Elsevier

Neuropharmacology

Volume 160, 1 December 2019, 107761
Neuropharmacology

Role of the PACAP system of the extended amygdala in the acoustic startle response in rats

https://doi.org/10.1016/j.neuropharm.2019.107761Get rights and content

Highlights

  • PACAP into either the CeA or the BNST increases ASR in rats.

  • VIP in the same brain regions has no effect on ASR.

  • PAC1R/VPAC2R antagonist PACAP(6–38) in either CeA or BNST has no per se effect on ASR.

  • PACAP(6–38) in CeA and the BNST blocks footshock-induced sensitization of ASR.

  • Footshock stress increases PACAP, but not VIP, immunoreactivity in both CeA and BNST.

Abstract

Anxiety-related disorders are the most prevalent mental disorders in the world and they are characterized by abnormal responses to stressors. Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide highly expressed in the extended amygdala, a brain macrostructure involved in the response to threat that includes the central nucleus of the amygdala (CeA) and the bed nucleus of the stria terminalis (BNST). The aim of this series of experiments was to systematically elucidate the role of the PACAP system of the CeA and BNST under both control, unstressed conditions and after the presentation of a stressor in rats. For this purpose, we used the acoustic startle response (ASR), an unconscious response to sudden acoustic stimuli sensitive to changes in stress which can be used as an operationalization of the hypervigilance present in anxiety- and trauma-related disorders. We found that infusion of PACAP, but not the related peptide vasoactive intestinal peptide (VIP), into either the CeA or the BNST causes a dose-dependent increase in ASR. In addition, while infusion of the antagonist PACAP(6–38) into either the CeA or the BNST does not affect ASR in non-stressed conditions, it prevents the sensitization of ASR induced by an acute footshock stress. Finally, we found that footshock stress induces a significant increase in PACAP, but not VIP, levels in both of these brain areas. Altogether, these data show that the PACAP system of the extended amygdala contributes to stress-induced hyperarousal and suggest it as a potential novel target for the treatment of stress-related disorders.

Introduction

Anxiety disorders, which affect approximately 40 million people and cost $42 billion a year in the United States, represent a massive public health issue (Greenberg et al., 1999; Kessler et al., 2005). Symptoms of anxiety disorders include excessive fear of stimuli or perceived threats which can manifest with hypervigilance and hyperarousal. All the components of the stress systems respond to states of threatened homeostasis (i.e. presentation of a stressor) by mobilizing adaptive responses aimed to maintain the equilibrium and preparing us for immediate or potential harm (Chrousos and Gold, 1992; Tovote et al., 2015). However, hyperactivation of these fundamental arousal and emotional systems may be key to the pathogenesis of conditions involving abnormal responses to stressors (Tsigos and Chrousos, 2002; Duval et al., 2015).

Two major brain structures are known to heavily modulate the behavioral response to stress: the central nucleus of the amygdala (CeA) and bed nucleus of the stria terminalis (BNST) (Davis et al., 1997). These brain regions are part of the extended amygdala, an anatomical construct which regulates the emotional component of the stress response (Koob and Le Moal, 2005; Alheid and Heimer, 1988). The CeA integrates sensory information from the environment, and sends direct projections to the BNST (Alheid and Heimer, 1988; Alheid et al., 1998; Oler et al., 2017). The CeA and the BNST then project information to various effector regions, such as the hypothalamus, the cortex, and the brainstem, to trigger appropriate responses, therefore coordinating the behavioral, autonomic and endocrine response to threats (Davis, 1992; Davis and Shi, 2000; Pitkanen et al., 2000; Zarrindast et al., 2008). Notably, in a non-pathological state, extended amygdala signaling is tapered appropriately to the severity of the present threat (Mathew et al., 2008), while hyperactivity of this brain macrostructure is hypothesized to play a critical role in the pathophysiology of anxiety and depressive disorders (Etkin et al., 2009; Etkin and Wager, 2007; Shin and Liberzon, 2010; Avery et al., 2016; Fox et al., 2015).

The CeA and BNST contain several important neuropeptides and neuropeptide receptors, which modulate their activity (Alheid, 2003; Fox and Shackman, 2019). One such neuropeptide is the pituitary adenylate cyclase-activating polypeptide (PACAP), a 38-amino acid peptide belonging to the secretin/glucagon/vasoactive intestinal polypeptide (VIP) superfamily. PACAP exerts its effects mainly via its cognate receptor PAC1 (PAC1R), which binds PACAP with an affinity of 1000-fold greater than VIP. On the other hand, VIP receptors (VPAC1R and VPAC2R) bind with PACAP and VIP with equal affinities (Vaudry et al., 2009). In the brain, PACAP and PAC1R are highly expressed in the hypothalamus, the brainstem, and the extended amygdala (Joo et al., 2004; Piggins et al., 1996). Dense PACAP-immunoreactive fibers are found in the dorsolateral BNST and in the capsular and lateral parts of the CeA subdivision (Piggins et al., 1996; Hannibal, 2002). PACAP inputs to the CeA and BNST are non-local projections originating from the parabrachial nucleus (Missig et al, 2014, 2017).

