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

https://doi.org/10.1016/j.nlm.2018.11.011Get rights and content

Highlights

  • PACAP increased activity-regulated cytoskeleton associated protein after fear conditioning.

  • Significant increases were seen in the extended amygdala but not the hippocampus.

  • Expression was co-localized with PKCdelta in the extended amygdala.

  • Freezing behavior was significantly reduced in PACAP treated rats one day after fear conditioning.

Abstract

The stress-related neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) is implicated in neuromodulation of learning and memory. PACAP can alter synaptic plasticity and has direct actions on neurons in the amygdala and hippocampus that could contribute to its acute and persistent effects on the consolidation and expression of conditioned fear. We recently demonstrated that intracerebroventricular (ICV) infusion of PACAP prior to fear conditioning (FC) results in initial amnestic-like effects followed by hyper-expression of conditioned freezing with repeated testing, and analyses of immediate-early gene c-Fos expression suggested that the central nucleus of the amygdala (CeA), but not the lateral/basolateral amygdala (LA/BLA) or hippocampus, are involved in these PACAP effects. Here, we extend that work by examining the expression of the synaptic plasticity marker activity-regulated cytoskeleton-associated protein (Arc/Arg 3.1) after PACAP administration and FC. Male Sprague-Dawley rats were implanted with cannula for ICV infusion of PACAP-38 (1.5 µg) or vehicle followed by FC and tests for conditioned freezing. One hour after FC, Arc protein expression was significantly elevated in the CeA and bed nucleus of the stria terminalis (BNST), interconnected structures that are key elements of the extended amygdala, in rats that received the combination of PACAP + FC. In contrast, Arc expression within the subdivisions of the hippocampus, or the LA/BLA, were unchanged. A subpopulation of Arc-positive cells in both the CeA and BNST also express PKCdelta, an intracellular marker that has been used to identify microcircuits that gate conditioned fear in the CeA. Consistent with our previous findings, on the following day conditioned freezing behavior was reduced in rats that had been given the combination of PACAP + FC—an amnestic-like effect—and Arc expression levels had returned to baseline. Given the established role of Arc in modifying synaptic plasticity and memory formation, our findings suggest that PACAP-induced overexpression of Arc following fear conditioning may disrupt neuroplastic changes within populations of CeA and BNST neurons normally responsible for encoding fear-related cues that, in this case, results in altered fear memory consolidation. Hence, PACAP systems may represent an axis on which stress and experience-driven neurotransmission converge to alter emotional memory, and mediate pathologies that are characteristic of psychiatric illnesses such as post-traumatic stress disorder.

Introduction

Pituitary adenylate cyclase-activating polypeptide (PACAP) belongs to the secretin/glucagon superfamily of peptides and exists in two biologically active forms (as 38- and 27-amino acid peptides) found in peripheral tissues and brain (Vaudry et al., 2009). PACAP-38 is the predominant form in the brain and shares identical amino acid sequence homology in species including mice, rats, sheep, and humans, indicating strong evolutionary conservation (Montero, Yon, Kikuyama, Dufour, & Vaudry, 2000). Although many biological and behavioral functions have been ascribed to PACAP actions in the CNS, a role for this neuropeptide as a modulator of learning and memory is emerging (Borbely et al., 2013, Zhou et al., 2002). The high density of PACAPergic afferents and PACAP-type-I receptors (PAC1) within the extended amygdala (e.g. central nucleus of the amygdala [CeA] and bed nucleus of the stria terminalis [BNST]; Alheid and Heimer, 1988, Hannibal, 2002, Joo et al., 2004, Piggins et al., 1996) suggests a role in modulating neural activity related to stress, anxiety, and fear-related learning (Hammack and May, 2014, Iemolo et al., 2016, Lebow and Chen, 2016). Along these lines, we have demonstrated that PACAP influences NMDA and AMPA-dependent synaptic transmission in the CeA (Cho et al., 2012), which receives heavy PACAPergic innervation from the parabrachial nucleus (Missig et al., 2014) and may be endogenously released in response to pain.

Recently, we demonstrated that intracerebroventricular (ICV) infusion of PACAP prior to fear conditioning can produce persistent blockade of conditioned freezing measured one or seven days after training (Meloni, Venkataraman, Donahue, & Carlezon, 2016). This effect is temporary, however, as freezing re-emerges with repeated testing and results in a hyper-expression of freezing one week later. PACAP also simultaneously elevates serum corticosterone (CORT) during fear conditioning, consistent with its role as a stress-related peptide and known stimulatory effects on corticotropin-releasing factor (CRF) neurons of the paraventricular nucleus of the hypothalamus (PVN; Agarwal et al., 2005, Tsukiyama et al., 2011). However, CORT levels return to normal one day later (the test day), suggesting that elevated CORT does not account for the expression of the amnestic-like effects. We also measured expression of the protein product of the immediate early gene c-Fos following PACAP administration and fear conditioning to map brain areas activated during consolidation of the fear memory to better understand brain systems involved in PACAP effects; we found significant elevations in c-Fos expression in the CeA and PVN, but reductions in other brain areas such as the lateral habenula, indicating effects at many different neural nodes.

