Pituitary adenylate cyclase-activating polypeptide (PACAP) signaling in the prefrontal cortex modulates cued fear learning, but not spatial working memory, in female rats
Introduction
Women are more than twice as likely as men to develop post-traumatic stress disorder (PTSD), yet the neurobiological basis of this sex difference is unknown (Kessler et al., 1995, Kilpatrick et al., 2013). Recently, dysregulation of pituitary adenylate cyclase-activating polypeptide (PACAP) signaling has been implicated in PTSD selectively in women (Ressler et al., 2011). Elevated PACAP blood levels and a single nucleotide polymorphism (SNP) in the PACAP receptor type-1 (PAC1R) gene are associated with hyperarousal symptoms in women but not men with PTSD (Ressler et al., 2011). The SNP is located within an estrogen response element of the gene and interferes with DNA binding of an estradiol-ERα complex, which suggests a link between ovarian hormones and altered PAC1R expression in these patients (Mercer et al., 2016). PACAP is a highly-conserved peptide important for mediating adaptive physiological stress responses, and alterations in PACAP signaling may contribute to the development or maintenance of PTSD in women (Hammack and May 2015). PACAP may also contribute to PTSD by modulating the formation of emotional memories. PACAP and its receptors are widely distributed throughout the central nervous system, including areas important for memory such as the hippocampus, amygdala, cingulate and frontal cortices, and thalamus (for a review see Vaudry et al., 2009). This raises the possibility that PACAP signaling may normally contribute to fear memory encoding in these areas. Indeed, the PAC1R genetic polymorphism is associated with increased reactivity of fear circuitry to threat-related cues and impaired discrimination of threat and safety cues in women with PTSD (Ressler et al., 2011, Stevens et al., 2014). Currently, very little is known about the neurobiology of PACAP signaling in emotional learning, especially cued fear learning. Preclinical studies suggest that PACAP signaling influences contextual fear memory. Mice lacking the PAC1R either globally or in the hippocampus and neocortex showed impaired contextual fear conditioning (Otto et al., 2001). PACAP delivered intracerebroventricularly in rats enhanced the consolidation of a passive avoidance memory at low doses (Sacchetti et al., 2001) and temporarily impaired contextual fear memory at high doses (Meloni et al., 2016). PACAP delivered directly into the hippocampus enhanced the consolidation of a contextual fear memory (Schmidt et al., 2015). Endogenous PACAP may also contribute to the consolidation of contextual fear memory given that blocking the activation of the PAC1R with the antagonist PACAP6-38 in the hippocampus or the amygdala attenuated the consolidation of contextual fear memory (Schmidt et al., 2015). With the exception of the Schmidt et al. study, the contribution of PAC1R signaling in individual brain regions to learning has not been explored. The prefrontal cortex in particular warrants closer investigation. Dysfunction within prefrontal cortical regions in humans is associated with aberrant cognitive and emotional regulation in PTSD, including heightened reactivity to threat-related stimuli, possibly through interaction with the amygdala (Fani et al., 2012, Shin et al., 2006, Taylor and Whalen, 2015). Moreover, abnormal activity in dorsal prefrontal cortical areas may even be a predisposing factor in the development of PTSD (Admon et al., 2013).
Here we tested the contribution of PAC1R signaling in the prefrontal cortex to memory formation using trace fear conditioning in female and male rats. Unlike standard cued fear conditioning, the trace variant of fear conditioning critically depends on the prefrontal cortex, which makes this variant useful for probing prefrontal contributions to emotional learning that may be altered in pathological fear and anxiety (Gilmartin et al., 2013b, Gilmartin et al., 2014, Gilmartin and Helmstetter, 2010, Guimarais et al., 2011). In trace fear conditioning, subjects learn to anticipate a shock that is delivered several seconds after a cue presentation. Successful acquisition is associated with sustained attention and prefrontal firing to the cue during the stimulus-free trace interval before the shock is delivered – a working-memory like function that depends on the dorsomedial prefrontal cortex in rats (Baeg et al., 2001, Gilmartin and McEchron, 2005, Gilmartin et al., 2013b, Han et al., 2003). We tested the importance of PAC1R in trace conditioning and spatial working memory using direct injections of PACAP6-38 into the prelimbic (PL) cortex. PAC1R is the primary target of this peptide in the PL given the apparent lack of vasoactive intestinal peptide receptor type-2 (VPAC2R) in rodent frontal cortices (Lein et al., 2007, Sheward et al., 1995, Usdin et al., 1994). Importantly, we conducted this study using both female and male rats. Preclinical studies of PACAP's role in memory have been limited to male subjects. However, the sex-specific link between the genetic polymorphism in the PAC1R gene in humans and PTSD demands the inclusion of female subjects in preclinical studies. We found that the administration of the PAC1R antagonist PACAP6-38 into the PL prior to conditioning attenuated the formation of cued fear memory in females, but not males. In contrast, the performance of a spatial working memory task was unaffected by PACAP6-38. Interestingly, females had significantly higher levels of mRNA for the PAC1R within the PL compared with males. These results show that PAC1R signaling within the prelimbic cortex has sex specific effects on the formation of cued fear memory and suggest that differences in PAC1R transcripts may contribute to this sex difference.
Section snippets
Subjects and surgery
Adult female (Experiments 1, 2, and 4) and male (Experiments 3 and 4) Long-Evans rats (225 g females; 325 g males; Envigo, Indianapolis, IN.) were used in this study. Rats were housed individually and received food and water ad libitum. All procedures were in accordance with the National Institutes of Health guidelines and approved by the Marquette University Institutional Animal Care and Use Committee. After three days of handling, rats in Experiments 1-3 underwent cannula-implantation surgery
Experiment 1: prefrontal PAC1R and fear learning in females
All forty-eight female rats implanted with bilateral cannulae had successful placements in the PL. Placements for the Saline and PACAP6-38 2 mM dose groups are shown in Fig. 1b. One rat was excluded from all analyses due to having some tissue damage at the injection site (PACAP6-38 1 mM group).
Pre-training injection of the PAC1R antagonist PACAP6-38 impaired the formation of cued, but not contextual fear memory (Fig. 2). The administration of PACAP6-38 did not affect the within-session
Discussion
Administration of the PAC1R antagonist PACAP6-38 directly into the PL dose-dependently impaired the formation of associative fear memory. This effect was specific to females and to cued fear learning. Females had greater expression of mRNA for the PAC1R in the PL compared with males, regardless of training, and expression levels varied with the estrous cycle. These results suggest that PACAP signaling in the prefrontal cortex is a means by which fear to threat-predictive cues is strengthened in
Conflicts of interest
The authors declare no competing financial interests.
Acknowledgments
This work was supported by a Regular Research Grant from the Marquette University Committee on Research. We would like to thank Dr. R. Spencer (UW-Madison) for his helpful advice when setting up the delayed alternation paradigm. We would like to thank D. Durigan and B. Natwora for assistance with histology, and M. Hurley and the S. Choi lab for RT-qPCR primer design and validation. We also thank Dr. D. Wheeler for helpful comments on the manuscript.
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2022, Neurobiology of Learning and MemoryCitation Excerpt :Freezing was used as the measure of conditional fear during all training and testing sessions and was defined as the cessation of all movement except that needed for respiration (Fanselow & Bolles, 1979). Freezing was scored automatically using FreezeScan 2.0 (CleverSys; Reston, VA) as described previously (Kirry et al., 2018). We routinely observe the behavior of the rats during each session in order to identify behaviors that could interfere with freezing or represent an alternative conditional response, such as darting.