HPA-axis response after CVS. These data are in contrast to Ostrander, et al. who found blunting of the HPA-axis response after 7 day recovery from a nearly identical CVS regimen (Ostrander MM et al, 2006). However, the

Ostrander study used the Sprague-Dawley rat strain, while these studies used the Long-Evans strain and the discrepant results may be due to genetic differences in stress sensitivities between strains.

71

A

B

Figure 7: Rats exposed to chronic variable stress exhibit sensitized plasma corticosterone response to a novel acute acoustic stressor. (A) Acute stress response testing schedule schematic (B) Plasma corticosterone levels at 30, 60 and 120 min after initiation of stressor at early (left panel) or delayed (right panel) recovery post CVS. Values represent mean ± SEM; n=10-12 animals per group, * p< 0.05 versus control group

72 2.7 Discussion

Chronic variable stress (CVS) induces physiological changes

As expected, Chronic Variable Stress resulted in a significant failure to gain weight in stressed

animals. Even after a recovery period, stressed animals failed to gain weight to match controls. Failure

to gain weight was highly replicable as it occurred in all experiments and is consistent with other

reports in stress literature (Ostrander MM et al., 2006;Weinberg DH et al., 2009). In this study, there

was a significant increase in adrenal size, indicative of chronic stimulation by ACTH. This is also

reported in other studies of HPA-axis activation (Blanchard RJ et al., 1998;Konkle ATM et al.,

2003;Ostrander MM et al., 2006;Weinberg DH et al., 2009). Elevated levels of circulating

glucocorticoids are associated with reductions in thymus size. Changes in thymus size are less

consistent in the literature, but thymic atrophy has been previously reported in the Long-Evans strain

(Weinberg DH et al., 2009). Thymus and adrenal size normalized after recovery; presumably because

HPA-axis stimulation decreased after the stressors ended. Taken together, these data indicate that the

CVS paradigm was highly stressful and relevant as a putative trauma.

CVS impairs fear memory extinction and potentiates fear recall

The data on fear conditioning, extinction and reminder shock-induced reinstatement of

extinguished fear revealed significantly sensitized fear responses in animals recovering from CVS.

This expanded model of fear learning and reinstatement is relevant in screening PTSD-like behaviors.

PTSD has been described as a disorder of impaired learning and processing of fear memories.

Furthermore, the reinstatement by reminder shock was added to simulate re-exposure to unconditioned

stimuli post extinction, which is pertinent to chronic traumatization situations.

73 Extinction trials indicated an overall compromised extinction in the CVS group, although significant differences were only evident during late phase. A significant potentiation of fear to a low intensity reminder shock was observed in the CVS group. Since the reminder shock followed extinction training, it is possible that the exaggerated fear reaction to reminder is a result of a deficit in extinction retention, a phenomenon reported in PTSD patients (Milad MR et al., 2008). Significantly lower freezing in unstressed control group after reminder shock may indicate a recall of extinction learning, resulting in a desensitized fear response relative to the initial unconditioned response.

Impaired recall of extinction of conditioned fear in chronically stressed animals has also been reported in previous studies (Garcia R et al., 2007;Miracle AD et al., 2006). Post initial shock freezing, a measure of unconditioned fear, was similar in both groups. However, reinstated fear following reminder shock was enhanced only in the CVS group. The data are in agreement with previous work on potentiation of sensitized response to reminders of shock environment (Louvart H et al., 2005). This pattern of response may explain why mild stressors cause reactions more appropriate to the original traumatic stressor in PTSD individuals. Although fear behaviors were not measured at early 16h recovery point (due to practical limitations), it is important to note that CVS induced fear memory deficits are observed after 7 days of recovery. Thus, the CVS model has utility for investigating traumatization-induced emergence of impaired fear extinction, fear reinstatement and recall.

CVS exposure induces delayed expression of fearful arousal

Context and environment may play an important role in the expression of sensitized behavior.

The aversiveness of the EPM experience was manipulated by testing under both standard low light and more stressful bright light conditions. Increased time spent in open arms accompanied by increased freezing, motor activity and rearing was observed under bright light testing. This unusual constellation

74 of behaviors may represent fearful arousal, and is different from the non-specific CVS induced hyperlocomotion described previously (Strekalova T et al., 2005). Hyperaroused behavior was not evident early after CVS in this case, whereas non-specific hyperlocomotion was observed immediately following CVS cessation in other studies (D'Aquila PS et al., 1994;McEuen JG et al., 2008;Strekalova

T et al., 2005).

Furthermore, freezing behavior significantly increased in CVS animals at delayed recovery, which suggests aroused behavior is accompanied by fear rather than reduced anxiety, as concluded in other reports (D'Aquila PS et al., 1994). Increased freezing was also accompanied by an increase in rearing behavior. Taken as a whole, this collection of behaviors in consistent with an emergent reduction in arousal threshold.

Under low light illumination, open arm time in the CVS animals was increased compared to controls when tested at early recovery. This effect did not persist at delayed recovery and is therefore not pertinent to posttraumatic phenomenon. In agreement with our observation, chronic variable stress induced increases in open arm exploration are reported during (D'Aquila PS et al., 1994) and immediately following CVS (McEuen JG et al., 2008;Strekalova T et al., 2005).

Startle response is blunted in early recovery from CVS

Based on previous findings of exaggerated startle response in stressed animals, it was anticipated that the startle response would be potentiated by chronic stress and that potentiation would be maintained or enhanced in the later test time point. A number of possible reasons may explain why enhanced startle response was not detected in this experiment. In human studies, increase in heart rate following noise stimulus has been the most consistent physiologic finding, although startle response

75 has also been significant in many of studies (Orr SP et al., 1995a;Orr SP et al., 1997a). Potentially,

CVS alters heart rate responses that were not an outcome measure in this study.

Chronic stress induced effects on cardiovascular responses are supported by Chalmers et al, who reported dramatically altered heart-rate response to acoustic startle (Chalmers DV et al., 1974).

Heart rate response in controls showed the anticipated rapid increase in heart rate and gradual decline in response over multiple trials (Chalmers DV et al., 1974). Pre-shocked animals showed a rapid deceleration in heart rate over the initial tones, followed by a steady increase over the remainder of the test that was significantly elevated over controls by the 8th trial (Chalmers DV et al., 1974). This same study reported no significant changes in startle amplitude of pre-shocked animals over controls and a decrease in inter-trial activity, suggestive of increased freezing.

A second study also using footshock stress over 5 consecutive days showed an inhibition of startle in shocked animals and no change in startle amplitude between the first block of 5 tones and the last block of 5 tones (Pijlman FTA et al., 2003). In both of these studies, as in ours, the animals were not habituated to the chamber prior to the day of testing.

Rat strains vary physiologically and behaviorally in their response to stress (Conti LH and

Printz MP, 2003;Faraday MM, 2002). In the Long-Evans strain, Faraday reported no differences in acoustic startle amplitude following chronic restraint stress (Faraday MM et al., 1999;Faraday MM,

2002). However, other studies have reported enhanced acoustic startle following social defeat (Pulliam

JVK et al., 2009), and predator stress (Adamec RE et al., 1999). A confounding factor in previous work may be the measurement of startle in animals previously tested for other behavioral outcomes.

This calls into question whether the increase in startle amplitude was a function of the predator exposure, or an accumulation of anxiety from multiple prior tests conducted in a relatively short period of time (Adamec RE et al., 1999). Given the relatively few studies on acoustic startle using the Long-

76 Evans strain, and inconsistent results in those studies, it is possible that other measures of sympathetic activation, such as heart rate response, may be a more appropriate outcome measure in the Long-Evans strain.

Persistent sensitization of Hypothalamic Pituitary Adrenotropic responses during recovery from CVS

Studies of basal cortisol levels in PTSD are inconsistent (reviewed in (de Kloet CS et al.,

2006;Orr SP et al., 1995)). Discrepancies may be a result of differences in sample collection methods

(e.g. plasma vs saliva), choice of control (e.g. traumatized non-PTSD or untraumatized), gender of the sample population, and time of day for cortisol measurement. Due to the lack of consensus in methodology and results in previous work, there is no definitive determination of dysregulation in basal HPA-axis activity. Fewer studies have looked at the HPA-axis response following an acute stressor; however both trauma reminders (Elzinga B et al., 2003) and non-trauma related stressors elevated plasma cortisol levels higher in PTSD patients than in traumatized-non-PTSD controls

(Bremner JD et al., 2003a;Elzinga B et al., 2003)

Induction of the HPA-axis was investigated via measurement of circulating corticosterone released following an acute stressor at early and delayed recovery. Rats exposed to CVS had significantly elevated CORT levels 30 min following an acoustic stressor at both early and delayed recovery. This observation is in agreement with previous studies with other chronic stress paradigms

(Bhatnagar S and Dallman M, 1998;Ulrich-Lai YM et al., 2007), supporting the hypothesis that a previous exposure to chronic stress enhances the potential capability of HPA axis to respond to further challenges (Armario A, 2006). Sensitization was not dependent on the nature of acute stressor since exposure to novel environment also led to HPA sensitization in animals exposed to CVS.

