Protein kinase A (PKA) is an inactive tetrameric holoenzyme consisting of two catalytic (C) subunits each bound to a regulatory (R) subunit dimer. The four R subunits RIα, RIβ, RIIα, and RIIβ, coded by different genes, characterize the subtypes PKA-I and -II (1). Different genes also code for the four catalytic (C) subunits Cα, Cβ, Cγ, and protein kinase X (PRKX), which are expressed (like the R subunits) in a cell- and tissue-specific manner (2). When the R and C subunits form the PKA tetramer (R2C2), there is no PKA catalytic activity. Additionally, R2C2 is bound and, thus, compartmentalized within the cell by A-kinase anchoring proteins (AKAPs) that direct PKA-signaling to specific cell regions and/or organelles (3).

Protein kinase A is considered the primary target for cyclic AMP (cAMP) in the cell, is widely distributed, and serves as the principal effector mechanism for G-protein-coupled receptors (GPCRs) linked to adenylate cyclase (4). The seven-transmembrane domain GPCRs sense molecules outside the cell and activate inside signal transduction pathways to induce cellular responses. The majority of receptors for proteins, biogenic amines, protons, hormones, neurotransmitters, and neuromodulators elicit their responses through guanine nucleotide-binding proteins (G-proteins). The G-protein α subunits, encoded by 16 distinct genes, confer receptor–effector specificity to G-proteins. The γ subunits, encoded by 12 genes, have G protein-specific recognition sites and bind tightly to β subunits. The β subunits, encoded by five genes, also contain a common binding site for α subunit recognition. In their inactive state, the α and βγ subunits are bound to guanosine diphosphate (GDP) and can interact with effectors (Figure 1). This interaction releases GDP in an exchange with guanosine triphosphate (GTP) leading to the generation of α-GTP and a βγ subunit dimer (5, 6). Of the various Gα isoforms, the activation of G_sα stimulates adenylyl cyclase resulting in the production of cAMP. By contrast, the Giα mediates the inhibition of adenylyl cyclase (7, 8).

The increased intracellular cAMP, serving as a second messenger, binds to the regulatory PKA subunits, leading to the disassociation of the tetrameric PKA holoenzyme into an R2-cAMP 4 dimer and two monomers of free catalytic subunits (9). Then, the PKA C subunits catalyze the transfer of phosphates from ATP to serine and threonine residues of targeted intracellular proteins modifying hormonal and neurotransmitter responses, desensitizing receptors related to cortisol biosynthesis, cell differentiation, and synaptic plasticity, and activating or repressing gene transcription (10–12). The principal target of C subunits that translocate to the nucleus is phosphorylation of cAMP-responsive nuclear factors (13) that regulate the expression of genes containing cAMP-responsive elements binding proteins (CREBs) (14) (Figure 1). Phosphorylated CREB binds to CRE nucleotide sequences in DNA as a dimer, recruiting CREB-binding protein (CBP) and p300 cofactors to form larger transcriptional complexes, and catalyzes histone acetylation regulating target genes. Activated CREBs coordinate various neuronal functions, development, and synaptic plasticity (15). Through a feedback mechanism, activated by RIα, cytosolic phosphodiesterases (PDEs) terminate the signals generated by cAMP by hydrolyzing cAMP into 5′AMP (16). As shown in knockout (KO) mouse studies, these four genes function in a tissue- and cell-type-specific manner to regulate accurately the activity of the C subunits (2).

Corticotropin (ACTH)-releasing hormone (CRH)-induced intracellular increase in calcium and cAMP (and PKA activation) through binding to its seven-transmembrane receptor activates several transcription factors including CREB, c-fos, and JunB, which subsequently activate the pro-opiomelanocortin promoter (17). ιnnervation to CRH neurons is provided by fibers containing the pituitary adenylate cyclase-activating polypeptide (PACAP) (18). It is possible that stress induces secretion of PACAP in the paraventricular nucleus (PVN), thereby stimulating CRH gene expression via activation of the cAMP/PKA system (19). Especially, amygdala and lower brainstem contributions to the augmentation of the stress response have been identified as sites of pituitary PACAP innervation (19). Thus, CRH, released by stress signals, stimulates a pulsatile secretion of adrenocorticotropic hormone (ACTH) with peak levels seen before waking and declining at night.

