Circulating PACAP levels are associated with altered imaging measures of entorhinal cortex neurite density in posttraumatic stress disorder.

Introduction: Pituitary adenylate cyclase-activating polypeptide (PACAP) regulates plasticity in brain systems underlying arousal and memory and is associated with posttraumatic stress disorder (PTSD). Research in animal models suggests that PACAP modulates entorhinal cortex (EC) input to the hippocampus, contributing to impaired contextual fear conditioning. In PTSD, PACAP is associated with higher activity of the amygdala to threat stimuli and lower functional connectivity of the amygdala and hippocampus. However, PACAP-affiliated structural alterations of these regions have not been investigated in PTSD. Here, we examined whether peripheral PACAP levels were associated with neuronal morphology of the amygdala and hippocampus (primary analyses), and EC (secondary) using Neurite Orientation Dispersion and Density Imaging.Methods: Sixty-four (44 female) adults (19 to 54 years old) with DSM-5 Criterion A trauma exposure completed the Clinician-Administered PTSD Scale (CAPS-5), a blood draw, and magnetic resonance imaging. PACAP38 radioimmunoassay was performed and T1-weighted and multi-shell diffusion-weighted images were acquired. Neurite Density Index (NDI) and Orientation Dispersion Index (ODI) were quantified in the amygdala, hippocampus, and EC. CAPS-5 total score and anxious arousal score were used to test for clinical associations with brain structure.Results: Higher PACAP levels were associated with greater EC NDI (ß = 0.0099, q = 0.032) and lower EC ODI (ß = -0.0073, q = 0.047), and not hippocampal or amygdala measures. Neither EC NDI nor ODI was associated with clinical measures.Conclusions: Circulating PACAP levels were associated with altered neuronal density of the EC but not the hippocampus or amygdala. These findings strengthen evidence that PACAP may impact arousal-associated memory circuits in PTSD.

PACAP was associated with altered entorhinal cortex neurite density in PTSD.PACAP was not associated with altered neurite density in amygdala or hippocampus.PACAP may impact arousal-associated memory circuits.

Circulating PACAP levels are associated with altered imaging measures of entorhinal cortex neurite density in posttraumatic stress disorder.

Background: Pituitary adenylate cyclase-activating polypeptide (PACAP) regulates plasticity in brain systems underlying arousal and memory and is associated with posttraumatic stress disorder (PTSD). Research in animal models suggests that PACAP modulates entorhinal cortex (EC) input to the hippocampus, contributing to impaired contextual fear conditioning. In PTSD, PACAP is associated with higher activity of the amygdala to threat stimuli and lower functional connectivity of the amygdala and hippocampus. However, PACAP-affiliated structural alterations of these regions have not been reported. Here, we examined whether peripheral PACAP levels were associated with neuronal morphology of the amygdala and hippocampus (primary analysis), and EC (secondary analysis) using Neurite Orientation Dispersion and Density Imaging. Methods: Sixty-four (44 female) adults (19 to 54 years old) with DSM-5 Criterion A trauma exposure completed the Clinician-Administered PTSD Scale (CAPS-5), a blood draw, and magnetic resonance imaging. PACAP38 radioimmunoassay was performed and T1-weighted and multi-shell diffusion- weighted images were acquired. Neurite Density Index (NDI) and Orientation Dispersion Index (ODI) were quantified in the amygdala, hippocampus, and EC. CAPS-5 total score and anxious arousal score were used to test for clinical associations with brain structure. Results: Higher PACAP levels in blood were associated with greater EC NDI (ß=0.31, q=0.034) and lower EC ODI (ß=-0.30, q=0.042) and not hippocampal or amygdala measures. Neither EC NDI nor ODI was associated with clinical measures. Conclusions: Circulating PACAP levels were associated with altered neuronal density of the EC but not hippocampus or amygdala. These findings strengthen evidence that PACAP may impact arousal- associated memory circuits.

The functional heterogeneity of PACAP: Stress, learning, and pathology.

