Amy D. Dietrich

A key role for orexin in panic anxiety

Panic disorder is a severe anxiety disorder with recurrent, debilitating panic attacks. In individuals with panic disorder there is evidence of decreased central γ-aminobutyric acid (GABA) activity as well as marked increases in autonomic and respiratory responses after intravenous infusions of hypertonic sodium lactate. In a rat model of panic disorder, chronic inhibition of GABA synthesis in the dorsomedial-perifornical hypothalamus of rats produces anxiety-like states and a similar vulnerability to sodium lactate–induced cardioexcitatory responses. The dorsomedial-perifornical hypothalamus is enriched in neurons containing orexin (ORX, also known as hypocretin), which have a crucial role in arousal, vigilance and central autonomic mobilization, all of which are key components of panic. Here we show that activation of ORX-synthesizing neurons is necessary for developing a panic-prone state in the rat panic model, and either silencing of the hypothalamic gene encoding ORX (Hcrt) with RNAi or systemic ORX-1 receptor antagonists blocks the panic responses. Moreover, we show that human subjects with panic anxiety have elevated levels of ORX in the cerebrospinal fluid compared to subjects without panic anxiety. Taken together, our results suggest that the ORX system may be involved in the pathophysiology of panic anxiety and that ORX antagonists constitute a potential new treatment strategy for panic disorder.

Neuropeptide Y in the Amygdala Induces Long-Term Resilience to Stress-Induced Reductions in Social Responses But Not Hypothalamic–Adrenal–Pituitary Axis Activity or Hyperthermia

Resilience to mental and physical stress is a key determinant for the survival and functioning of mammals. Although the importance of stress resilience has been recognized, the underlying neural mediators have not yet been identified. Neuropeptide Y (NPY) is a peptide known for its anti-anxiety-like effects mediated via the amygdala. The results of our current study demonstrate, for the first time that repeated administration of NPY directly into the basolateral nucleus of the amygdala (BLA) produces selective stress-resilient behavioral responses to an acute restraint challenge as measured in the social interaction test, but has no effect on hypothalamic–adrenal–pituitary axis activity or stress-induced hyperthermia. More importantly, the resilient behaviors observed in the NPY-treated animals were present for up to 8 weeks. Antagonizing the activity of calcineurin, a protein phosphatase involved in neuronal remodeling and present in NPY receptor containing neurons within the BLA, blocked the development of long-term, but not the acute increases in social interaction responses induced by NPY administration. This suggests that the NPY-induced long-term behavioral resilience to restraint stress may occur via mechanisms involving neuronal plasticity. These studies suggest one putative physiologic mechanism underlying stress resilience and could identify novel targets for development of therapies that can augment the ability to cope with stress.

Phosphorylation of Ser640 in muscle glycogen synthase by DYRK family protein kinases

Glycogen synthase, a key enzyme in the regulation of glycogen synthesis by insulin, is controlled by multisite phosphorylation. Glycogen synthase kinase-3 (GSK-3) phosphorylates four serine residues in the COOH terminus of glycogen synthase. Phosphorylation of one of these residues, Ser640 (site 3a), causes strong inactivation of glycogen synthase. In previous work, we demonstrated in cell models that site 3a can be phosphorylated by an as yet unidentified protein kinase (3a-kinase) distinct from GSK-3. In the present study, we purified the 3a-kinase from rabbit skeletal muscle and identified one constituent polypeptide as HAN11, a WD40 domain protein with unknown function. Another polypeptide was identified as DYRK1A, a member of the dual-specificity tyrosine phosphorylated and regulated protein kinase (DYRK) family. Two isoforms of DYRK, DYRK1A and DYRK1B, co-immunoprecipitate with HAN11 when coexpressed in COS cells indicating that the proteins interact in mammalian cells. Co-expression of DYRK1A, DYRK1B, or DYRK2 with a series of glycogen synthase mutants with Ser/Ala substitutions at the phosphorylation sites in COS cells revealed that protein kinases cause phosphorylation of site 3a in glycogen synthase. To confirm that DYRKs directly phosphorylate glycogen synthase, recombinant DYRK1A, DYRK2, and glycogen synthase were produced in bacterial cells. In the presence of Mg-ATP, both DYRKs inactivated glycogen synthase by more than 10-fold. The inactivation correlated with phosphorylation of site 3a in glycogen synthase. These results indicate that protein kinase(s) from the DYRK family may be involved in a new mechanism for the regulation of glycogen synthesis.

