Brain neuropeptide Y and corticotropin-releasing hormone in mediating stress and anxiety

Background

Under acute conditions, stress invokes a response that may be beneficial for survival. However, when activated chronically, stress responses can cause damage and be the basis for, as well as accelerate, disease conditions. ^1–4 Stress as a term refers to processes involving perception, appraisal and response to threatening, challenging and/or possibly harmful stimuli. There exists a division of stressful stimuli into emotional, or psychological, stimuli and physiological stimuli. Examples of stimuli that are considered emotional/psychological include social interactions/conflicts, inescapable situations, death of a loved one and so forth. Physiological stimuli can be hypo/hyperthermia, an immune challenge, starvation and also drug withdrawal.

The internal stress response can be related by the term allostasis and refers to the process of re-establishing homeostasis in response to a challenge. ^5,6 Homeostasis refers to consistency of internal parameters within a normal range, while allostasis describes the body's way to keep stable outside the normal range by changing internal systems to match external demands. ^5,6 Allostasis links the brain with the endocrine system as well as our immune system to coordinate appropriate responses to an (external) stressor. ⁷

In addition to being intrinsically harmful, (chronic) stress has also been shown to increase vulnerability to addiction. Drug intake and withdrawal impose a stress on internal systems leading to a disruption of the homeostatic state. Furthermore, repeated exposure to and withdrawal from drug use leads to increased sensitivity to stress and an increased behavioral stress response. ⁸ In addition, stress-induced relapse is a model frequently used in preclinical settings and involves exposure to a stressor (foot-shock for example), which then leads to the reinstatement of a previously extinguished behavior, drug use (for example, see reference ⁹ ).

The terms stress, fear and anxiety are at times used interchangeably; however, they are considered different in the area of anxiety research. As stated above, stress refers to a real or inferred threat affecting numerous systems in the body leading to a response, and may be defined as a force changing a (previously) static condition. Fear may be the result of the stress response itself and is associated with a specific event or stressor. Anxiety cannot be related to a specific event, but may be due to chronic stress or fear. It is a more general response and may be indicative of changes on both neuronal and system-wide levels. Anxious individuals emotionally anticipate an aversive situation.

Approximately one in four individuals will experience an anxiety-related disorder during their lifetime. ¹⁰ This together with well-known issues such as side-effects and limited efficacy for existing anxiolytics makes the need for new treatments urgent. Neuropeptide systems may offer opportunities for such treatment development. Neuropeptides have been suggested to only be released if the firing of a neuron exceeds a certain level, i.e. frequency-dependent release. ¹¹ Furthermore, neuropeptides, when compared with classical neurotransmitters, can act over longer distances and may thus be used to recruit large neuronal populations to produce a concerted physiological effect. ¹² Additionally, the accessibility of neuropeptides is higher due to limited reuptake mechanisms as compared with classical neurotransmitters.

In this brief review, we discuss the involvement of two neuropeptide systems: corticotropin-releasing hormone (CRH) and neuropeptide Y NPY) in stress-related behavior and anxiety, and the putative opposing roles of these peptides in the behavioral responses to stress.

Corticotropin-releasing hormone

CRH is a 41-amino-acid neuropeptide that is released by the hypothalamus upon exposure to a stressful stimulus. It mediates autonomic, ^13,14 neuroendocrine ¹⁴ and behavioral responses to stress. ^15,16 CRH activates the hypothalamic-pituitary-adrenal axis (HPA) by stimulating release of adrenocorticotropic hormone (ACTH) from the pituitary, which in turn stimulates release of cortisol/corticosterone (human/rodent) from the adrenal cortex. The stress-induced activation of the HPA axis is dependent on CRH. ¹⁷ In addition to HPA axis activation, CRH participates in extrahypothalamic behavioral responses to stress. ¹⁸ The neuroanatomical substrates for CRH, in addition to the hypothalamus, include, among others, the amygdala, the bed nucleus of the stria terminalis and the locus coeruleus. ^19–21

CRH is a member of a peptide family including Urocortin, Urocortin II and Urocortin III. ^22,23 The effects of these peptides are mediated via two G-protein (G_s)-coupled receptors: CRH-R1 and CRH-R2. ²⁴ Agonist activation of these receptors leads to increased intracellular concentrations of cyclic AMP (cAMP). CRH-R1 is the primary receptor for CRH and participates in mediation of the behavioral stress response. ^25,26