This study did not replicate the induction of HPA axis hypoactivity reported in a previous study using a similar CVS paradigm (Ostrander MM et al., 2006). These differences may be attributed to

77 strain differences between the two investigations; the current study used Long Evans while the previous study used Sprague Dawley rats. Adamec reported similar results in a study measuring CORT in the Long-Evans and Wistar strains following predator exposure; while Wistar rats showed a blunted

CORT response seven days after predator exposure, the predator-exposed Long-Evans rats had higher basal and peak CORT levels than unexposed Long-Evans rats after seven days (Adamec RE,

1997;Ostrander MM et al., 2006). The finding of elevated corticosterone levels in CVS rats following acute stress is consistent with previous findings in PTSD patients who exhibit significantly elevated cortisol levels in situations provoking anticipatory anxiety as well as on exposure to trauma-related stimuli (Bremner JD et al., 2003a;Elzinga B et al., 2003).

78

Effects of CVS on NPY expression

79 3.0 The effects of chronic variable stress on NPY peptide in select brain regions

NPY has been proposed as a putative resiliency peptide. Following maternal separation, a

model of early life stress, NPY was depleted into adulthood (Jimenez-Vasquez PA et al., 2001;Park HJ

et al., 2005). In humans, trauma exposure is reported to decrease basal plasma NPY levels (Morgan III

CA et al., 2003). However, successful recovery from PTSD is associated with higher levels of plasma

NPY (Yehuda R et al., 2006a). Furthermore, NPY levels were found to correlate to the degree of

symptom improvement (Yehuda R et al., 2006a). NPY is known to antagonize the effects of CRH,

acting directly in limbic forebrain structures to help regulate emotional state and in brainstem

structures to regulate neuroendocrine and neurotransmitter systems (Sajdyk TJ et al., 2004).

Importantly, NPY is significantly reduced in the cerebrospinal fluid of combat-PTSD volunteers (Sah

R et al., 2009).

We hypothesized that CVS induced dysregulation of NPY may be associated with fear memory

and emotional arousal related behaviors observed in these behavioral studies. Assessment of regional

NPY expression was performed following CVS using ELISA and immunohistochemistry.

Measurements were performed during early and delayed recovery to investigate whether persistent

dysregulation of NPY accompanied CVS associated deficits.

As previously described, NPY regulates excitatory neurotransmission in a number of PTSD-

relevant brain regions. NPY also antagonizes the effects of CRH, indirectly regulating both the HPA-

axis and noradrenergic systems. NPY is known to co-localize with cathecholaminergic neurons in the

brainstem and affects both behavior and the autonomic nervous acting within brainstem nuclei (Everitt

BJ et al., 1984;Thiele TE et al., 2000)

Tyrosine-hydroxylase (TH) is the rate-limiting enzyme in the production of catecholamines.

TH is routinely used as a marker of catecholaminergic cells. TH mRNA in the locus coeruleus is

80 increased following chronic social stress (Everitt BJ et al., 1984;Watanabe Y et al., 1995), chronic

isolation (Angulo JA et al., 1991) and chronic immobilization (Makino S et al., 2002;Rusnak M et al.,

1998). In rats, chronic stress increased TH-immunoreactivity in prefrontal cortex and LC (Miner LH et

al., 2006;Watanabe Y et al., 1995). Very few studies included recovery periods longer than 24 hours.

However, TH-immunoreactivity increased in amygdala of hamsters following 14 days of social stress

and remained elevated for at least four days (Wommack JC and Delville Y, 2002). In rats, TH mRNA

in the LC was decreased three days after cessation of chronic mild stress (Duncko R et al., 2001).

Since catecholamines are potential modulators of fear and arousal behaviors and co-localize with NPY

in brainstem nuclei measurement of TH expression was performed in addition to NPY to evaluate

potential dysregulation of catecholaminergic systems.

Methods

ELISA for regional assessment of NPY peptide concentration in brain tissue

Animals used for NPY ELISA were sacrificed by rapid decapitation. Brains were immediately

dissected from the skull and flash frozen in isopentane on dry ice. The brains were stored at -80°C;

once frozen. The brains were not allowed to thaw until transfer into acid for extraction of the NPY

peptide. For this experiment, the regions of interest were the amygdala, prefrontal cortex and

hippocampus. To dissect out amygdala and hippocampus from the brain, two cuts were made on a

cryostat to isolate a 1.5mm slice from approximately bregma –2.12 to bregma –3.6. Once dissected

from the brain slice, regions were stored at -80°C until extraction.

Amygdala and prefrontal cortices were homogenized in .2ml of .2M HCl and hippocampi were

homogenized in .3ml of .2ml HCl. Ten microliter aliquots were removed for later analysis of total

protein concentrations. The homogenates were boiled for 5 minutes and cooled on ice. Remaining

81 supernatants were then lysophilized overnight in a speed vac to ensure complete drying. Dried extracts were stored at -80°C.

Following protein extraction, NPY ELISA was performed on frozen samples using a peptide enzyme immunoassay (Peninsula Laboratories, San Carlos, CA) according to the manufacturer’s protocol. Peptide concentration was determined from plotting optical density of unknown samples against a 10 point standard curve. Total protein was determined by Bradford protein assay. Six samples from each group, CVS and control, were tested at both early and delayed recovery times. ELISA data is expressed as nanograms of NPY per milligram total protein.

Subjects from the CVS-recovery-fear conditioning and extinction experiment were used for immunohistochemistry. One hour after completion of the re-test for contextual fear reactivation, animals were perfused transcardially with 3.7% formaldehyde. The brains were removed and post- fixed overnight in 3.7% formaldehyde, then transferred to a 30% sucrose solution. Tissue was sectioned on a freezing stage microtome at 20um and stored in cryoprotectant at -20°C.

Immunohistochemistry was performed on free-floating sections on six control animals and seven CVS animals.

The neuropeptide Y primary antibody (Immunostar, Hudson WI) was used at a 1:3000 concentration and incubated overnight on a rocking platform at 4°C. Tyrosine Hydroxylase antibody

(Millipore, Billerica, MA) was used at a 1:1000 concentration and incubated 1 hour at room temperature then overnight on a shaker at 4°C. The secondary antibody for NPY and TH antibodies was biotinylated anti-rabbit IgG. For both antibodies, specific antibody staining was amplified using

TSA-biotin amplification regents (PerkinElmer, Boston, MA).

Images were taken at 5x magnification. Digital images were captures using an Axiocam camera and imaging software Measurements were taken between approximately bregma 3.2 to bregma 2.2 for

82 the medial prefrontal cortex, bregma -1.8 to bregma -3.6 for amygdala, bregma -9.8 to bregma -10.3

for locus coeruleus, and bregma -13.24 to bregma -14.08 for the NTS according to the Paxinos and

Watson brain atlas of the rat brain (Paxinos G and Watson C, 1998). Measurements were taken using

Axiovision 4.4 or Image J software and data is expressed as percent area stained or in densitometric

units. For percent area stained, upper and lower thresholds are defined and staining intensity within

these thresholds are counted in the region defined. Staining density above the upper threshold is

eliminated as artifact and below the lower threshold eliminated as background. Measurements in

densometric units use a similar in principal; however in these measurements backgound is taken as a

separate measurment and subtracted from the measurement in the region of interest. Densometric

measurements were used for the medial amygdala because the differences in staining intensity between

CVS and control animals were large enough to make defining thresholds that did not saturate in the

more intensely stained tissue nearly impossible. Side by side testing of the two methods in the

prefrontal cortex yielded equivalent results.

Statistical Analysis

Data for NPY ELISA at early and delayed recovery timepoints was conducted by 2-way

ANOVA followed by Bonferroni post-hoc test. There was no a priori hypothesis as to NPY expression

at the 15-day timepoint, therefore immunohistochemical data was analyzed control vs CVS using two-

tailed, unpaired t-test. Criterion for statistical significance was p<.05.