Pituitary adenylate cyclase-activating peptide (PACAP) is a highly conserved and widely expressed neuropeptide that has emerged as a key regulator of multiple neural and behavioral processes. PACAP systems, including the various PACAP receptor subtypes, have been implicated in neural circuits of learning and memory, stress, emotion, feeding, and pain. Dysregulation within these PACAP systems may play key roles in the etiology of pathological states associated with these circuits, and PACAP function has been implicated in stress-related psychopathology, feeding and metabolic disorders, and migraine. Accordingly, central PACAP systems may represent important therapeutic targets; however, substantial heterogeneity in PACAP systems related to the distribution of multiple PACAP isoforms across multiple brain regions, as well as multiple receptor subtypes with several isoforms, signaling pathways, and brain distributions, provides both challenges and opportunities for the development of new clinically-relevant strategies to target the PACAP system in health and disease. Here we review the heterogeneity of central PACAP systems, as well as the data implicating PACAP systems in clinically-relevant behavioral processes, with a particular focus on the considerable evidence implicating a role of PACAP in stress responding and learning and memory. We also review data suggesting that there are sex differences in PACAP function and its interactions with sex hormones. Finally, we discuss both the challenges and promise of harnessing the PACAP system in the development of new therapeutic avenues and highlight PACAP systems for their critical role in health and disease.

Circulating PACAP levels are associated with increased amygdala-default mode network resting-state connectivity in posttraumatic stress disorder.

The pituitary adenylate cyclase-activating polypeptide (PACAP) system is implicated in posttraumatic stress disorder (PTSD) and related amygdala-mediated arousal and threat reactivity. PTSD is characterized by increased amygdala reactivity to threat and, more recently, aberrant intrinsic connectivity of the amygdala with large-scale resting state networks, specifically the default mode network (DMN). While the influence of PACAP on amygdala reactivity has been described, its association with intrinsic amygdala connectivity remains unknown. To fill this gap, we examined functional connectivity of resting-state functional magnetic resonance imaging (fMRI) in eighty-nine trauma-exposed adults (69 female) screened for PTSD symptoms to examine the association between blood-borne (circulating) PACAP levels and amygdala-DMN connectivity. Higher circulating PACAP levels were associated with increased amygdala connectivity with posterior DMN regions, including the posterior cingulate cortex/precuneus (PCC/Precun) and left angular gyrus (lANG). Consistent with prior work, this effect was seen in female, but not male, participants and the centromedial, but not basolateral, subregions of the amygdala. Clinical association analyses linked amygdala-PCC/Precun connectivity to anxious arousal symptoms, specifically exaggerated startle response. Taken together, our findings converge with previously demonstrated effects of PACAP on amygdala activity in PTSD-related processes and offer novel evidence for an association between PACAP and intrinsic amygdala connectivity patterns in PTSD. Moreover, these data provide preliminary evidence to motivate future work ascertaining the sex- and subregion-specificity of these effects. Such findings may enable novel mechanistic insights into neural circuit dysfunction in PTSD and how the PACAP system confers risk through a disruption of intrinsic resting-state network dynamics.

Exercise reduces the anxiogenic effects of meta-chlorophenylpiperazine: The role of 5-HT2C receptors in the bed nucleus of the stria terminalis.

Introduction: Two weeks of voluntary exercise in group-housed mice produces a reduction in anxiety-like behaviors across a number of different measures, including a reduction in the anxiety levels typically produced by the anxiogenic serotonergic drug m-chlorophenylpiperazine (mCPP), an agonist at 5-HT2C/2b receptors. We have previously demonstrated that 2-weeks of voluntary exercise blunted the anxiogenic effects of systemic mCPP, and we have also shown that mCPP infused into the bed nucleus of the stria terminalis (BNST) is anxiogenic. Here we follow up on these reports. Methods: In Experiment 1 we infused several doses of mCPP into the BNST with or without the 5-HT2C antagonist SB242084. In Experiment 2, we administered mCPP into amygdala subregions and the dorsal hippocampus to investigate site specificity. In Experiment 4 we lesioned the BNST and subsequently infused mCPP systemically, and in Experiment 4 we used RNAscope® to assess BNST 5-HT2C transcripts following wheel running. Results: BNST mCPP infusion increased acoustic startle responding, which was by 5-HT2C antagonism, while neither mCPP infused into the amygdala nor hippocampus was anxiogenic. Lesions of the BNST prevented the anxiogenic effect of systemically administered mCPP. Lastly, exercise reduced 5-HT2C transcripts in the BNST. Discussion: These results suggest that the BNST is a critical site of action for the effects of exercise on mCPP. Together these data suggest that exercise may reduce 5-HT2C receptor function in the BNST, which may, in part, explain some of the anxiolytic effects associated with wheel running.

