Elsevier

Neuropharmacology

Volume 133, 1 May 2018, Pages 145-154
Neuropharmacology

Pituitary adenylate cyclase-activating polypeptide (PACAP) signaling in the prefrontal cortex modulates cued fear learning, but not spatial working memory, in female rats

https://doi.org/10.1016/j.neuropharm.2018.01.010Get rights and content

Highlights

  • Prefrontal PAC1 receptor signaling contributes to the strength of cued, but not contextual, fear memory.

  • The effects of blocking prefrontal PAC1 receptor signaling are specific to females.

  • Females exhibit greater PAC1 receptor mRNA expression compared with males.

  • Prefrontal PAC1 receptor signaling is a potential neurobiological substrate of PTSD in women.

Abstract

A genetic polymorphism within the gene encoding the pituitary adenylate cyclase- activating polypeptide (PACAP) receptor type I (PAC1R) has recently been associated with hyper-reactivity to threat-related cues in women, but not men, with post-traumatic stress disorder (PTSD). PACAP is a highly conserved peptide, whose role in mediating adaptive physiological stress responses is well established. Far less is understood about the contribution of PACAP signaling in emotional learning and memory, particularly the encoding of fear to discrete cues. Moreover, a neurobiological substrate that may account for the observed link between PAC1R and PTSD in women, but not men, has yet to be identified. Sex differences in PACAP signaling during emotional learning could provide novel targets for the treatment of PTSD. Here we investigated the contribution of PAC1R signaling within the prefrontal cortex to the acquisition of cued fear in female and male rats. We used a variant of fear conditioning called trace fear conditioning, which requires sustained attention to fear cues and depends on working-memory like neuronal activity within the prefrontal cortex. We found that cued fear learning, but not spatial working memory, was impaired by administration of a PAC1R antagonist directly into the prelimbic area of the prefrontal cortex. This effect was specific to females. We also found that levels of mRNA for the PAC1R receptor in the prelimbic cortex were greater in females compared with males, and were highest during and immediately following the proestrus stage of the estrous cycle. Together, these results demonstrate a sex-specific role of PAC1R signaling in learning about threat-related cues.

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.

References (67)

  • G. Missig et al.

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

    Neuropharmacology

    (2014)
  • H. Monyer et al.

    Developmental and regional expression in the rat brain and functional properties of four NMDA receptors

    Neuron

    (1994)
  • J.M. Resch et al.

    Inhibition of food intake by PACAP in the hypothalamic ventromedial nuclei is mediated by NMDA receptors

    Physiol. Behav.

    (2014)
  • C.W. Roman et al.

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

    Psychoneuroendocrinology

    (2014)
  • B. Sacchetti et al.

    Pituitary adenylate cyclase-activating polypeptide hormone (PACAP) at very low dosages improves memory in the rat

    Neurobiol. Learn. Mem.

    (2001)
  • S.D. Schmidt et al.

    PACAP modulates the consolidation and extinction of the contextual fear conditioning through NMDA receptors

    Neurobiol Learn Mem

    (2015)
  • W.J. Sheward et al.

    The distribution of vasoactive intestinal peptide2 receptor messenger RNA in the rat brain and pituitary gland as assessed by in situ hybridization

    Neuroscience

    (1995)
  • L.M. Shin et al.

    An fMRI study of anterior cingulate function in posttraumatic stress disorder

    Biol. Psychiatr.

    (2001)
  • G.W. Wang et al.

    Disconnection of the hippocampal-prefrontal cortical circuits impairs spatial working memory performance in rats

    Behav. Brain Res.

    (2006)
  • M. Wang et al.

    NMDA receptors subserve persistent neuronal firing during working memory in dorsolateral prefrontal cortex

    Neuron

    (2013)
  • R. Yaka et al.

    Pituitary adenylate cyclase-activating polypeptide (PACAP(1-38)) enhances N-methyl-D-aspartate receptor function and brain-derived neurotrophic factor expression via RACK1

    J. Biol. Chem.

    (2003)
  • E.H. Baeg et al.

    Fast spiking and regular spiking neural correlates of fear conditioning in the medial prefrontal cortex of the rat

    Cereb Cortex

    (2001)
  • C.V. Buhusi et al.

    Timing for the absence of a stimulus: the gap paradigm reversed

    J. Exp. Psychol. Anim. Behav. Process.

    (2000)
  • A. Burgos-Robles et al.

    Sustained conditioned responses in prelimbic prefrontal neurons are correlated with fear expression and extinction failure

    J. Neurosci.

    (2009)
  • R.L. Butcher et al.

    Plasma concentration of LH, FSH, prolactin, progesterone and estradiol-17beta throughout the 4-day estrous cycle of the rat

    Endocrinology

    (1974)
  • K.A. Corcoran et al.

    Activity in prelimbic cortex is necessary for the expression of learned, but not innate, fears

    J. Neurosci.

    (2007)
  • S.G. Cull-Candy et al.

    Role of distinct NMDA receptor subtypes at central synapses

    Sci. STKE

    (2004)
  • M.S. Fanselow et al.

    Naloxone and shock-elicited freezing in the rat

    J. Comp. Physiol. Psychol.

    (1979)
  • M.M. Farnham et al.

    PACAP causes PAC1/VPAC2 receptor mediated hypertension and sympathoexcitation in normal and hypertensive rats

    Am. J. Physiol. Heart Circ. Physiol.

    (2012)
  • M. Figiel et al.

    Pituitary adenylate cyclase-activating polypeptide (PACAP), a neuron-derived peptide regulating glial glutamate transport and metabolism

    J. Neurosci.

    (2000)
  • T.M. Furlong et al.

    The role of prefrontal cortex in predictive fear learning

    Behav. Neurosci.

    (2010)
  • C. Gao et al.

    Hippocampal NMDA receptor subunits differentially regulate fear memory formation and neuronal signal propagation

    Hippocampus

    (2010)
  • M.R. Gilmartin et al.

    Trace and contextual fear conditioning require neural activity and NMDA receptor-dependent transmission in the medial prefrontal cortex

    Learn. Mem.

    (2010)
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