Results

As a predicted resilience factor, it was hypothesized that neuropeptide Y levels would be

depleted in stress-responsive brain regions following traumatic stress and depletion would persist into

recovery. Data from the NPY ELISA assay are presented in Fig. 1. Neuropeptide Y content in

83 hippocampus was significantly depleted at the early recovery time point in CVS animals. Hippocampal neuropeptide Y of CVS animals returned to control levels after a seven day recovery. Although there was a trend towards lower NPY content in early recovery amygdala of CVS animals, NPY was significantly depleted in later recovery, consistent with expectations. In the prefrontal cortex, NPY content showed a profound increase in CVS animals at the delayed recovery timepoint.

84

Figure 1: Post CVS neuropeptide Y peptide concentrations in forebrain limbic regions at early and delayed recovery using ELISA. A significant depletion of NPY is observed in the amygdala at delayed recovery, while elevated NPY concentrations are observed in the medial prefrontal cortex at delayed recovery. Early decrement in hippocampal NPY normalizes at delayed time point. *=p<.05 n=6 per group

85

3.1 Immunohistochemical analysis of subregional NPY expression

Immunohistochemistry was performed on tissue from the fear conditioning experiment. CVS

animals were allowed a seven-day recovery period and all animals, CVS and controls, underwent the

fear conditioning-extinction-reactivation procedure. Elapsed time for CVS animals from the

completion of the stress regimen was 15 days. Subnuclei within the amygdala and prefrontal cortex

were analyzed independently to gain a more refined determination of NPY expression within these

regions.

To extend the time-frame for changes in neuropeptide Y expression, and to get a more detailed

morphological picture of NPY expression with brain regions, immunohistochemistry for NPY was

performed in tissue from fear conditioning animals. Figure 2 identifies the stress responsive regions in

the rat brain and their locations. Prefrontal cortex and amygdala, the two regions showing emergent

changes in NPY peptide content, and brainstem noradrenergic nuclei that express NPY were selected

for immunohistochemical analysis.

Figure 2: Illustration of the rat brain. Location of key stress-responsive brain regions are identified.

86

Neuropeptide Y remained significantly depleted in amygdala following recovery and fear conditioning. The basolateral, central and medial nuclei of the amygdala showed substantial reductions in NPY immunoreactivity (see Figure 3). The ELISA data indicated a delayed increase in

NPY peptide in the prefrontal cortex. In this study, there were no discernable differences in NPY immunoreactivity between CVS and control animals in the infralimbic, prelimbic or anterior cingulated (see figure 4). The delayed depletion of neuropeptide Y in amygdalar nuclei controlling emotional responses and fear memory may be relevant to the development of exaggerated fear conditioning and fear recall.

87

Control CVS

B

C

Figure 3: Reduced amygdalar neuropeptide Y immunoreactivity in CVS animals after 7 day recovery - 7 day fear conditioning/extinction procedure. A: basolateral amygdala B: central amygdala C: medial amygdala *=p<.05 n=6 per group

88

Dysregulation of noradrenergic signaling is thought to underlie multiple symptom clusters in

PTSD including emotional memory dysfuntion and hyperarousal. Antagonism of CRH signaling in the brainstem may be a way which NPY can regulate tonic noradrenergic release. Although NPY injected into the brainstem and endogenous over-expression in brainstem regions both reduce anxiety-like behaviors in rodents, the effects of chronic stress on expression of NPY in the brainstem has not been examined. Measurement of NPY immunoreactivity was performed in the locus coeruleus (LC) and nucleus tractus solitarius (NTS). There were no significant differences in NPY expression between

CVS and control animals (figure 5).

89

Control CVS

Figure 4: NPY immunoreactivity in the medial prefrontal cortex does not differ between CVS and control animals after 7 day recovery and 7 day fear-conditioning/extinction procedure A: infralimbic PFC B; prelimbic PFC and C: anterior cingulate n=6 per group, except anterior cingulate control where n=5

90

Control CVS

Figure 5: NPY immunoreactivity does not differ in locus coeruleus or solitary tract between CVS and control animals after 7 day recovery-7 day fear-conditioning/extinction procedure A: locus coeruleus B: nucleus tractus solitarius n=6 per group

91 3.2 Tyrosine-hydroxylase immunohistochemistry

Long-term dysregulation of catecholaminergic systems is suspected in posttraumatic

stress disorder. Tyrosine hydroxylase (TH) is an enzyme marker of dopaminergic, noradrenergic

and adrenergic cells. Persistent up-regulation of tyrosine hydroxylase following chronic stress

and recovery would suggest long-lasting changes in excitatory transmitter systems. Although,

previous studies have found increases in TH-immunoreactivity following chronic stress lasting a

few days, more long-term changes have not been reported. To evaluate the long-term effects of

CVS on catecholamine sysnthesis, immunohistochemistry was performed on tissue from fear

conditioned animals.

No TH immunoreactivity was visible in the prefrontal cortex (data not shown). In the

amygdala, only the central nucleus showed significant TH expression. As would be expected,

both the locus coeruleus (LC) and nucleus of the solitary tract (NTS) showed high TH

expression. Analysis between CVS and control animals was performed on the central amygdala,

the LC and the NTS. No differences between CVS animals and controls were detected in either

the central amygdala or the locus coeruleus (figure 6 A and B). In the NTS, CVS animals had

significantly more TH staining than control animals (figure 6 C). The increase in TH staining

was not due to an increase in the number of neuronal cell bodies, as these did not differ

significantly between CVS and control animals (data not shown).

92

Control CVS

Figure 6: Tyrosine-hydroxylase immunoreactivity in A: central amygdala and B: locus coeruleus does not differ between CVS and control animals after 7 day recovery and 7 day fear-conditioning/extinction procedure. C: tyrosine-hydroxylase is significantly elevated in CVS animals in the nucleus tractis solitaris after 7 day recovery and 7 day fear-conditioning/extinction procedure. *=p<.05 n=6 per group

93

3.3 Discussion

Selective and persistent depletion of NPY in the amygdala

Both the NPY ELISA and immunohistochemistry data support a selective and persistent

depletion of NPY in the amygdala as a consequence of the chronic variable stress experience. This is

consistent with previous reports of depletion of neuropeptide Y following stress in rats (Jimenez-

Vasquez PA et al., 2001;Park HJ et al., 2005).

Until now, only a single study has looked NPY in an animal model of PTSD (Cui H et al.,

2008). The authors found an increase in NPY expression in the basolateral complex of the amygdala,

but a sharp decrease in the central nucleus (Cui H et al., 2008). The authors were unable to explain the

apparent contradiction to previous reports on the effects of acute stress in the basolateral amygdala and

the anxiolytic properties of NPY (Thorsell A et al., 1998) but speculated that the increase in NPY

content was related to the neuronal hypertrophy in the BLA also reported (Cui H et al., 2008). As

increases in NPY are also associated with recuperation from trauma (Yehuda R et al., 2006a) and

habituation or behavior adaptation to stress (Thorsell A et al., 1999), the authors were potentially

capturing adaptive coping responses to single-prolonged stress. Further studies in the chronic variable

stress model looking at amygdalar NPY expression would be required to determine at what point NPY

begins to recover in the amygdala or if the observed depletion is long term. At this time, it can not be

ruled out that the extended depletion of NPY in the amygdala 14 days post CVS was due in part to the

effects of the intervening fear conditiong and extinction procedure.

An apparent transient increase in NPY peptide levels in the prefrontal cortex was observed

seven days after CVS termination that was not seen in the immunohistochemical experiments

performed at 14d post CVS. There are no previous reports on the effects of acute or chronic stress on

expression of NPY in PFC. Increased NPY in the PFC may suppress neuronal excitation (Bacci A et

94 al., 2002). As the PFC-amygdalar connections are inhibitory GABA-ergic interneurons, suppressed

PFC activity is permissive to amygdalar excitation (Ghashghaei HT and Barbas H, 2002;Quirk GJ et al., 2003;Karagiannis A et al., 2009). Both decreased mPFC activation and hyperactivity in the amygdala are consistent findings in PTSD imaging (Shin LM et al., 2004a).

Increased NPY in the prefrontal cortex was not replicated in the immunohistochemistry experiment. It is possible that NPY levels in the prefrontal cortex recover in the intervening time frame to the extent that the sensitivity of the immunohistochemistry was not sufficient to detect differences or that differences in intracellular NPY content were not identifiable.