Human literature has shown that a single nucleotide polymorphism (SNP) in a putative estrogen response element (ERE) within the PAC1R gene was shown to predict stress disorder (PTSD) symptom severity in a human population of highly traumatized females (Ressler et al., 2011). This SNP leads to less efficient binding of estradiol activated estrogen receptor alpha at the PAC1R gene ERE, resulting in reduced expression of PAC1R and increasing risk for PTSD (Mercer et al., 2016). Recent preclinical literature has also implicated PACAP as a strong mediator of the stress response. Knockout mice studies have shown that several effects of stress, including HPA-axis activation and anxiety-like behavior, are dependent on PACAP signaling (Hashimoto et al., 2001; Lehmann, 2013; Stroth and Eiden, 2010). Central administration of PACAP to rodents evokes a stress-like response, activates the HPA axis, and induces depressive-like behavior (Agarwal et al., 2005; Dore et al., 2013; Stroth et al., 2011; Seiglie et al., 2015). Specifically in the extended amygdala, PACAP microinfusion into the CeA produces anxiety-like behavior in exploration-based tests (Missig et al., 2014; Iemolo et al., 2016) as well as increased passive-coping in a shock-probe fear test (Legradi et al., 2007). PACAP microinfusion into the BNST also reduces exploratory behavior and increases acoustic startle reactivity (Hammack et al., 2009; Roman et al., 2014).

A behavioral test that has proven very useful in the investigation of the neural mechanisms of hypervigilance is the acoustic startle response (ASR). The ASR is a rapid reflex to an abrupt auditory stimulus that is mediated by very well defined neuronal pathways in the cochlear nucleus of the brainstem and spinal cord (Lee et al., 1996). The response to these stimuli is very well conserved across species and it consists in the rapid contraction of the facial and skeletal muscles (LeDoux, 1995). Patients with PTSD exhibit an exaggerated ASR (Butler et al., 1990; Grillon et al., 1998; Morgan et al., 1996), and in rats, ASR is a dependable measure of current emotional state and anxiety levels (Davis, 1993; Davis et al., 2010). Another advantage of using ASR is that it has a non-zero baseline, which allows bidirectional changes to be detected (Gerber et al., 2014). Importantly, drugs that are used as clinical anxiolytics, like diazepam and alprazolam, decrease startle reflex amplitude (Abduljawad et al., 1997; Rodriguez-Fornells et al., 1999), while drugs such as yohimbine promote anxiogenic-like effects and increase startle amplitude (Morgan et al., 1993). It has been shown that ASR can be increased as an unconditioned effect of an acute stressor (footshock), with potentiation occurring within minutes of administration (Davis, 1989); this sensitization is also observed following various chronic stress paradigms (Hammack et al., 2009; Gewirtz et al., 1998), and it is likely to reflect a heightened state of anxiety as a result of the exposure. Importantly, unlike fear-conditioning paradigms, classical sensitization of ASR by footshock is a non-associative form of learning (e.g. there are no cues involved), and it is therefore hypothesized to recruit different systems and circuits (Koch and Schnitzler, 1997).

While the effects of PACAP administration in the BNST on ASR have been described, whether PACAP or PAC1R antagonism within the CeA modulates ASR is unknown. In addition, whether the PACAP/PAC1R system of either the BNST or the CeA plays a role in the effects of an acute stressor has not yet been reported. The aim of the present study was, therefore, to systematically elucidate the role of the PACAP system of the CeA and the BNST in the ASR, under both unstressed conditions and following an acute stressor.

Section snippets

Subjects

Adult, male Wistar rats (Charles River, Wilmington, MA), weighing 301–325g upon arrival, were single-housed in wire-topped, plastic cages in a 12 h:12 h reverse light cycle, humidity- and temperature-controlled vivarium, with food and water available ad libitum. Experimental tests were conducted during the rats’ dark cycle. Procedures adhered to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the Principles of Laboratory Animal Care and were approved by

PACAP, but not VIP, administered into the CeA increases ASR

As shown in Fig. 1A, PACAP microinfusion into the CeA significantly affected ASR (Noise level: F(2, 20) = 35.82, p < 0.001; Dose: F(2, 20) = 3.63, p = 0.045; Noise level x Dose: F(4, 40) = 3.18, p = 0.02); post hoc analysis showed that both the 0.1 and the 0.3 μg/rat dose were effective at increasing ASR across noise intensities. Fig. 1B shows the effect of intra-CeA PACAP when the 3 noise intensities are collapsed; the highest dose of PACAP increased ASR 34.9% above vehicle.