The current study was designed to enable a deeper level of resolution of brain areas and potential mechanisms involved in PACAP effects on fear memory consolidation by examining a known molecular marker of synaptic plasticity and memory formation: activity-regulated cytoskeleton-associated protein (Arc, also known as Arg3.1; Nikolaienko et al., 2017, Steward et al., 2015, Tzingounis and Nicoll, 2006). Like c-Fos, Arc is an immediate-early gene product and is not only a regulatory transcription factor (Korb, Wilkinson, Delgado, Lovero, & Finkbeiner, 2013), but also an effector molecule directly involved in activity-dependent structural remodeling in dendritic spines, AMPA receptor trafficking, and LTP/LTD/homeostatic plasticity (Bramham et al., 2008, Li et al., 2015, Shepherd and Bear, 2011). Brain areas examined for PACAP-dependent effects on Arc expression include those known to be involved in cue and context dependent fear conditioning such as the amygdala (including CeA) and dorsal hippocampus (Huff et al., 2006, Lonergan et al., 2010, Plath et al., 2006). We also examined the BNST, which is implicated in fear learning (Goode & Maren, 2017), although there are limited reports using Arc as a readout for fear-induced neuroplastic changes in this structure (e.g. Pelrine et al., 2016, Ravinder et al., 2013).

As with our previous studies (Meloni et al., 2016), our rationale for using ICV administration of PACAP rather than localized delivery (e.g. Legradi et al., 2007, Roman et al., 2014) was to investigate how PACAP actions in multiple brain areas might simultaneously interact to affect Arc expression and subsequent learning of aversive contingencies, as might be expected under circumstances that activate PACAP systems (e.g., stress). The complex interactions of multiple brain areas recruited during fear conditioning (Maren et al., 2013, Zelikowsky et al., 2014), and modified by the effects of exogenously applied PACAP, may produce a different behavioral outcome to that seen with brain-specific infusions alone (Schmidt et al., 2015). Because systemic PACAP does not cross the blood-brain barrier, ICV administered PACAP recapitulates a condition where elevated levels of endogenous PACAP—putatively increased by exposure to stress or pain—affects multiple brain areas undergoing neuroplastic changes as a consequence of exposure to trauma (i.e., the learning-inducing event). Given that preclinical fear conditioning paradigms have been useful tools to help understand the neurobiology of psychiatric conditions such as PTSD (Mahan & Ressler, 2012), where dysfunction within PACAP systems has been implicated (Ressler et al., 2011), the current study may have useful face and construct validity for studying these illnesses as they appear in humans.

Section snippets

Animals

Male Sprague-Dawley rats (250 g) were obtained from Charles River Labs; all rats used in these studies came from the same room at the same facility (Raleigh, NC). They were housed in groups of four and acclimated to the McLean Hospital vivarium for six weeks until surgery. Rats were maintained on 12/12 h light dark cycles and food and water were provided ad libitum. Experiments were performed from 10 a.m. to 4p.m. Sample sizes were determined on the basis of our previous work using the

Arc localization

Fig. 2 illustrates representative sections through the brain areas examined for Arc expression in animals that received either VEH or PACAP (1.5 µg) and were fear conditioned (non-fear conditioned animals that received VEH or PACAP not shown). Arc-positive cells in the CeA (primarily restricted to the lateral division; CeL) and BNST (restricted to lateral dorsal subnucleus corresponding to the oval nucleus) were plentiful in PACAP-treated animals but rarely observed in VEH-treated animals.

Discussion

We show that ICV PACAP given prior to fear conditioning results in significant elevations in Arc levels in the CeA and BNST, but not the dorsal hippocampus or LA/BLA complex. PACAP alone or fear conditioning alone were not sufficient to produce this effect; rather, the combination of PACAP plus fear conditioning (PACAP + FC) was necessary to elevate Arc in these regions. PACAP + FC-induced Arc expression was heaviest in the lateral parts of the BNST, roughly corresponding to the oval nucleus of

Acknowledgments

This research was funded by National Institutes of Health grant MH097860 (WAC).

Conflicts of interest

WAC has served as a paid consultant for Psy Therapeutics within the past 2 years. The other authors report no disclosures relevant to this work.

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