No significant changes in NPY expression were detected in the brain stem. Previous reports have found increased prepro-NPY in the locus coeruleus following chronic, but not acute, air puff stress in the Wistar-Kyoto strain (McDougall SJ et al., 2005). However restraint stress in the same strain or in the Sprague-Dawley strain did not affect prepro-NPY or the number of NPY expressing neurons in either the LC or NTS (Krukoff TL et al., 1999;Sweerts BW et al., 2001). Makino, et al reported increases in locus coeruleus mRNA following chronic treatment with the anti-depressant desipramine during and after four days of immobilization stress (Makino S et al., 2000). A noise stressor decreased expression of NPY mRNA in the locus coeruleus of the Wistar strain, an effect abolished by application of footshock prior to the noise test (de Lange RPJ et al., 2008). In total, previous reports suggest that changes in NPY expression in the locus coeruleus are dependent on strain and stressor type. Previously published work also indicates that expression of NPY is not significantly affected by either acute or chronic stressors in the NTS. From our observations it appears that brain stem NPY may not contribute to the physiological/behavioral consequences of CVS.

95

Selective up-regulation of TH in the nucleus tractus solitarius following CVS and recovery

Tyrosine hydroxylase (TH) is an enzyme marker of dopaminergic, noradrenergic and adrenergic cells. Persistent up-regulation of tyrosine hydroxylase following chronic stress and recovery would suggest long-lasting changes in excitatory transmitter systems. A significant upregulation of

TH-immunoreactivity was observed in the NTS of CVS exposed rats. This is the first observation of stress-induced enduring changes in expression of TH in the NTS. Tyrosine-hydroxylase is the initiating step for the synthesis of dopamine, norepinephrine and epinephrine. There is evidence of stress- induced regulation of TH in other brain regions. TH expression is dramatically up-regulated in the LC following both acute and chronic air-puff (McDougall SJ et al., 2005), social stress (Watanabe Y et al.,

1995), isolation stress (Angulo JA et al., 1991) and immobilization stress (Rusnak M et al., 1998), but was not tested after a recovery. Brief recovery periods suggested prolonged increases in TH following chronic social stress (Wommack JC and Delville Y, 2002). Chronic mild stress had the opposite effect, decreasing TH mRNA when tested after three days (Dunko R et al., 2001).

There is one report of suppressed expression of TH mRNA in the LC following chronic cold stress (21 days at 5°C) lasting seven days after return to room temperature, however by 14 days TH levels recover to control levels (Featherby T and Lawrence AJ, 2004). In our hands no differences in

TH expression were detected. This may be due to differences in mRNA expression in the previous study versus peptide expression in our hands. More rapid turnover of TH enzyme could result in no net effect in TH protein. Additionally, all studies reporting extended increases or decreases in TH expression used stressors of at least two weeks duration. It is possible that a one-week CVS procedure is not sufficient to induce lasting changes in TH expression, at least in the LC.

96 Further studies are required to determine whether the observed increase in TH expression indicates up-regulation of dopaminergic, noradrenergic or adrenergic systems. An alternative explanation may be that the fear conditioning procedure suppressed TH expression in the NTS of control animals, an effect abolished by the prior experience of CVS.

Visibility of TH-immunoreactive fibers in the central nucleus of the amygdala has been previously reported (Honkaniemi J, 1992). No significant differences were observed in TH immunoreactivity in the central nucleus between CVS and control animals indicating there are no long- term effects of CVS on TH expression in the amygdala.

The neurochemical data described in this section suggests that the CVS model induces highly selective deficits in neuropeptide Y expression. Furthermore, amygdalar depletion of NPY is temporally consistent with PTSD-relevent behavioral changes observed following CVS.

97

Summary and Conclusions

98 4.0 CVS as a model of chronic traumitization

Understanding the long-term effects of chronic stress on physiologic and behavioral stress

responses is essential for understanding the pathological mechanisms underlying stress-related

disorders, including PTSD. This study investigated the early and delayed expression of behavioral and

neuroendocrine outcomes of chronic variable stress exposure, a potential model for chronic

traumatization. The CVS model is unique among current rodent models of trauma, consisting of

multiple, discrete and unpredictable “traumas” within an extended traumatization period. Further

investigations probed a biological mechanism, NPY depletion, which could potentially contribute to

the physiological and behavioral effects of chronic acute stress.

The spectrum of data collected in this study point to a pronounced negative impact of CVS on

delayed processing of emotional information, manifested as impaired extinction, behavioral hyper-

responsiveness to a reminder stimulus, hyperarousal in response to intense (but not mild) emotional

stimuli, and persistent sensitization of physiological stress responses. It is important to note that certain

behavioral outcomes were unique to the CVS and recovery paradigm. In contrast to the “avoidant”

anxiety behaviors observed in acute stress associated models (reviewed in (Stam R, 2007)), we

observed the delayed emergence of “aroused” behavior associated with fear.

In these experiments rats exposed to repeated unpredictable stressors exhibit emergence of

behavioral responses consistent with enhanced fear reactivity. Importantly, behavioral impairments are

not expressed during the early “peri-traumatic” recovery time point, but rather emerge at long latencies

after cessation of stress. The data suggest that pronounced behavioral plasticity is precipitated by

prolonged exposure to unpredictable stress. Collectively, these measurements are consistent with the

constellation of symptoms associated with posttraumatic stress syndrome, such as re-experiencing, and

99 arousal to fearful contexts. The CVS-recovery paradigm may therefore be useful in simulating trauma

outcomes following chronic traumatization pertinent to repeated combat stress.

Contemporaneous with the observed behavioral changes, there was selective depletion of

neuropeptide Y in the amygdala. While further studies are needed to directly link NPY depletion with

increased arousal following chronic stress, these data strongly suggest that a persistent depletion of

NPY after chronic stress may contribute to the etiology of stress and anxiety disorders, as discussed in

the following section.

4.1 Integrating CVS induced behavioral changes with NPY dysregulation

Selective depletion of NPY in the amygdala is highly consistent with an “amygdalocentric”

model of PTSD (see figure 6). The persistence of NPY depletion in the amygdala and PTSD-relevant

behavioral changes supports CVS as a potential model of posttraumatic stress disorder. This is

supported by studies identifying reduced NPY in the cerebrospinal fluid (CSF) of PTSD patients (Sah

R et al., 2009). Loss of NPY inhibition is permissive to increased CRH release and elevated levels of

CRH have been reported in the CSF of PTSD patients (Baker DG et al., 1999). The opposing actions

of NPY and CRH, particularly in the amygdala, help determine emotional and arousal states (Sadjyk

TJ et al., 2004). Furthermore, CRH stimulates both the HPA-axis and sympathetic stress responses;

consistent with findings of increased cortisol (Baker DG et al., 2005) and norepinephrine (Geracioti TJ

et al., 2001) in the CSF of PTSD patients.

Chronic variable stress initiates a delayed and sustained depletion of neuropeptide Y in the

amygdala. The fear conditioning procedure began at a time when depletion of NPY in the amygdala of

CVS animals would be significant. Loss of NPY-mediated inhibition of excitatory transmission and

LTP, consolidation of fear-memory in the amygdala could be more profound. This is supported by the

100 fear conditioning data indicating more entrenched fearful memories. The reduced threshold for

arousal-like behavior on the EPM also coincided with depletion of amygdalar NPY. Future studies are

needed to determine whether NPY supplementation in the amygdala can alleviate the effects of CVS

on fear memory consolidation and arousal behavior.

ELISA data indicated a dramatic increase in NPY content in the PFC in CVS animals at seven

days post-CVS. This finding may be of relevance to the phenomenon of extinction and recall, given

that NPY may promote inhibitory tone in the PFC. Although differences were not detected after the

fear conditioning-extinction-reactivation procedure, elevated NPY in the PFC during the extinction

process on days 9-12 post-CVS could impair consolidation and later recall of extinction learning. The

contributions of prefrontal cortical NPY to the crosstalk between the PFC and amygdala have not yet

been studied and may provide new information on the reciprocal influences between cognition and

emotion.

4.2 Potential pharmacotherapeutic relevance of NPY for stress associated disorders

The effects of NPY and related compounds in pharmacologically validated animal models

suggest that receptors of the NPY system could be attractive targets for drug development in search of

novel treatments for stress induced anxiety disorders such as PTSD. The core symptom of PTSD is the

lack of long term adaptation to the effects of extreme traumatic stress, and it is apparent from all

evidence reported above that the NPY system plays an important role in stress responsiveness,

adaptation, and resilience as well as anxiety. An attractive strategy for NPY agonist or peptide

administration is via the intra-nasal spray formulations. Further preclinical studies and human trials

are necessary to directly link the NPY system to psychopathological effects of stress pertinent to

disorders such as PTSD.