Activation of Lateral Parabrachial Nucleus (LPBn) PACAP-Expressing Projection Neurons to the Bed Nucleus of the Stria Terminalis (BNST) Enhances Anxiety-like Behavior.

Anxiety disorders are among the most common psychiatric disorders, and understanding the underlying neurocircuitry of anxiety- and stress-related behaviors may be important for treatment. The bed nucleus of the stria terminalis (BNST) has been studied for its role in many stress-related pathologies, such as anxiety, pain, depression, and addiction. Our prior work has demonstrated that pituitary adenylate cyclase-activating polypeptide (PACAP) receptor activation in the BNST mediates many of the behavioral consequences of chronic stress. While the BNST contains local PACAP-expressing neurons, a major source of afferent PACAP is the lateral parabrachial nucleus (LPBn), and excitotoxic lesions of the LPBn substantially decreasess PACAP immunostaining in the BNST. Here, we first assessed Cre-dependent reporter expression by injecting AAV2-hSyn-DIO-mCherry into the LPBn of PACAP-IRES-Cre mice for circuit mapping studies and identified PACAP projections to the BNST, lateral capsular central nucleus of the amygdala (CeLC), and ventromedial hypothalamus (VMH). In a second study, we assessed the effects of chemogenetically activating LPBn PACAP afferents in the BNST by injecting AAV2-hSyn-DIO-hM3D(Gq)-mCherry into the LPBn of PACAP-IRES-Cre mice for Cre-dependent expression of excitatory designer receptors exclusively activated by designer drugs (DREADDs). Before behavioral testing, clozapine-N-oxide (CNO), the selective agonist of our DREADD, was infused directly into the BNST. We found that after specific activation of LPBn PACAP afferents in the BNST, mice had increased anxiety-like behavior compared with controls, while total locomotor activity was unaffected. These results indicate that activation of PACAPergic LPBn projections to the BNST may play an important role in producing anxiety-like behavior.

Chemoarchitecture of the bed nucleus of the stria terminalis: Neurophenotypic diversity and function.

The bed nucleus of the stria terminalis (BNST) is a compact but neurophenotypically complex structure in the ventral forebrain that is structurally and functionally linked to other limbic structures, including the amygdala nuclear complex, hypothalamic nuclei, hippocampus, and related midbrain structures, to participate in a wide range of functions, especially emotion, emotional learning, stress-related responses, and sexual behaviors. From a variety of sensory inputs, the BNST acts as a node for signal integration and coordination for information relay to downstream central neuroendocrine and autonomic centers for appropriate homeostatic physiological and behavioral responses. In contrast to the role of the amygdala in fear, the BNST has gained wide interest from work suggesting that it has main roles in mediating sustained responses to diffuse, unpredictable and/or long-duration threats that are typically associated with anxiety-related responses. Further, some BNST subregions are highly sexually dimorphic which appear contributory to the differential stress and social interactive behaviors, including reproductive responses, between males and females. Notably, maladaptive BNST neuroplasticity and function have been implicated in chronic pain, depression, anxiety-related abnormalities, and other psychopathologies including posttraumatic stress disorders. The BNST circuits are predominantly GABAergic-the glutaminergic neurons represent a minor population-but the complexity of the system results from an overlay of diverse neuropeptide coexpression in these neurons. More than a dozen neuropeptides may be differentially coexpressed in BNST neurons, and from variable G protein-coupled receptor signaling, may inhibit or activate downstream circuit activities. The mechanisms and roles of these peptides in modulating intrinsic BNST neurocircuit signaling and BNST long-distance target cell projections are still not well understood. Nevertheless, an understanding of some of the principal players may allow assembly of the circuit interactions.