Adrenocorticotropic hormone exerts its effects on the adrenal cortex by binding to a specific receptor (ACTHR) that is the melanocortin-2 receptor (MC2R, coded by the MC2R gene). MC2R is a GPCR linked to G_sα and, thus, ACTH binding triggers activation of adenylate cyclase that catalyzes the conversion of adenosine triphosphate to cAMP (20). The elevation of cAMP is followed by increased PKA activity at the adrenal cortex that results in increased steroidogenesis and the production of glucocorticoids (GCs). Negative feedback on pituitary ACTH secretion is exerted by cortisol at both the hypothalamic and anterior pituitary levels. Importantly, hypothalamic cAMP-inducing CRH might also counterbalance excessive stimulatory stress effects on the hypothalamic–pituitary–adrenal (HPA) axis and maintain immuno-neuroendocrine homeostasis (21).

Other peptide hormones that are secreted and function in an endocrine manner also act as ligands and signal via a wide range of GPCRs. Such peptide hormones, mostly bind within the transmembrane domain, include the growth-regulating hormones somatostatins, parathyroid hormone, angiotensin, HCRTR2, oxytocin, calcitonin, C5a anaphylatoxin, cannabinoids, follicle-stimulating hormone, gonadotropin-releasing hormone, neurokinin, thyrotropin-releasing hormone, and the cholecystokinin peptide hormone system. GPCRs are responsive to hormones, calcium, and neurotransmitters allowing them to form the largest family of validated drug targets (22).

This review focuses on the role of the cAMP-signaling pathway and its mediator, PKA, in the pathogenesis of disorders related to fear learning and anxiety. Anxiety disorders are associated with abnormalities in neural processing of threat-related stimuli (fear learning), which is regulated by cAMP/PKA pathway.

Preclinical studies provide evidence that the pathogenesis of mood disorders, such as anxiety and depression, involves alterations in the plasticity of neuronal pathways (23, 24). Also, clinical studies demonstrate that chronic stress and depression alter brain structures (i.e., cell number and density, cell body size, neuronal and glial density in frontal cortical and hypothalamus) and that result in functional changes (25–29). In addition, studies of suicide subjects report alterations in levels of serotonin and norepinephrine, their metabolites, and receptors, in the brain and peripheral tissues, as well as intracellular signaling pathways (30, 31). Post-mortem studies demonstrate disruptions in cAMP/PKA/CREB/Rap1/BDNF in the brains of suicide subjects that are modulated by stress and GCs (32). There is a paucity of data about PKA activity in brain areas other than the frontal cortex from post-mortem brains of depressed and suicidal subjects. The cAMP/PKA signaling pathway in the central nervous system is well-characterized and has a crucial role for various physiological responses that are important for cell survival, synaptic plasticity, and gene expression (33, 34). Alterations in synaptic and structural plasticity are associated with mood disorders, including generalized anxiety disorder, depression, and suicide.

Studies using inhibitors or activators of PKA helped to elucidate its role in memory formation. Inhibition of protein synthesis or PKA activity blocks LTP in the hippocampus and interferes with memory consolidation for fear in the amygdala (70–72). Also, infusion of PKIs into basolateral amygdala immediately following fear-conditioning training dose-dependently blocked consolidation of fear memory (24-h post training) but not short-term memory (4-h) (73). Infusion of inhibitor Rp-cAMP into the CEA decreased CREB function and decreased neuropeptide Y expression and provoked anxiety-like behavior and alcohol intake in non-preferring rats (74).

In addition, studies using PDE4 inhibitors help to elucidate the molecular mechanisms involved with the behavioral response (anxiolytic-like) to inhibitors of PKA, which depend in part on neurogenic action and activation of GC receptor in the hippocampus (75, 76). There is ample data to support the role of the cAMP/PKA pathway in the mediation of antidepressant/anxiolytic activity (i.e., rolipram, fluoxetine, and clozapine increase cAMP and pCREB expression) in the hippocampus; however, our understanding of the specific effects on neurogenesis is evolving.

Studies with G_sα (Gnas) transgenic mice have shown that increased cAMP signaling is associated with an anxiety-like phenotype (77). Zhang et al. (78) reported that mice with reduced PDE 4B activity, the enzyme that degrades cAMP and interrupts the negative feedback of PKA pathway resulting in increased PKA activity, displayed anxiogenic behavior. In addition, transgenic mice with overexpression of the striatially enriched cAMP-producing adenyl cyclase 5 showed increased anxiety-related behavior (79). Results of the studies reviewed above indicate that increased cAMP signaling is associated with an anxiety-like phenotype, and provide indirect evidence that an increase in PKA activity may be associated with an increased risk for anxiety. Also, studies of mice with genetic deletion of specific PDE4 subtypes have reported anxiogenic behavior, suggesting that PDE4 may be involved in the regulation of anxiety (78, 80, 81).