101

Figure 1: A model of NPY actions in the development of PTSD pathophysiology. Depletion of NPY in the amygdala is permissive for amygdalar hyperexcitability leading to increased emotional reactivity, enhanced acquisition of emotional memory and hyperarousal symptoms

102

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127

Appendix 1: Enhanced fear recall and emotional arousal in rats recovering from

chronic variable stress

128

Enhanced fear recall and emotional arousal in rats recovering from chronic variable stress

Jennifer McGuire1, James P. Herman1, Paul S Horn2, Floyd R. Sallee1 and Renu Sah1*

1Department of Psychiatry, 2 Department of Mathematical Sciences,

University of Cincinnati Medical Center, Cincinnati, OH 45267, USA

Running Title: Chronic Variable Stress, fear recall, emotional arousal, HPA

*Address correspondence to:

Renu Sah, Department of Psychiatry, University of Cincinnati, Genome Research Institute, 2170 East Galbraith Road Cincinnati, Ohio 45237

129 Tel: 513-558-5129 Fax: 513-558-2288 Email: [email protected]

Summary

Emergence of posttraumatic stress-like behaviors following chronic trauma is of interest given the rising prevalence of combat-related posttraumatic stress disorder (PTSD). Stress associated with combat usually involves chronic traumatization, composed of multiple, single episode events occurring in an unpredictable fashion. In this study, we investigated whether rats recovering from repeated trauma in the form of chronic variable stress (CVS) express posttraumatic stress-like behaviors and dysregulated neuroendocrine responses. Cohorts of Long Evans rats underwent a 7d CVS paradigm followed by behavioral and neuroendocrine testing during early (16 hr post CVS) and delayed (7d) recovery time points. A fear conditioning-extinction-reminder shock paradigm revealed that CVS induces exaggerated fear recall to reminder shock that is associated with impaired extinction and potentiation of conditioned fear. Rats with CVS experience also expressed a delayed expression of fearful arousal under aversive context, however, social anxiety was not affected during post-CVS recovery. Persistent sensitization of the hypothalamic-pituitary-adrenocorticotropic response to a novel acute stressor was observed in CVS exposed rats. Collectively, our data are consistent with the constellation of symptoms associated with posttraumatic stress syndrome, such as re-experiencing, and arousal to fearful contexts. The CVS-recovery paradigm may be useful to simulate trauma outcomes following chronic traumatization that is often associated with repeated combat stress.

Keywords: chronic variable stress, fear memory, arousal, anxiety, HPA, PTSD

130

Introduction

Posttraumatic stress disorder (PTSD) is a stress-linked disorder that affects a substantial proportion of the population, and is particularly prevalent in combat veterans. According to a recent study by army scientists at the Walter Reed Medical Center, PTSD was diagnosed in as many as 18 percent of

U.S. veterans from Iraq (Hoge et al., 2007). PTSD can be triggered by acute or chronic exposure to traumatic events, with intensity and duration of trauma considered important determinants in the trajectory of posttraumatic symptoms (Buydens-Branchey et al., 1990).

There are several animals models of PTSD in the literature, including acute predator exposure

(odor and physical presence of a predator), electric tails shocks, single prolonged stress, and inescapable stress, among others (reviewed in (Stam, 2007; and references within). However, efforts to develop paradigms that model chronic stress-induced emergence of posttraumatic stress-like behaviors have been limited (Servatius et al., 1995; Wakizono et al., 2007; Zoladz et al., 2008). Since combat-related PTSD usually follows exposure to chronic and unpredictable trauma, we are interested in modeling the emergence of PTSD-like behaviors following exposure to an unpredictable stress regimen (chronic variable stress-recovery) (CVS-R). The CVS-R model is based on generation of deficits arising after repeated exposure to variable stressors and is pertinent to chronic trauma exposure such as that found in combat operations. More importantly, previous work indicates a delayed emergence of neuroendocrine deficits in rats following CVS (Ostrander et al., 2006). The current study investigates whether exposure to CVS invokes the expression of potentiated fear memories, anxiety to aversive and social encounters, and dysregulation of HPA function. Our results indicate that

CVS exposure produces a delayed and selective expression of exaggerated fear memory recall and impaired extinction, as well as context-dependent enhancement of fear behaviors. These behavioral

131 sequelae are accompanied by persistent sensitization of neuroendocrine stress responses. Our data are consistent with chronic stress induction of late emerging and persistent physiological and behavioral deficits characteristic of PTSD.

Methods

Subjects

A total of 168 male Long-Evans rats (275-300g) were used for all experiments. Animals were purchased from Harlan (Indianapolis, IN) and singly housed in a climate-controlled vivarium on a 12-

12 light dark cycle (lights on 6 a.m.). Except for brief periods during the chronic stress procedure, all animals had ad libitum access to food and water. All experiments and chronic variable stress protocols were conducted during the lights-on period. All procedures involving animals were approved by the

Institutional Animal Care and Use Committee of the University of Cincinnati. To investigate the effects of CVS on specific outcomes and avoid cross sensitization and conditioning effects between different measures, separate cohorts of animals were used for each behavioral endpoint and for neuroendocrine outcomes.

Experimental Plan

Figure 1 illustrates the temporal layout of the experimental plan. After a one week acclimation period animals were either exposed to CVS or handled as controls for seven days. All testing was performed at early (16 h post CVS) and delayed (7d) recovery stages, except for fear conditioning and extinction studies where only the 7d delayed recovery time point was assessed, due to temporal overlap of 16 h extinction and 7 d conditioning experiments that needed to be performed within the same context.

132 Chronic variable stress (CVS) Paradigm

The chronic variable stress was a modification of that described previously (Ostrander et al.,

2006). Subjects were randomly assigned to handled control and chronic stress groups. CVS animals underwent twice daily, morning and afternoon exposure to alternating stressors for seven days.

Additionally, experimental animals were housed in mouse cages overnight on two occasions during this period. Morning and afternoon stressors were administered between 0900 - 1100 h and 1400 -

1600 h, respectively. Overnight stressors began immediately after cessation of afternoon stressors and terminated at the initiation of the next day’s morning stressor. Stressors were administered in a randomized order with each stressor represented an equivalent number of times except restraint and cold room exposures that were conducted thrice. Stressors included i) cold swim (10m, 16-18°C), ii) warm swim (20m, 30-32°C), iii) hypoxia (30m, 8%O2), iv) 1h shaker (100rpm), v) 1h cold room and vi) 1h restraint. Cold and warm swim consisted of trials of 5-8 animals per trial. For hypoxia, all animals were tested at the same time in hypoxia chambers divided in the center, with 5-8 animals on a side. During the shaker stress, animals were grouped 5-8 per cage in neutral cages. For the cold room animals were paired in cages without bedding. For the restraint stressor, the animals’ home cages were moved out of the housing room and the animals were placed in restrainers and returned to their home cage.

Physiological Measures

To determine the efficacy of the CVS paradigm several physiological measures were assessed.

Body weight was recorded at the initiation of the experiment prior to CVS, at the early recovery (16h) and delayed recovery (7d). Thymus and adrenal glands were removed and weighed for comparison

133 with unstressed group. Physiological measures from separate cohorts exposed to behavioral testing were not different from each other, so these data were pooled.

Fear conditioning

Animals were allowed to recover from CVS for 7d, and were exposed to fear conditioning paradigm thereafter (see Fig 2A for schematic). All animals underwent a contextual conditioning paradigm to investigate three phases of training: fear conditioning, extinction and post extinction recall of fear memory. All of these phases were conducted in the same context. Exposure to a post extinction re-shock was chosen to investigate whether re-exposure to mild trauma reminder post extinction can produce sensitized fear responses and recall, a phenomenon observed in individuals with PTSD (Milad et al., 2008). On day 1, each animal was placed in the Gemini Shock apparatus (San Diego Instruments) and acclimated to the chamber for 3 minutes, then received 3 shocks of 1mA intensity, 1s duration administered 1 minute apart. Animals were recorded for post shock freezing for 3 minutes. The animals were placed in the chamber the next five days and recorded for 5 minutes without shocks to measure fear conditioning and extinction. Seven days after the initial shock training, the animals were placed in the same context and after 1 minute received a single, 1mA, 1s shock. Behavior was recorded for 3 minutes post shock. We measured sensitized fear (post shock freezing after initial shocks), conditioned fear and extinction (freezing on exposure to context with no shock) and fear memory recall (post shock freezing after mild trauma reminder shock). Freezing was defined as the absence of all movement except that necessary for respiration (Fanselow, 1980).