PACAP orchestration of stress-related responses in neural circuits.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a pleiotropic polypeptide that can activate G protein-coupled PAC1, VPAC1, and VPAC2 receptors, and has been implicated in stress signaling. PACAP and its receptors are widely distributed throughout the nervous system and other tissues and can have a multitude of effects. Human and animal studies suggest that PACAP plays a role responding to a variety of threats and stressors. Here we review the roles of PACAP in several regions of the central nervous system (CNS) as they relate to several behavioral functions. For example, in the bed nucleus of the stria terminalis (BNST), PACAP is upregulated following chronic stress and may drive anxiety-like behavior. PACAP can also influence both the consolidation and expression of fear memories, as demonstrated by studies in several fear-related areas, such as the amygdala, hippocampus, and prefrontal cortex. PACAP can also mediate the emotional component of pain, as PACAP in the central nucleus of the amygdala (CeA) is able to decrease pain sensitivity thresholds. Outside of the central nervous system, PACAP may drive glucocorticoid release via enhanced hypothalamic-pituitary-adrenal axis activity and may participate in infection-induced stress responses. Together, this suggests that PACAP exerts effects on many stress-related systems and may be an important driver of emotional behavior.

PAC1 Receptor Internalization and Endosomal MEK/ERK Activation Is Essential for PACAP-Mediated Neuronal Excitability.

Pituitary adenylate cyclase activating polypeptide (PACAP, Adcyap1) activation of PAC1 receptors (Adcyap1r1) can significantly increase the excitability of diverse neurons through differential mechanisms. For guinea pig cardiac neurons, the modulation of excitability can be mediated in part by PAC1 receptor plasma membrane G protein-dependent activation of adenylyl cyclase and downstream signaling cascades. By contrast, PAC1 receptor-mediated excitability of hippocampal dentate gyrus granule cells appears independent of membrane-delimited AC/cAMP/PKA and PLC/PKC signaling. For both neuronal types, there is mechanistic convergence demonstrating that endosomal PAC1 receptor signaling has prominent roles. In these models, neuronal exposure to Pitstop2 to inhibit ß-arrestin/clathrin-mediated PAC1 receptor internalization eliminates PACAP modulation of excitability. ß-arrestin is a scaffold for a number of effectors especially MEK/ERK and notably, paradigms that inhibit PAC1 receptor endosome formation and ERK signaling also blunt the PACAP-induced increase in excitability. Detailed PAC1 receptor internalization and endosomal ERK signaling mechanisms have been confirmed in HEK PAC1R-EGFP cells and shown to be long lasting which appear to recapitulate the sustained electrophysiological responses. Thus, PAC1 receptor internalization/endosomal recruitment efficiently and efficaciously activates MEK/ERK signaling and appears to represent a singular and critical common denominator in regulating neuronal excitability by PACAP.

The Role of Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) Signaling in the Hippocampal Dentate Gyrus.

Pituitary adenylate cyclase-activating polypeptide (PACAP, ADCYAP1) dysregulation has been associated with multiple stress-related psychopathologies that may be related to altered hippocampal function. In coherence, PACAP- and PAC1 receptor (ADCYAP1R1)-null mice demonstrate changes in hippocampal-dependent behavioral responses, implicating the PACAPergic system function in this structure. Within the hippocampus, the dentate gyrus (DG) may play an important role in discerning the differences between similar contexts, and DG granule cells appear to both highly express PAC1 receptors and receive inputs from PACAP-expressing terminals. Here, we review the evidence from our laboratories and others that PACAP is an important regulator of activity within hippocampal circuits, particularly within the DG. These data are consistent with an increasing literature implicating PACAP circuits in stress-related pathologies such as post-traumatic stress disorder (PTSD) and implicate the hippocampus, and in particular the DG, as a critical site in which PACAP dysregulation can alter stress-related behaviors.

Pituitary adenylate cyclase-activating polypeptide-induced PAC1 receptor internalization and recruitment of MEK/ERK signaling enhance excitability of dentate gyrus granule cells.

Pituitary adenylate cyclase activating polypeptide (PACAP; ADCYAP1) is a pleiotropic neuropeptide widely distributed in both the peripheral and central nervous systems. PACAP and its specific cognate PAC1 receptor (ADCYAP1R1) play critical roles in the homeostatic maintenance of multiple physiological and behavioral systems. Notably, maladaptations in the PACAPergic system have been associated with several psychopathologies related to fear and anxiety. PAC1 receptor transcripts are highly expressed in granule cells of the dentate gyrus (DG). Here, we examined the direct effects of PACAP on DG granule cells in brain slices using whole cell patch recordings in current clamp mode. PACAP significantly increased the intrinsic excitability of DG granule cells via PAC1 receptor activation. This increased excitability was not mediated by adenylyl cyclase/cAMP or phospholipase C/PKC activation, but instead via activation of an extracellular signal-regulated kinase (ERK) signaling pathway initiated through PAC1 receptor endocytosis/endosomal signaling. PACAP failed to increase excitability in DG granule cells pretreated with the persistent sodium current blocker riluzole, suggesting that the observed PACAP effects required this component of the inward sodium current.