On the other hand,

Discussion

The main findings of the present study were that PACAP microinfusion into either the CeA or the BNST increased ASR in rats, while the related peptide VIP in the same brain regions had no effect. In addition, the PAC1R/VPAC2R antagonist PACAP(6–38) into either the CeA or the BNST had no per se effect on baseline ASR, while it blocked footshock-induced sensitization of ASR. Finally, footshock stress increased PACAP levels in both the CeA and BNST, without altering VIP levels.

Rats microinfused

Funding

This publication was made possible thanks to grant numbers MH093650 (VS), AA025038 (VS), and DA030425 (PC) from the National Institute of Mental Health (NIMH), the National Institute on Alcohol and Alcoholism (NIAAA), and the National Institute on Drug Abuse, the Peter Paul Career Development Professorship (PC), and the Boston University's Undergraduate Research Opportunities Program (UROP). Its contents are solely the responsibility of the authors and do not necessarily represent the official

Acknowledgments

We thank Angela Ho, Diane Tang, and Hannah Bae for their technical help.

References (112)

  • S.E. Hammack

    Chronic stress increases pituitary adenylate cyclase-activating peptide (PACAP) and brain-derived neurotrophic factor (BDNF) mRNA expression in the bed nucleus of the stria terminalis (BNST): roles for PACAP in anxiety-like behavior

    Psychoneuroendocrinology

    (2009)
  • S.C. Heinrichs

    Brain penetrance, receptor occupancy and antistress in vivo efficacy of a small molecule corticotropin releasing factor type I receptor selective antagonist

    Neuropsychopharmacology

    (2002)
  • M. Koch et al.

    The acoustic startle response in rats--circuits mediating evocation, inhibition and potentiation

    Behav. Brain Res.

    (1997)
  • M.L. Lehmann

    PACAP-deficient mice show attenuated corticosterone secretion and fail to develop depressive behavior during chronic social defeat stress

    Psychoneuroendocrinology

    (2013)
  • Q. Liu et al.

    Pituitary adenylate cyclase-activating polypeptide: postnatal development in multiple brain stem respiratory-related nuclei in the rat

    Respir. Physiol. Neurobiol.

    (2019)
  • E.G. Meloni

    PACAP increases Arc/Arg 3.1 expression within the extended amygdala after fear conditioning in rats

    Neurobiol. Learn. Mem.

    (2019)
  • G. Missig

    Parabrachial nucleus (PBn) pituitary adenylate cyclase activating polypeptide (PACAP) signaling in the amygdala: implication for the sensory and behavioral effects of pain

    Neuropharmacology

    (2014)
  • G. Missig

    Parabrachial pituitary adenylate cyclase-activating polypeptide activation of amygdala endosomal extracellular signal-regulated kinase signaling regulates the emotional component of pain

    Biol. Psychiatry

    (2017)
  • S.D. Norrholm et al.

    Behavioral effects of local microinfusion of pituitary adenylate cyclase activating polypeptide (PACAP) into the paraventricular nucleus of the hypothalamus (PVN)

    Regul. Pept.

    (2005)
  • C.W. Roman

    PAC1 receptor antagonism in the bed nucleus of the stria terminalis (BNST) attenuates the endocrine and behavioral consequences of chronic stress

    Psychoneuroendocrinology

    (2014)
  • N. Stroth et al.

    Stress hormone synthesis in mouse hypothalamus and adrenal gland triggered by restraint is dependent on pituitary adenylate cyclase-activating polypeptide signaling

    Neuroscience

    (2010)
  • G. Telegdy et al.

    The action of pituitary adenylate cyclase activating polypeptide (PACAP) on passive avoidance learning. The role of transmitters

    Brain Res.

    (2000)
  • K.A. Abduljawad

    Effects of clonidine and diazepam on the acoustic startle response and on its inhibition by 'prepulses' in man

    J. Psychopharmacol.

    (1997)
  • S. Ahrens

    A central extended amygdala circuit that modulates anxiety

    J. Neurosci.

    (2018)
  • G.F. Alheid

    Extended amygdala and basal forebrain

    Ann. N. Y. Acad. Sci.