Central administration of CRH is anxiogenic in several animal behavioral tests such as the novel open-field, ²⁷ acoustic startle ²⁸ and operant conflict tests. ²⁹ Whether the centrally mediated, anxiogenic behavioral effects of CRH are dependent on the HPA axis, or completely independent thereof requires further discussion. Lesions of the central nucleus of the amygdala, but not the hypothalamic paraventricular nucleus (PVN), disrupt CRH-potentiated fear responses, ³⁰ and peripheral blockade of ACTH/glucocorticoids does not affect the behavioral response. ³¹ Furthermore, central administration of CRH in dexamethasone-treated rats still elicits an anxiogenic effect. ³² However, adrenalectomy abolishes the locomotor activity-inducing effects of central CRH administration. ³³ In preclinical studies, stress increases CRH release in the amygdala ^34,35 as well as CRH mRNA expression. ^26,36 Furthermore, in non-human primate models of early adverse events chronic elevation of cerebrospinal fluid (CSF) CRH is seen, ³⁷ and patients with post-traumatic stress disorder (PTSD) have significant elevation of CSF CRH, ³⁸ indicating significant activation of the extrahypothalamic CRH system in these conditions.

Given the above studies, administration of CRH-R1 receptor antagonists may offer an effective treatment for stress-induced anxiety disorders. Indeed, treatment with CRH-R1 receptor antagonists does result in decreased levels of experimental anxiety in rats with a ‘high anxiety’ phenotype, ³⁹ in mice following exposure to a chronic mild stress paradigm, ⁴⁰ as well as in human subjects diagnosed with major depression or PTSD. ^41–43 Furthermore, a CRH-R1 antagonist was effective in blocking alcohol withdrawal-induced anxiety on the elevated plus-maze as well as stress-induced reinstatement of alcohol seeking. ⁴⁴ Similar to other potent stressors, acute alcohol withdrawal induces anxiety-like responses that are correlated with increased CRH levels in the central nucleus of the amygdala and in the bed nucleus of the stria terminalis, ⁴⁵ sites mediating the behavioral responses to stress. Accordingly, anxiogenic effects of alcohol withdrawal are reversed by CRH-R1 antagonism. ^44,46 It should be noted that limited to no effect of CRH-R1 antagonists upon administration to naïve/unstressed animals was seen, suggesting that the CRH system may have limited tone under baseline (non-challenged) conditions.

Neuropeptide Y

NPY is a 36-amino-acid peptide widely expressed in the mammalian nervous system, with high levels in brain regions such as the hypothalamus, in particular the arcuate and the paraventricular nuclei, the hippocampal formation, the amygdala and septum. Following its isolation, ^47,48 NPY has been extensively studied with particular attention to anxiety/stress-related behavior and feeding.

To date, all NPY receptors cloned belong to the superfamily of G-protein-coupled receptors, but differ in their ligand affinity profiles. NPY receptors are coupled via G_i/o proteins to several downstream signaling pathways, including inhibition of adenylyl cyclase, activation of mitogen-activated protein kinase, regulation of intracellular calcium (Ca²⁺) concentrations and activation of G-protein-coupled, inwardly rectifying potassium (K⁺) channels. Currently, there are four subtypes of NPY receptors known to mediate biological responses: Y1, Y2, Y4 and Y5. The predominantly postsynaptic Y1 receptor requires the intact NPY sequence for recognition and activation, and has been proposed as the subtype mediating antianxiety actions of NPY. ⁴⁹ The presynaptic Y2 receptor subtype is in addition activated by C-terminal fragments of NPY, such as NPY ^13–36 and NPY ^3–36 . ^50,51 The Y4 receptor has low affinity for NPY and is primarily the target for pancreatic polypeptide (PP), another member of the PP family of peptides. ⁵² The Y5 receptor was initially thought to be the receptor regulating NPY's effect on feeding behavior and Y5 receptor antagonists have been evaluated for putative obesity treatment, possibly in combination with compounds aimed at other NPY receptors such as the Y1. ^53,54