Anxiety-Associated Behaviors

Elevated Plus Maze

134 The EPM test was based on previous studies (Pellow et al., 1985). To study the expression of anxiety and innate fear we assessed behavior on the EPM under two conditions, a “low threat” minimal light setting and an “aversive” bright light setting. CVS and controls were tested on the EPM apparatus at early (16 hr) or delayed (7 d) recovery time points post chronic variable stress. The apparatus comprised of a PVC maze with two open (40 x 10) and two enclosed (40 x 10 x 20). The arms radiated from a 10cm central square. The entire apparatus is elevated 60cm off the floor. For testing, each animal was placed on the center square of the maze facing the same open arm. Behavior was recorded from an overhead ceiling camera for 5 minutes. Video files were captured and saved for later scoring. A number of behavioral measures were analyzed, which included standard measures of exploration and anxiety-like behavior as well as additional measures of fear and arousal. Parameters were scored manually and using the Topscan program CleverSys (CleverSys Inc. Reston, VA) for certain endpoints such as locomotor activity. Several parameters were scored; arm time (open and closed) arm entries (open and closed), rearing, grooming, freezing, stretch-attend posture (SAP)/risk assessment, head dips, and general locomotor activity.

Social Interaction

The Social Interaction test procedure was based on previous studies (Sanders and Shekhar,

1995), with modifications. CVS and handled controls animals were tested at early and delayed recovery time intervals (n=12/group). In addition to the experimental cohorts neutral “interactor” animals were used for providing social experience to control and CVS animals. For acclimation, all the animals were brought into the testing room and handled daily during the stress and recovery period prior to testing. Handling consisted of lifting the animals and allowing them to rest on a gloved hand for several seconds. The testing arena consisted of a standard rat shoebox cage without bedding. On

135 the day before testing, all animals were weighed and experimental animals were weight matched to an interactor to within 5% of body weight. For identification purposes, the interactor animals were marked on their white sides with a non-toxic black marker. All animals were habituated individually to freely explore the arena for 10 min. The following day, experimental animals were placed in the test arena and the interactor animal was introduced immediately thereafter. Test cages were videotaped for

10 minutes. Interactions were scored by two observers blinded to experimental conditions. Active and total interaction, grooming, freezing, rearing and fighting was scored. Active interaction was determined when the experimental animal (CVS or control) was actively engaged with the interactor, sniffing, following, climbing over or fighting.

Hypothalamic Pituitary Adrenocortical Axis (HPA) response to acute stressor

Corticosterone responses were measured following a 108 dB acoustic stimulus administered at unexpected intervals, which served as an acute stressor. Separate cohorts of CVS and handled control rats were tested at early and delayed recovery time intervals (n= 12/group). Peripheral blood was collected in unrestrained animals at 30 minutes, 60 minutes and 120 minutes from the time the animals were exposed to the stimulus. These timepoints were selected to measure peak or near peak levels of corticosterone (30 minutes) as well as the termination of the response by feedback inhibition. Plasma corticosterone was measured using the ImmuChem Double Antibody Corticosterone 125I RIA kit (MP

Biomedicals, Orangeburg, NY) according to the manufacturer’s protocol. Corticosterone concentration was calculated using AssayZap software (Biosoft, Cambridge, UK).

Statistical Analysis

Data are shown as mean ± SEM and were analyzed by two-factorial ANOVA, student t test, or non parametric test where applicable. Physiological data are reported as percent changes or adjusted

136 (normalized to body weight) data. These were analyzed by two-way ANOVA with Stress (Control,

CVS) and Recovery Time (Early, Delayed) as between-subject factors. Fear conditioning data expressed as percent freezing were analyzed by two factorial ANOVA using stress and recovery time as between subject variables. EPM and social interaction data were analyzed by non parametric

Wilcoxon Rank Sum test for treatment group differences. Time course of corticosterone responses were analyzed by repeated measures two way ANOVA with stress as between subject factor and time as a within-subject factor. Statistical significance was taken as p<0.05

Results

Effects of chronic variable stress on physiological measures

The efficacy of the CVS paradigm was verified by assessment of several physiological parameters (Table 1). Body weight gain was significantly lower in CVS exposed animals compared to controls [stress, F(1, 94)= 16.12; p<0.05] during recovery. An overall effect of recovery time was found on body weights [time, F(2,94) = 44.88; p<0.05]. There was also a significant stress x time interaction

[stress x time, F(2,94) = 4.507 ; p<0.05]. Post hoc analyses revealed that the CVS animals displayed lower body weight gain at both 16 hr and 7 d recovery time points (p<0.05).

As previously noted, CVS increased adjusted adrenal weight (stress, F(1,30) = 8.85; p<0.05) and decreased thymus weight (stress, F(1,35) = 10.71; p<0.05), consistent with stress-induced adrenal hypertrophy and thymic atrophy. Significant stress by recovery time interactions were observed for both organs (adrenal: F(1,30) = 6.25; p<0.05, thymus: F(1,35) = 5.926; p<0.05). Post hoc analysis revealed that CVS-induced adrenal hypertrophy was significant at early 16h recovery (p<0.05) point but was normalized at the 7d time interval, consistent with post-stress withdrawal of HPA axis drive.

137 Sensitized fear recall and impaired fear extinction in CVS animals

A contextual fear conditioning paradigm was used to assess the impact of chronic variable stress on fear memory related behaviors (see Fig 2A for schematic). We measured post shock freezing following initial conditioning and reminder shocks to gauge sensitized fear (initial shock) and conditioned fear recall on re-exposure (reminder shock). Freezing to context was measured on five consecutive days following conditioning (Test 1-4) to assess conditioned fear (Test 1) and extinction

(Test 2-4). Conditioned fear measured 24 hr post shock (Test1) revealed significant increase in freezing in CVS animals during the first two minutes (Fig 2B), followed by similar freezing response during the following time interval. Significant effects of stress [F(1,55) =6.66; p<0.05] and time [F (4,55)

=6.37; p<0.05] were observed, with no significant stress x time interaction. Post hoc analysis revealed significantly increased freezing at the 2 minute interval (Fig 2B). To assess extinction of contextually conditioned fear, freezing time in the chamber was measured in the absence of shock for four consecutive days (Fig 2C). Two way repeated measures ANOVA revealed a significant main effect of stress [F(1,30) = 5.909; p<0.05] and time [F(3,30) = 26.76; p<0.05]; no stress x time interaction was noted.

CVS animals showed a delayed extinction profile as compared to controls however, differences in freezing between CVS and control animals reached statistical significance on Test 4 (p<0.05) as revealed by posthoc analysis. Interestingly, following extinction rats with CVS experience exhibited a sensitized response to a mild reminder shock as compared to the control group (Fig 2D). Two way analysis of variance revealed a significant main effect of stress [stress, F(1,22) = 5.924; p<0.05]; with no significant effect of time or an interaction between stress and time. Post hoc analysis revealed significantly increased freezing in the CVS group after the reminder shock as compared to the control group (t= 2.756; p<0.05). Although the intensity of reminder shock was lower than the initial shock

(single versus three shocks, respectively), the freezing response of CVS animals was the same. On the

138 other hand, control animals elicited reduced freezing to the reminder shock as compared to the initial shock, a response appropriate in magnitude to the shock intensity. No significant differences in freezing were observed between the two groups after the initial conditioning shocks.

Delayed emergence of fearful arousal in CVS animals on elevated plus maze

CVS and control animals were tested on the EPM at early and delayed recovery period (Fig

3A). Testing was performed under low and bright light conditions to investigate effects on anxiety- like behaviors in contexts that are mildly or highly aversive in nature. All indicators of anxiety and arousal were scored and assessed between groups at early and delayed recovery time points. We observed striking differences in behavioral outcomes between the two testing conditions. Under aversive bright light conditions a delayed emergence of behaviors representing fear associated arousal was observed in CVS animals with 7d recovery. As shown in Fig 3B, an increase in open arm time was observed which trended towards statistical significance (p= 0.08). This was accompanied by a significant increase in freezing (p=0.049), motor activity (p=0.023) and rearing (p=0.055) as determined by the Wilcoxon non parametric statistical analysis (see Fig 3C). Grooming was significantly increased in CVS animals at early recovery (p=0.004); however, open arm time was not significantly different between CVS and control animals at this time point.

In contrast to bright light conditions, testing on the EPM under low light produced increased open time times at early recovery (p<0.05), accompanied by a significant increase in grooming

(p<0.05, data not shown); these effects were normalized after 7d recovery. However, other parameters

(motor activity and rearing) were not significantly different between CVS and control cohorts (data not shown). Animals did not show any freezing responses under low light conditions. Risk assessment behaviors were not different between groups at either recovery time intervals (data not shown).

