PACAP regulation of central amygdala GABAergic synapses is altered by restraint stress
Introduction
Anxiety disorders are one of the most prevalent forms of mental illness, and manifest as excessive fear, anxiety or avoidance of perceived threats from within the individual or in the surrounding environment (Craske and Stein, 2016). Pituitary adenylate cyclase-activating polypeptide (PACAP) signaling acts as a master regulator of the brain's emotional response to anxiety and stress, and is thought to be specifically recruited after psychological stress (vs. physical stress) to activate cellular processes and circuits associated with threat exposure (Hammack and May 2015; Ramikie and Ressler, 2016).
PACAPergic signaling in the central nervous system (CNS) is primarily activated by the neuropeptide PACAP-38, which is expressed 10–100-fold more abundantly than its truncated form PACAP-27 (Arimura et al., 1991). Chronic stress induces PACAP expression in several rodent brain regions that regulate anxiety, including the bed nucleus of the stria terminalis (BNST) and the paraventricular nucleus of the hypothalamus (PVN) (Hammack et al., 2009), while intracerebroventricular infusion of PACAP-38 activates the hypothalamic pituitary adrenal (HPA) axis, and increases the stress responses and anxiety-like behavior of rats (Agarwal et al., 2005; Dore et al., 2013). PACAP-38 mainly produces these effects by binding selectively and with high affinity to its cognate receptor PAC1, though it can also bind the vasoactive intestinal polypeptide receptors 1 and 2 (VPAC1 and VPAC2) (Shivers et al., 1991). Accordingly, chronic stress and fear-conditioning paradigms induce PAC1 expression in multiple rodent brain regions, including the basolateral amygdala (BLA), BNST, medial prefrontal cortex and PVN (Andero et al., 2014; Hammack et al., 2009; Ressler et al., 2011), while PAC1 deletion (i.e. PAC1 knock-out mice) reduces startle behavior and anxiety-like responses (Otto et al., 2001a, 2001b). Of note, recent studies have linked human allelic variants and site-specific methylation of the gene encoding PAC1 (ADCYAP1R1) with an altered patient susceptibility to post-traumatic stress disorder (PTSD) (Almli et al., 2013; Ressler et al., 2011; Uddin et al., 2013), though some studies are conflicting (Chang et al., 2012).
Amygdala dysfunction is implicated in several anxiety disorders (Shin and Liberzon, 2010), with the central nucleus of the amygdala (CeA), in particular, acting as a hub for negative emotional processing (Gilpin et al., 2015). The CeA is a primarily inhibitory nucleus, with an interconnected network of γ-aminobutyric acid (GABA) interneurons and GABA projection neurons that can inhibit each other via axon collaterals (Haubensak et al., 2010; Lopez de Armentia and Sah, 2004; Marek et al., 2013; Pape and Pare, 2010); the lateral (CeL) and capsular (CeC) subdivisions of the CeA receive glutamatergic input from the BLA and lateral amygdala, and send GABAergic afferents to its medial subdivision (CeM) (Jolkkonen and Pitkanen, 1998; Pitkanen et al., 1995; Savander et al., 1995). The CeM, which also receives excitatory input from the BLA, serves as the major output nucleus of the entire amygdala complex and sends GABAergic projections to downstream effector regions that regulate stress and fear responses (e.g BNST, dorsal vagal complex of the brainstem, lateral hypothalamus, locus coeruleus and periaqueductal gray (Gilpin et al., 2015);). The CeA is innervated by PACAPergic fibers from the lateral parabrachial nucleus and dorsal vagal complex of the brainstem (Cho et al., 2012; Missig et al., 2014), and PAC1 is expressed throughout the CeA (Joo et al., 2004; Piggins et al., 1996). PACAP-38 infusion directly into the rodent CeA activates the animals’ HPA axes (Iemolo et al., 2016), increases their anxiety-like behaviors (Iemolo et al., 2016; Missig et al., 2014), shifts their stress-coping behaviors from active to passive modes (Legradi et al., 2007), and heightens their nociceptive sensitivity (Missig et al., 2014). Moreover, intra-CeA infusion of a PAC1 competitive antagonist (PACAP(6-38)) reduces anxiogenic and nociceptive responses in a model of chronic neuropathic pain (Missig et al., 2017).
Despite the clear role of CeA PACAP/PAC1 signaling in mediating anxiogenic and stress coping behaviors, its mechanisms of action within the CeM (the major output nucleus for the entire amygdala) are not well understood. Therefore, in the present study, rats were subjected to either restraint stress (a primarily psychological stressor (Gray et al., 2015)) or control conditions, and PACAPergic regulation of CeM cellular function was assessed using immunohistochemistry and whole-cell patch-clamp electrophysiology. Overall our data demonstrate that PACAP-38 acts via PAC1 to enhance inhibition within the CeM and that psychological stress can modulate this PACAPergic influence over local GABA synaptic activity.
Section snippets
Materials and methods
The Scripps Research Institute (TSRI) Institutional Animal Care and Use Committee (IACUC) and Boston University Medical Campus IACUC approved all protocols involving the use of experimental animals in this study.
Restraint stress does not alter basal GABA transmission in the rat CeM
To investigate whether psychological stress alters basal CeM activity we recorded GABAA-mediated spontaneous inhibitory postsynaptic currents (sIPSCs) in naïve rats and rats subjected to either a single (1 h) or repeated (1 h per day for 3 consecutive days) restraint stress followed by a 1 h post-stress recovery period (Fig. 1A). There were no significant differences in baseline sIPSC frequencies, amplitudes, rise times and decay times across all three animal groups (frequency: F(2, 87) = 1.30,
Discussion
PACAPergic signaling directs the brain's emotional response to psychological stress (Hammack and May 2015; Ramikie and Ressler, 2016) and, in particular, its actions in the CeA are crucial to anxiogenic and stress coping behaviors (Iemolo et al., 2016; Legradi et al., 2007; Missig et al., 2014). Therefore, we investigated the effects of restraint stress on PACAP's regulation of neuronal activity in the medial subdivision of the CeA (CeM; the major output nucleus for the entire amygdala
Conclusions
Here we report that the neuropeptide PACAP acts via its receptor PAC1 to tonically regulate GABA release in the CeM of naïve rats, demonstrating a critical role for this peptidergic system in maintaining local inhibitory control within the major output nucleus for the entire amygdala. This influence is stress-sensitive, supporting a critical role for CeA PACAP/PAC1 signaling in mediating an acute psychological stress response. Therefore, the PACAPergic system's dynamic regulation of CeA
Acknowledgements
Support for this study was provided by the National Institute on Alcohol Abuse and Alcoholism grants R01 AA021491 (MR), R01 AA015566 (MR), R01 AA006420 (MR), R01 AA025038 (VS), R01 AA024439 (VS), R37 AA017447 (MR), U01 AA013498 (MR), K99 AA025408 (FPV), and T32 AA007456 (MQS) and by the National Institute of Mental Health grant R01 MH093650 (VS), as well as the Pearson Center for Alcoholism and Addiction Research. The authors declare no conflict of interest. This is TSRI manuscript number 29710.
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