Parallel signaling pathways of pituitary adenylate cyclase activating polypeptide (PACAP) regulate several intrinsic ion channels.

Pituitary adenylate cyclase activating polypeptide (PACAP), acting through its cognate receptors PAC1, VPAC1, and VPAC2, is a pleiotropic signaling neuropeptide of the vasoactive intestinal peptide/secretin/glucagon family. PACAP has known functions in neuronal growth, development, and repair, and central PACAP signaling has acute behavioral consequences. One of the ways in which PACAP may affect neuronal function is through the modulation of intrinsic membrane currents to control neuronal excitability. Here, we review the evidence of PACAP-dependent modulation of calcium- and voltage-gated potassium currents, hyperpolarization-activated cation currents, calcium currents, and voltage-gated sodium currents. Interestingly, PACAP signaling pathways diverge into parallel pathways to target different ionic currents for modulation, though single pathways are not limited to modulating just one target ionic current. Despite the various targets of modulation, the weight of the evidence suggests that PACAP signaling most commonly leads to a net increase in neuronal excitability. We discuss possible mechanisms by which PACAP signaling leads to the modulation of intrinsic membrane currents that may contribute to changes in behavior.

Pituitary Adenylate Cyclase-Activating Peptide (PACAP) Signaling and the Dark Side of Addiction.

While addiction to drugs of abuse represents a significant health problem worldwide, the behavioral and neural mechanisms that underlie addiction and relapse are largely unclear. The concept of the dark side of addiction, developed and explored by George Koob and colleagues, describes a systematic decrease in reward-related processing following drug self-administration and subsequent recruitment of anti-reward (i.e., stress) systems. Indeed, the activation of central nervous system (CNS) stress-response systems by drugs of abuse is contributory not only to mood and anxiety-related disorders but critical to both the maintenance of addiction and relapse following abstinence. In both human and animal studies, compounds that activate the bed nucleus of the stria terminalis (BNST) have roles in stress-related behaviors and addiction processes. The activation of pituitary adenylate cyclase-activating peptide (PACAP) systems in the BNST mediates many consequences of chronic stressor exposure that may engage in part downstream corticotropin-releasing hormone (CRH) signaling. Similar to footshock stress, the BNST administration of PACAP or the PAC1 receptor-specific agonist maxadilan can facilitate relapse following extinction of cocaine-seeking behavior. Further, in the same paradigm, the footshock-induced relapse could be attenuated following BNST pretreatment with PAC1 receptor antagonist PACAP6-38, implicating PACAP systems as critical components underlying stress-induced reinstatement. In congruence with previous work, the PAC1 receptor internalization and endosomal MEK/ERK signaling appear contributory mechanisms to the addiction processes. The studies offer new insights and approaches to addiction and relapse therapeutics.

Pituitary Adenylate Cyclase-Activating Peptide in the Bed Nucleus of the Stria Terminalis Mediates Stress-Induced Reinstatement of Cocaine Seeking in Rats.

Stressors often contribute to difficulties in maintaining behavior change following a period of abstinence, and may play a significant role in drug relapse. The activation of pituitary adenylate cyclase-activating peptide (PACAP) systems in the bed nucleus of the stria terminalis (BNST) mediates many consequences of chronic stressor exposure. Here we ask whether PACAP is also involved in producing reinstatement in a model of stress-induced relapse to drug taking. Rats self-administered cocaine for 1 h daily over 10 days that was followed by 20 days of extinction training in which lever pressing no longer produced cocaine. In experiment 1, quantitative PCR (qPCR) was performed at several stages to determine transcript levels of PACAP and corresponding receptors. Reinstatement of cocaine seeking was then tested after footshock exposure in different groups of rats that were pretreated with vehicle solution, a PAC1 receptor antagonist (experiment 2), or a PACAP agonist (experiment 3) without footshock. In experiment 1, cocaine self-administration increased BNST PACAP transcript levels similar to what we have previously reported with chronic stress. In experiment 2, intra-BNST infusions of the PAC1/VPAC2 antagonist, PACAP 6-38, prevented footshock-induced reinstatement of extinguished cocaine seeking. In experiment 3, intra-BNST PACAP infusion reinstated previously extinguished cocaine-seeking behavior in the absence of footshock. Cocaine self-administration elevated BNST PACAP, and BNST PACAP receptor activation was necessary and sufficient for stress-induced reinstatement of cocaine seeking. These data suggest that BNST PACAP systems may be viable targets for relapse prevention.