Prior studies in our lab showed that transgenic mice with a downregulated Prkar1a gene (tTA/X2AS, antisense transgene) (82) exhibited behavioral abnormalities, including anxiety (83) and depression. A KO mouse heterozygous for a null allele of Prkar1a was developed in our lab as a model to investigate Carney complex that is caused by heterozygous inactivating PRKAR1A mutations (84), which results in increased PKA signaling in all cells where this gene is expressed. We hypothesized that a transgenic mouse model with downregulation of Prkar1a would provide a research tool to evaluate the effect of altered PKA expression on anxiety-like behaviors.

In support of our hypothesis, we found that downregulation of the regulatory subunit of PKA in mice led to an augmentation of anxiety-like behavior supporting the role of PKA in modulating anxiety-like behaviors. Compared with WT mice, Prkar1a^+/− mice had higher basal and stimulated (cAMP) PKA activity levels in the central and basolateral amygdala, brain areas known to have a critical role in the processing of sensory information related to anxiety and emotion as well as regulation of arousal level (85).

Since activity in neural circuits prior to or immediately after stimuli may influence PKA activity and LTP and, therefore, may affect fear learning; we then investigated the rodent defensive response in the Prkar1a^+/− mouse hypothesizing that Prkar1a^+/− mice would exhibit an atypical response to threat detection (37, 39). As predicted, we found that in contrast to the response of WT mice, Prkar1a^+/− mice failed to exhibit behavioral changes (exploratory or defensive) to distinguish between predator versus control odor. The behavioral changes paralleled significant differences found in PKA activity between WT and Prkar1a^+/− mice in the amygdala, prefrontal cortex, and ventromedial hypothalamus (86). Our findings are consistent with results of electrophysiological studies showing that changes in amygdala circuitry and dendritic morphology affect fearful responses and correlate with BLA transmission and that the degree of anxiogenic effect of predator stress is positively associated with the degree of potentiation of amygdala circuitry (48, 87, 88). Also, since the function of the prefrontal cortex is to inhibit prepotent behavioral and promote task relevant behaviors, alterations in PKA activity in the prefrontal cortex may also have contributed to the atypical response to threat detection in the Prkar1a^+/− mice (86).

We introduced half-null alleles of Prkaca^+/− into the Prkar1a^+/− mice, hypothesizing abrogation of the excess Cα activity caused by R1α haploinsufficiency. The phenotype of Prkaca^+/− mice was characterized by attenuation but not elimination of the anxiety phenotype noted in Prkar1a heterozygote mice. Measurement of PKA activity in various brain areas showed increased PKA activity in the amygdala in Prkar1a^+/− compared with Prkaca^+/− or WT, and in part compared with Prkar1a^+/−/Prkaca^+/− mice. The alteration of PKA activity in these transgenic mice was not a ubiquitous effect, since PKA activity was found to be similar between heterozygotes and WT mice in some brain areas (e.g., prefrontal cortex, hippocampus, paraventricular hypothalamus, cerebellum, and neural sensory areas). These findings highlight the importance of even modest changes in PKA activity in modulating anxiety-like behaviors and also that catalytic subunit activity is not the sole determinant of PKA’s cAMP-signaling effects (89, 90). It is also possible that compensatory mechanisms in remaining PKA subunits and PDE4 may be a factor in areas not showing any differences in PKA activity between Prkar1a^+/− and Prkar1a^+/−/Prkaca^+/− and WT or Prkaca^+/− mice.

In this review, we highlighted the association of abnormal neural processing of threat-related stimuli and anxiety disorders, which is significantly influenced by the cAMP/PKA pathway, among others. Animal models have helped to elucidate the molecular pathways that have an important role in anxiety; however, there are limitations, so cautious interpretation is appropriate. A recently developed mouse of R1a deficiency provides a unique model to investigate the direct effect of increased PKA activity on the acquisition and expression of learned fear.

Results of clinical studies support the finding that alterations in PKA and some of its substrates are associated with various psychiatric disorders, including anxiety, depression, obsessive–compulsive and bipolar disorders, schizophrenia, and panic disorder (91–97). Also, in adult patients with PRKAR1A mutations, we reported an increased incidence of psychiatric disorders, including anxiety, depression, and bipolar disorder (in that order), and for children with PRKAR1A mutations an increased incidence of learning disorders, attention deficit hyperactivity disorder, anxiety, and depression (in that order) (98). Recent animal studies support the hypothesis that selective gene intervention in the cAMP/PKA system may constitute a promising anxiolytic target.

All authors contributed equally to the writing (MK and GB) and editing (MK, GB, CS, and TW) of this manuscript.

The opinions or assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the Department of Defense (DoD) or the Uniformed Services University of the Health Sciences (USUHS). NICHD, USUHS, or DOD had no further role in the study design; in the collection, analysis, and interpretation of the data; in the writing of the report, or in the decision to submit the paper for publication.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.