Early indications of anxiolytic/sedative properties of NPY included synchronization of electroencephalographic activity, ⁵⁵ the prevention of stress-induced ulcers and suppression of locomotor activity. ^56,57 Central administration of exogenous NPY produces an anxiolytic phenotype in numerous animal models of experimental anxiety. ^58–60 Antianxiety effects of NPY have been shown to rely in part on the activation of Y1 receptors in the amygdala. ^61–63 Intracerebroventricular injections of NPY or Y1 receptor agonists, but not Y2 receptor agonists, are anxiolytic in behavioral models of experimental anxiety. ⁶⁴

In agreement with antistress effects observed following central administration of NPY, a role for endogenous NPY in the control of stress- and anxiety-related behaviors is suggested by several findings. Acute physical restraint, which promotes experimental anxiety, suppresses NPY mRNA and peptide levels within the amygdala and cortex. ⁶⁵ In contrast, repeated exposure to the same stressor once daily for 10 d leads to a complete behavioral and endocrine habituation, accompanied by an upregulation of amygdala NPY expression. ⁶⁶ Furthermore, chronic overexpression of NPY generates a stress-insensitive and ‘anxiolytic’ phenotype. ^67,68 Genetic variations in human NPY expression affects stress response and emotion. ^69,70 We have therefore proposed that an upregulation of NPY expression may contribute to the behavioral adaptation to stress. This extends a hypothesis that NPY may act to ‘buffer’ behavioral effects of stress-promoting signals. ⁷¹

Evidence for interactions between CRH and NPY systems

Cannon (1927) ⁷² pioneered the concept of a specific site regulating emotional responses. Later work by Papez (1937) ⁷³ and Klüver and Bucy (1937) ^74,75 confirmed a role for, among other regions, the amygdala in emotional regulation. Studies in human subjects ^76,77 as well as animals ^78,79 have since shown that activation of the amygdala is crucial for the mediation of behavioral and emotional responses associated with anxiety. Lesioning of the amygdala in turn leads to decreased levels of experimental anxiety and decreased sensitivity to stress. ^80,81 A role has been proposed for the amygdala to integrate inputs from the varying brain regions to produce a coherent behavioral response to fear and anxiety. In part this response is mediated by neuropeptides including, but not limited to, NPY and CRH.

NPY and CRH have indeed been demonstrated to have presence in many brain regions relevant for the behavioral and emotional response to stressful stimuli and/or fear/anxiety-producing events. In 1994, Heilig et al. ⁷¹ proposed opposing roles for NPY and CRH within the amygdala to maintain emotional balance following stress exposure. NPY was suggested to be released subsequently to increases in amygdalar CRH producing an opposing action on the stress system, facilitating return to a homeostatic (or allostatic) balance.

As discussed above, numerous studies have indicated anxiolytic and anxiogenic effects of NPY and CRH, respectively, upon administration into the CNS. In addition, studies have revealed overlapping biological substrates, primarily within the amygdala, for these effects. Both CRH and NPY receptors have been found within the amygdala. ^82–85 The basolateral nucleus (BLA) is of particular interest since CRH, as well as Urocortin I, elicit long-term anxiety-like responses upon administration into the BLA. ^86,87 However, the opposite behavioral response is seen upon BLA administration of NPY. ⁶³ Furthermore, pretreatment with NPY into the BLA prior to administration of Urocortin blocks the anxiogenic effect of the CRH-R1/2 receptor activation on anxiety-related behavior. ^12,88 This indicates a direct interaction of CRH and NPY systems within the BLA to modify anxiety-related behavior. The molecular mechanism for this interaction has been proposed to be differential regulation of cAMP levels. ⁸⁹ In addition, at the level of the hypothalamus, dose-dependent increases in NPY overflow from the PVN were observed following CRH administration. ⁹⁰ Further investigation into the clinical implications for these interactions is needed.

Concluding remarks

In conclusion, this review briefly discusses the importance of two neuropeptide systems, CRH and NPY, with regards to their individual and overlapping contribution to stress/anxiety-related behaviors and responses. Neuropeptide systems may offer more attractive treatment targets than the classical neurotransmitter systems due to the limited tonic activity in neuropeptide systems. Lack of tonic activity may generate treatments that have efficacy only during times of specific neuronal activity and may thus lead to state-specific treatments as well as a reduction in the appearance of side-effects.

The opposing roles of CRH and NPY in the regulation of stress/anxiety-related behaviors and responses may open the way for putative combination treatments aimed at targets within these systems.