Pituitary adenylate cyclase activating polypeptide (PACAP), stress, and sex hormones.

Stressor exposure is associated with the onset and severity of many psychopathologies that are more common in women than men. Moreover, the maladaptive expression and function of stress-related hormones have been implicated in these disorders. Evidence suggests that PACAP has a critical role in the stress circuits mediating stress-responding, and PACAP may interact with sex hormones to contribute to sex differences in stress-related disease. In this review, we describe the role of the PACAP/PAC1 system in stress biology, focusing on the role of stress-induced alterations in PACAP expression and signaling in the development of stress-induced behavioral change. Additionally, we present more recent data suggesting potential interactions between stress, PACAP, and circulating estradiol in pathological states, including PTSD. These studies suggest that the level of stress and circulating gonadal hormones may differentially regulate the PACAPergic system in males and females to influence anxiety-like behavior and may be one mechanism underlying the discrepancies in human psychiatric disorders.

Traumatic brain injury impairs sensorimotor function in mice.

INTRODUCTION: Understanding the extent to which murine models of traumatic brain injury (TBI) replicate clinically relevant neurologic outcomes is critical for mechanistic and therapeutic studies. We determined sensorimotor outcomes in a mouse model of TBI and validated the use of a standardized neurologic examination scoring system to quantify the extent of injury. MATERIALS AND METHODS: We used a lateral fluid percussion injury model of TBI and compared TBI animals to those that underwent sham surgery. We measured neurobehavioral deficits using a standardized 12-point neurologic examination, magnetic resonance imaging, a rotating rod test, and longitudinal acoustic startle testing. RESULTS: TBI animals had a significantly decreased ability to balance on a rotating rod and a marked reduction in the amplitude of acoustic startle response. The neurologic examination had a high inter-rater reliability (87% agreement) and correlated with latency to fall on a rotating rod (Rs = -0.809). CONCLUSIONS: TBI impairs sensorimotor function in mice, and the extent of impairment can be predicted by a standardized neurologic examination.

Parabrachial Pituitary Adenylate Cyclase-Activating Polypeptide Activation of Amygdala Endosomal Extracellular Signal-Regulated Kinase Signaling Regulates the Emotional Component of Pain.

BACKGROUND: Chronic pain and stress-related psychopathologies, such as depression and anxiety-associated abnormalities, are mutually reinforcing; however, the neuronal circuits and mechanisms that underlie this reinforcement are still not well understood. Pituitary adenylate cyclase-activating polypeptide (PACAP; Adcyap1) and its cognate PAC1 receptor (Adcyap1r1) are expressed in peripheral nociceptive pathways, participate in anxiety-related responses and have been have been linked to posttraumatic stress disorder and other mental health afflictions. METHODS: Using immunocytochemistry, pharmacological treatments and behavioral testing techniques, we have used a rodent partial sciatic nerve chronic constriction injury model (n = 5-8 per group per experiment) to evaluate PACAP plasticity and signaling in nociceptive and stress-related behaviors. RESULTS: We show that chronic neuropathic pain increases PACAP expression at multiple tiers along the spinoparabrachioamygdaloid tract. Furthermore, chronic constriction injury bilaterally augments nociceptive amygdala (in the central nucleus of the amygdala [CeA]) PACAP immunoreactivity, extracellular signal-regulated kinase phosphorylation, and c-Fos activation, in parallel with heightened anxiety-like behavior and nociceptive hypersensitivity. Acute CeA infusions with the PACAP receptor antagonist PACAP(6-38) blocked chronic constriction injury-induced behavioral responses. Additionally, pretreatments with inhibitors of mitogen-activated protein kinase enzymes or endocytosis to block endosomal PACAP receptor extracellular signal-regulated kinase signaling attenuated PACAP-induced CeA neuronal activation and nociceptive responses. CONCLUSIONS: Our data suggest that chronic pain-induced PACAP neuroplasticity and signaling in spinoparabrachioamygdaloid projections have an impact on CeA stress- and nociception-associated maladaptive responses, which can be ameliorated upon receptor antagonism even during injury progression. Thus, the PACAP pathway provides for an important mechanism underlying the intersection of stress and chronic pain pathways via the amygdala.