    (2003)
  • S.N. Avery et al.

    The human BNST: functional role in anxiety and addiction

    Neuropsychopharmacology

    (2016)
  • R.W. Butler

    Physiological evidence of exaggerated startle response in a subgroup of Vietnam veterans with combat-related PTSD

    Am. J. Psychiatry

    (1990)
  • S. Campeau et al.

    Involvement of the central nucleus and basolateral complex of the amygdala in fear conditioning measured with fear-potentiated startle in rats trained concurrently with auditory and visual conditioned stimuli

    J. Neurosci.

    (1995)
  • G.P. Chrousos et al.

    The concepts of stress and stress system disorders. Overview of physical and behavioral homeostasis

    J. Am. Med. Assoc.

    (1992)
  • S. Ciocchi

    Encoding of conditioned fear in central amygdala inhibitory circuits

    Nature

    (2010)
  • P. Cottone

    Feeding microstructure in diet-induced obesity susceptible versus resistant rats: central effects of urocortin 2

    J. Physiol.

    (2007)
  • S.E. Daniel et al.

    Stress modulation of opposing circuits in the bed nucleus of the stria terminalis

    Neuropsychopharmacology

    (2016)
  • M. Das et al.

    Hypothalamic and brainstem sources of pituitary adenylate cyclase-activating polypeptide nerve fibers innervating the hypothalamic paraventricular nucleus in the rat

    J. Comp. Neurol.

    (2007)
  • M. Davis

    Sensitization of the acoustic startle reflex by footshock

    Behav. Neurosci.

    (1989)
  • M. Davis

    The role of the amygdala in fear and anxiety

    Annu. Rev. Neurosci.

    (1992)
  • M. Davis

    Pharmacological analysis of fear-potentiated startle

    Braz. J. Med. Biol. Res.

    (1993)
  • M. Davis et al.

    Role of bed nucleus of the stria terminalis and amygdala AMPA receptors in the development and expression of context conditioning and sensitization of startle by prior shock

    Brain Struct. Funct.

    (2014)
  • M. Davis et al.

    Amygdala and bed nucleus of the stria terminalis: differential roles in fear and anxiety measured with the acoustic startle reflex

    Philos. Trans. R. Soc. Lond. B Biol. Sci.

    (1997)
  • M. Davis

    Phasic vs sustained fear in rats and humans: role of the extended amygdala in fear vs anxiety

    Neuropsychopharmacology

    (2010)
  • R. Dore

    CRF mediates the anxiogenic and anti-rewarding, but not the anorectic effects of PACAP

    Neuropsychopharmacology

    (2013)
  • E.R. Duval et al.

    Neural circuits in anxiety and stress disorders: a focused review

    Ther. Clin. Risk Manag.

    (2015)
  • S. Erb et al.

    Stress reinstates cocaine-seeking behavior after prolonged extinction and a drug-free period

    Psychopharmacology

    (1996)
  • A. Etkin et al.

    Functional neuroimaging of anxiety: a meta-analysis of emotional processing in PTSD, social anxiety disorder, and specific phobia

    Am. J. Psychiatry

    (2007)
  • A. Etkin

    Disrupted amygdalar subregion functional connectivity and evidence of a compensatory network in generalized anxiety disorder

    Arch. Gen. Psychiatr.

    (2009)
  • B. Gerber

    Pain-relief learning in flies, rats, and man: basic research and applied perspectives

    Learn. Mem.

    (2014)
  • M.A. Geyer et al.

    Measurement of startle response, prepulse inhibition, and habituation

    Curr Protoc Neurosci

    (2001)
  • P.J. Gilligan et al.

    Corticotropin releasing factor (CRF) receptor modulators: progress and opportunities for new therapeutic agents

    J. Med. Chem.

    (2000)
  • P.E. Greenberg

    The economic burden of anxiety disorders in the 1990s

    J. Clin. Psychiatry

    (1999)
  • G. Griebel

    4-(2-Chloro-4-methoxy-5-methylphenyl)-N-[(1S)-2-cyclopropyl-1-(3-fluoro-4-methylp henyl)ethyl]5-methyl-N-(2-propynyl)-1, 3-thiazol-2-amine hydrochloride (SSR125543A), a potent and selective corticotrophin-releasing factor(1) receptor antagonist. II. Characterization in rodent models of stress-related disorders

    J. Pharmacol. Exp. Ther.

    (2002)
  • C. Grillon

    Effect of darkness on acoustic startle in Vietnam veterans with PTSD

    Am. J. Psychiatry

    (1998)
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