139

CVS does not induce anxiety associated with social interaction

Exposure of CVS animals to a novel conspecific produced no significant effect on active interaction as compared to handled control animals at either the early or delayed recovery times. CVS animals spent 95.833 ± 5.681 mean ± SEM time (s) in active social interaction while control animals spent 98.417 ± 9.77 mean ± SEM time at 16 h. After 7 d recovery, active interaction time was 105.33

± 11.772 (CVS) and 117.10 ± 6.897 (control). Two-way ANOVA revealed no significant effects of stress or recovery time or a stress x time interaction. In addition to active interaction, time spent grooming and in aggressive encounters (fighting) was assessed and found to be similar between CVS and control groups (Fig 4). Interestingly, a significant increase in rearing frequency was observed in the CVS group only at the delayed recovery interval (p= 0.032).

Novel acute stressor induced neuroendocrine responses show persistent sensitization in CVS animals during recovery

Time course of plasma CORT response to an acoustic stimulus revealed that rats exposed to

CVS exhibited a significantly sensitized peak CORT response as compared to control animals both one day and seven days after stress cessation (Fig 5B). For early post CVS recovery, two way ANOVA analysis revealed a significant main effect of time from the onset of stressor [Time, F(2, 59) = 98.22; p<0.05], with trends for effects of stress [Stress, F(1,59) = 3.581; p=0.06] and stress by time interaction

[Stress x Time, F(2,59) = 3.07; p=0.0537]. Post hoc analysis indicated significantly elevated plasma

CORT levels at the 30-min time point in CVS animals (p<0.05). CVS rats tested at 7d recovery showed a significant main effect of time [Time, F(2, 55) = 48.57; p<0.05] and a trend for significance for stress x time [Stress x Time, F(2,55) = 2.986; p=0.0587]. Post hoc analysis indicated significantly

140 elevated plasma CORT levels at the 30-min time point in CVS animals (p<0.05). The significance of peak CORT responses at 30 min was also verified by unpaired t tests. CORT responses at 60 and 120 min following stress initiation were not significantly different between control and CVS group, suggesting that stress termination was not affected by the CVS exposure.

Discussion

The primary finding of these experiments is that rats exposed to repeated unpredictable stressors exhibit emergence of behavioral responses consistent with enhanced fear reactivity.

Importantly, behavioral impairments are not expressed during the early “peri-traumatic” recovery time point, but rather emerge at long latencies after cessation of stress. The data suggest that pronounced behavioral plasticity is precipitated by prolonged exposure to unpredictable stress.

CVS impairs fear memory extinction and potentiates fear recall

Although stress-evoked potentiation of conditioned fear has been investigated by previous paradigms (Iwamoto et al., 2007;Kohda et al., 2007;Rau et al., 2005;Siegmund and Wotjak,

2007;Yamamoto et al., 2008), there has been limited work on chronic stress effects on fear extinction

(Siegmund and Wotjak, 2007;Yamamoto et al., 2008), while reinstatement of fear following extinction has not been investigated to our knowledge. We included the testing of fear reinstatement by a reminder shock to simulate re-exposure to unconditioned stimuli post extinction, which is pertinent to chronic traumatization situations. Our data on fear conditioning, extinction and reminder shock- induced reinstatement of extinguished fear revealed significantly sensitized fear responses in animals recovering from CVS. Enhancement of conditioned fear observed by us is similar to that reported by

141 acute stress paradigms testing PTSD-like behaviors (Iwamoto et al., 2007;Rau et al., 2005;Siegmund and Wotjak, 2007;Yamamoto et al., 2008). Extinction trials over the next few days indicated an overall compromised extinction in the CVS group, although significant differences were only evident during late phase. Most interestingly, significant potentiation of fear to a low intensity reminder shock was observed in the CVS group. Since reminder shock followed extinction training, it is possible that the exaggerated fear reaction in CVS rats to a reminder is a result of impaired recall of extinguished fear or to a deficit in extinction retention. In the CVS animals, impaired extinction combined with suppressed recall of extinction may have potentially led to an exaggerated response to reinstated fear, similar in magnitude to the initial unconditioned fear response. Significantly lower freezing in unstressed control group after reminder shock may indicate a recall of extinction learning resulting in a desensitized fear response as compared to the initial unconditioned response. Post initial shock freezing, a measure of unconditioned fear, was similar in both groups, however, reinstated fear following reminder shock was enhanced only in the CVS group. Interestingly, one study reported impaired recall of extinction in rats exposed to chronic homotypic stress (Miracle et al., 2006).

Collectively, our data suggest that repeated exposure to stress may influence fear regulatory pathways, especially those controlling extinction and recall. Although fear behaviors were not measured at early

16h recovery point (due to practical limitations), it is important to note that CVS induced fear memory deficits are observed after 7 days of recovery, consistent with persistent changes in fear response dispositions.

CVS exposure induces delayed expression of fearful arousal

Since context and environment may play an important role in the expression of sensitized behavior, we controlled the degree of aversiveness of the EPM experience by testing under low and

142 bright light conditions. An interesting phenotype of fear arousal was observed under bright light testing. Increased time spent in open arms was observed and was accompanied by increased freezing, motor activity and rearing. We propose that these behaviors represent fear-associated arousal, and are different from non-specific CVS induced hyperlocomotion described previously (Strekalova et al.,

2005). First, it is important to note that this phenomenon was not evident early after CVS in our case, whereas hyperlocomotion associated arousal was observed immediately following chronic stress cessation in other studies (D'Aquila et al., 1994; McEuen et al., 2008; Strekalova et al., 2005). Thus, our CVS animals tested under bright light do not exhibit non-specific hyperactivity associated with

CVS exposure. Second, increased freezing behavior in CVS animals at delayed recovery suggests that increased locomotion is accompanied by fear-related behaviors. Third, increased freezing was also accompanied by an increase in rearing behavior. Rearing frequency is a measure of the stereotypical response to a change in the environment signifying exploration and emotionality (Gironi Carnevale et al., 1990; Thiel et al., 1998). Increased rearing in rats has also been found to be associated with anxiogenic behaviors (Borta and Schwarting, 2005). Our observations of increased rearing accompanied by fear and arousal may represent a fear associated hyperaroused state in the CVS group that is evident only after 7 day recovery post CVS.

We observed a significant increase in open arm time in the CVS animals under low light illumination as compared to controls when tested at early recovery, an effect that did not persist at delayed recovery and is therefore not pertinent to posttraumatic phenomenon. In agreement with our observation, chronic variable stress induced increase in open arm exploration has been reported previously during (D'Aquila et al., 1994) and immediately following CVS (McEuen et al.,

2008;Strekalova et al., 2005).

143 Previous reports on chronic stress effects on anxiety-like behavior tested on the elevated plus maze are contradictory. Studies have reported an increased incidence of anxiety-like behavior

(reduced open arm time) (Kim et al., 2009), decreased anxiety-like behavior (increased open arm time) response (D'Aquila et al., 1994; Kompagne et al., 2008) or no effect (Matuszewich et al., 2007). The interpretation of these reports is further complicated by the duration, type of stressors (homotypic versus heterotypic), illumination intensity during testing and strain of experimental animals. Most studies on chronic variable stress exposure and anxiety were performed early after stress cessation and correspond to the early recovery timepoint (e.g., 16 h) in this study. There are limited studies on the effects of chronic stress exposure on anxiety outcomes at delayed time points following chronic unpredictable stress cessation. In agreement with our data, one study tested rats at 1, 7 and 14 days following chronic unpredictable stress on the EPM under low intensity light but found no significant effects on anxiety-like behavior (Matuszewich et al., 2007). Aversiveness of the EPM context (light) appears to be crucial for trajectory of long term outcomes of chronic stress.

We also tested the emergence of social anxiety in animals exposed to CVS. No significant differences in active or passive social interaction, grooming or aggressive encounters were observed between control and CVS animals at either early or delayed recovery. Our data are in agreement with previous studies where social interaction was unchanged following emotional stress (Pijlman and van

Ree, 2002), but contrast with other studies where reduced social interaction is reported following exposure to a single predator (Adamec et al., 2007)or footshock (Louvart et al., 2005;Siegmund and

Wotjak, 2007) exposure. It is possible that differences in duration and modality of stressor may induce different outcomes in the SI test. Presence of an interactor animal led to significant increases in rearing behavior in CVS exposed animals which was observed only at delayed recovery. Although the exact explanation for the delayed increase in rearing is unclear, the results parallel changes in rearing

144 seen on the EPM under bright illumination, and may be associated with enhanced fear-related arousal in the novel social situation. The CVS paradigm does not appear to invoke social anxiety or avoidance associated behaviors. It should be noted that reduced social functioning is often observed in PTSD

(Solomon and Mikulincer, 2007), but is not identified as a consistent feature or defining symptom of the disorder.