The Effects of Prior Stress on Anxiety-Like Responding to Intra-BNST Pituitary Adenylate Cyclase Activating Polypeptide in Male and Female Rats.

Chronic or repeated exposure to stressful stimuli can result in several maladaptive consequences, including increased anxiety-like behaviors and altered peptide expression in anxiety-related brain structures. Among these structures, the bed nucleus of the stria terminalis (BNST) has been implicated in emotional behaviors as well as regulation of hypothalamic-pituitary-adrenal (HPA) axis activity. In male rodents, chronic variate stress (CVS) has been shown to increase BNST pituitary adenylate cyclase activating polypeptide (PACAP) and its cognate PAC1 receptor transcript, and BNST PACAP signaling may mediate the maladaptive changes associated with chronic stress. Here, we examined whether CVS would sensitize the behavioral and/or endocrine response to a subthreshold BNST PACAP infusion. Male and cycling female rats were exposed to a 7 day CVS paradigm previously shown to upregulate BNST PAC1 receptor transcripts; control rats were not stressed. Twenty-four hours following the last stressor, rats were bilaterally infused into the BNST with a normally subthreshold dose of PACAP. We found an increase in startle amplitude and plasma corticosterone levels 30 min following intra-BNST PACAP infusion in male rats that had been previously exposed to CVS. CVS did not enhance the startle response in cycling females. Equimolar infusion of the VPAC1/2 receptor ligand vasoactive intestinal polypeptide (VIP) had no effect on plasma corticosterone levels even in previously stressed male rats. These results suggest that repeated exposure to stressors may differentially alter the neural circuits underlying the responses to intra-BNST PACAP, and may result in different anxiety-like responses in males and females.

A microRNA negative feedback loop downregulates vesicle transport and inhibits fear memory.

The SNARE-mediated vesicular transport pathway plays major roles in synaptic remodeling associated with formation of long-term memories, but the mechanisms that regulate this pathway during memory acquisition are not fully understood. Here we identify miRNAs that are up-regulated in the rodent hippocampus upon contextual fear-conditioning and identify the vesicular transport and synaptogenesis pathways as the major targets of the fear-induced miRNAs. We demonstrate that miR-153, a member of this group, inhibits the expression of key components of the vesicular transport machinery, and down-regulates Glutamate receptor A1 trafficking and neurotransmitter release. MiR-153 expression is specifically induced during LTP induction in hippocampal slices and its knockdown in the hippocampus of adult mice results in enhanced fear memory. Our results suggest that miR-153, and possibly other fear-induced miRNAs, act as components of a negative feedback loop that blocks neuronal hyperactivity at least partly through the inhibition of the vesicular transport pathway.

Role of the bed nucleus of the stria terminalis in the acquisition of contextual fear at long or short context-shock intervals.

Rats received N-methyl-D-aspartate lesions of the bed nucleus of the stria terminalis (BNST) and then 10 aversive conditioning trials in which exposure to a context was paired with footshock. For half the animals, shock was presented 1 min after the onset of each context exposure; for the other half, shock was presented after 10 min. With the 1-min context duration, aversive conditioning (measured by freezing) was unaffected by BNST lesion. In contrast, at the 10-min duration, lesioned animals froze substantially less than sham controls. When 1-min-conditioned animals were left in the context for 10 min, freezing that was evident (though declining) throughout the test was not affected by the BNST lesion. When freezing over 10 min was similarly examined in the 10-min-conditioned animals, BNST lesions caused a deficit that was consistently evident over time. The results indicate that the BNST is involved in aversive conditioning to long-duration, but not merely contextual, conditional stimuli. Results may be less consistent with the view that BNST becomes activated after prolonged fear than the view that it is involved when a cue's onset has a remote temporal relation to shock.