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