Persistent sensitization of Hypothalamic Pituitary Adrenotropic responses during recovery from CVS

To investigate whether the sensitivity of neuroendocrine responses are altered by CVS trauma, we analyzed CORT response to an acute stressor at early and delayed recovery. Rats exposed to CVS had significantly elevated CORT levels 30 min following an acoustic stressor at both early and delayed recovery. This observation is in agreement with previous studies with other chronic stress paradigms

(Bhatnagar and Dallman, 1998; Ulrich-Lai et al., 2007), supporting that exposure to chronic stress enhances the capacity of the HPA axis to respond to further challenges (Armario, 2006). Sensitization was not dependent on the nature of acute stressor, since exposure to novel environment also led to

HPA sensitization in animals exposed to CVS (data not shown). Our current data did not replicate the induction of HPA axis hypoactivity reported by a previous study by our group using a similar CVS paradigm (Ostrander et al., 2006). These differences may be attributed to strain differences in the subjects (the current study used Long Evans while Sprague Dawley rats were employed in the previous study). In agreement with our observations, facilitation of HPA response was also observed following acute predator stress and chronic social unstability (Zoladz et al., 2008). Notably, previous models of posttraumatic stress-like behaviors employing more acute trauma have reported sensitized (Servatius et al., 1995; Weinberg et al., 1980) as well as blunted CORT responses to acute stress (Liberzon et al.,

1997; Kohda et al., 2007). Understanding the long term effects of stress on the HPA between various studies may often be complicated by the use of homotypic versus heterotypic acute stressors, the

145 duration and intensity of initial stressor, and most importantly whether HPA response is measured during or after the acute stressor (discussed in Armario et al., 2008). The finding that elevated corticosterone responses persist well after the cessation of CVS indicates long-lasting reorganization of stress pathways. These data are consistent with findings in PTSD patients, where cortisol responses are enhanced in situations provoking anticipatory anxiety (Bremner et al., 2003) as well as trauma- related stimuli (Elzinga et al., 2003).

CVS-R paradigm as a model of chronic traumatization?

In development of models pertinent to combat stress, it is necessary to simulate the phenomenon of chronic traumatization that is associated with combat exposure (Kaysen et al., 2009).

Moreover, it is essential to include multiple, single-incident traumatic events within the traumatization period that may be experienced in an unpredictable fashion (McFarlane and de Girolamo, 1996).

Within our paradigm we included stressors such as cold swim and hypoxia that would simulate “an event that involved actual threat of serious injury or threat to physical intergrity” a criterion for a traumatic event. Previous models of posttraumatic stress-like behaviors have ranged from acute stressors to subchronic models (Stam, 2007). Acuteness or brevity of stressors has been considered as an important feature in animals models for the induction of PTSD-like patho-physiology (Yehuda and

Antelman, 1993). However, it is important to simulate the repeated exposure, chronicity, and unpredictability, all of which are relevant to combat stress. An important feature of combat stress is the unpredictability and lack of control which are major factors in the development and expression of posttraumatic stress-like behaviors (Orr et al., 1990; Solomon et al., 1989). In our model, exposure of animal to variable stressors in a repeated and unpredictable manner satisfies these criteria and induces the emergence of altered fear memory and arousal. It is important to note that certain behavioral

146 outcomes were unique to the CVS-recovery paradigm. In contrast to the “avoidant” anxiety behaviors observed in acute stress associated models (reviewed in Stam R, 2007), we observed the delayed emergence of “aroused” behavior associated with fear.

Summary and Conclusions

This study attempted to investigate the early and delayed expression of behavioral and neuroendocrine outcomes of chronic variable stress exposure, a potential model for chronic traumatization. The spectrum of data collected in this study point to a pronounced negative impact of

CVS on later processing of emotional information, manifest as impaired extinction, behavioral hyper- responsiveness to a reminder stimulus, hyperarousal in response to intense (but not mild) emotional stimuli, and persistent sensitization of physiological stress responses. Collectively, these measurements are consistent with the constellation of symptoms associated with posttraumatic stress syndrome, such as re-experiencing, and arousal to fearful contexts. We propose that the CVS-recovery paradigm may be useful to simulate trauma outcomes following chronic traumatization that is often associated with repeated combat stress.

Acknowledgements

This work was supported by NIH grant MH083213 (RS) and MH069625 (JPH). The NIMH had no further role in study design, data collection, analysis, and interpretation of data, writing of the report or the decision to submit the paper for publication. Technical assistance of Benjamin Packard and James

“Brad” Chambers is greatly appreciated. Dr. Sallee is a member of the board of directors and equity holder in P2Dinc; he also serves as a consultant to Impax Labs, Otsuka Pharmaceutical Development and Shire Pharmaceutical. Other authors have no disclosures.

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Figure Legends

Fig. 1: Schematic of experimental timeline. Rats were acclimated to the vivarium for one week after arrival. Rats were weighed and assigned to CVS or control groups. Rats were exposed to a one week

CVS paradigm or were gently handled during the period of CVS exposure. Independent cohorts of control and CVS exposed animals were tested for various behavioral tests or neuroendocrine response at either early (16 hr) or delayed (7d) post CVS recovery.

Fig 2: CVS animals express sensitization of conditioned fear and fear memory recall as well as impaired extinction. (A) Fear conditioning, extinction and reinstatement training and testing schedule.

At 7 days post CVS rats were administered three shocks for conditioning followed by measurement of conditioned fear and extinction (Test 1-Test 4). To assess fear memory recall, a single reminder shock was administered 48 hr after the last extinction test. Post shock freezing and contextual freezing in the absence of shock was measured for 5 min. (B) Percent freezing ± SEM per minute in groups of control and CVS rats exposed to context 24 hr after conditioning. (C) Extinction of conditioned fear in groups of control and CVS rats between Test 1 to Test 4. Mean percent freezing ± SEM over five minutes was measured. (D) Mean post shock percent freezing ± SEM following initial conditioning shocks and reminder shock in control and CVS rats. * p<0.05 versus control responses (n= 6-7 per group)

152 Fig 3: Elevated Plus Maze testing revealed a delayed expression of fear associated arousal in rats exposed to CVS. (A) EPM testing schedule schematic. Testing at early and delayed recovery was conducted under bright light or low light conditions using separate cohorts of CVS and control animals for each condition. (B) Open arm time in CVS and Control groups tested under bright (left) and low

(right) light conditions. Data as shown as mean ± SEM represented as percentage of control. (C)

Panel shows motor activity, freezing, grooming and rearing outcomes measured for bright light conditions in CVS and control rats at early and delayed recovery. Data are represented as mean ±

SEM values

* p<0.05 versus control; # trends for significance p=0.08 (open arm time) and p=0.053 (rearing) n = 10-12 animals/group.

Fig 4: Social interaction is not affected by CVS exposure. (A) Social Interaction testing schedule schematic. (B) Panel shows active interaction, rearing, grooming and duration of fights in CVS and control rats at early and delayed recovery. Data are represented as mean ± SEM values; n = 12 animals/group, * p<0.05 versus control

Fig 5: Rats exposed to chronic variable stress exhibit sensitized plasma corticosterone response to a novel acute acoustic stressor. (A) Acute stress response testing schedule schematic (B) Plasma corticosterone levels at 30, 60 and 120 min after initiation of stressor at early (left panel) or delayed

(right panel) recovery post CVS. Values represent mean ± SEM; n=10-12 animals per group, * p<

0.05 versus control group

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Table 1: Physiological measures during recovery from CVS

Recovery Early (16 hr) Delayed (7d)

BW change (%) Control 6.0 ± 0.74 13.65 ± 1.65 CVS 0.215 ± 0.08 * 8.40 ± 0.85 *

Thymus Wt (mg/gm BW) Control 111.23±8.6 97.42±8.40 CVS 86.9±8.40 * 96.69±9.49

Adrenal Wt (mg/gm BW) Control 17.27±0.76 16.30±1.74 CVS 19.40±1.05 * 16.45±1.49

Data represent mean ± SEM from 12-20 rats per group. Rats were exposed to CVS for 7 day or were unstressed controls. At each recovery point, body weights were determined prior to any experimental manipulation. Time points reflect the length of time after cessation of stress. * CVS is significantly different from corresponding control group (p<0.05)

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