Anxiety-like behavior is modulated by a discrete subpopulation of interneurons in the basolateral amygdala

The basolateral amygdala (BL) is a putative site for regulating anxiety, where inhibition and excitation respectively lead to decreases and increases in anxiety-like behaviors. The BL contains local networks of GABAergic interneurons that are subdivided into classes based on neurochemical content, and are hypothesized to regulate unique functional responses of local glutamatergic projection neurons. Recently it was demonstrated that lesioning a portion of the BL interneuronal population, those interneurons that express neurokinin1 receptors (NK(1r)), resulted in anxiety-like behavior. In the current study, these NK(1r) expressing cells of the BL are further phenotypically characterized, demonstrating approximately 80% co-expression with GABA thus confirming them as GABAergic interneurons. These NK(1r) interneurons also colocalize with two distinct populations of BL interneurons as defined by the neuropeptide content. Of the NK(1r) positive cells, 41.8% are also positive for neuropeptide Y (NPY) and 39.7% of the NK(1r) positive cells are also positive for cholecystokinin (CCK). In addition to enhancing the phenotypic characterization, the extent to which the NK(1r) cells of amygdala nuclei contribute to anxiety-like responses was also investigated. Lesioning the NK(1r) expressing interneurons, with a stable form of substance P (SSP; the natural ligand for NK(1r)) coupled to the targeted toxin saporin (SAP), in the anterior and posterior divisions of the BL was correlated to increased anxiety-like behaviors compared to baseline and control treated rats. Furthermore the phenotypic and regional selectivity of the lesions was also confirmed.

Orexin-A induces anxiety-like behavior through interactions with glutamatergic receptors in the bed nucleus of the stria terminalis of rats

The hypothalamic neuropeptide orexin (ORX) has been implicated in anxiety, and anxiety-like behaviors. The purpose of these studies was to determine the role of ORX, specifically orexin-A (ORX-A) in the bed nucleus of the stria terminalis (BNST) on anxiety-like behaviors in rats. Rats injected with ORX-A into the BNST displayed greater anxiety-like measures in the social interaction and elevated plus maze tests compared to vehicle treated controls. Such anxiety-like behaviors were not observed when the ORX-A injections were adjacent to the BNST, in the medial septum. The anxiety-inducing effects of direct infusions of ORX-A into the BNST may be a consequence of increased activation of BNST neurons. In BNST slice preparations using patch-clamp techniques, ORX-A induced membrane depolarization and generation of action potentials in a subset of BNST neurons. The anxiety-inducing effects of ORX-A in the BNST also appear to be dependent on NMDA-type glutamate receptor activity, as pre-injecting the NMDA antagonist AP5 into the BNST blocked anxiogenic effects of local ORX-A injections. Injections of AMPA-type receptor antagonists into the BNST prior to ORX-A resulted in only a partial attenuation of anxiety-like behaviors.

Topographical distribution of corticotropin-releasing factor type 2 receptor-like immunoreactivity in the rat dorsal raphe nucleus: co-localization with tryptophan hydroxylase

Corticotropin-releasing factor (CRF) and CRF-related neuropeptides are involved in the regulation of stress-related physiology and behavior. Members of the CRF family of neuropeptides bind to two known receptors, the CRF type 1 (CRF₁) receptor, and the CRF type 2 (CRF₂) receptor. Although the distribution of CRF₂ receptor mRNA expression has been extensively studied, the distribution of CRF₂ receptor protein has not been characterized. An area of the brain known to contain high levels of CRF₂ receptor mRNA expression and CRF₂ receptor binding is the dorsal raphe nucleus (DR). In the present study we investigated in detail the distribution of CRF₂ receptor immunoreactivity throughout the rostrocaudal extent of the DR. CRF₂ receptor-immunoreactive perikarya were observed throughout the DR, with the highest number and density in the mid-rostrocaudal DR. Dual immunofluorescence revealed that CRF₂ receptor immunoreactivity was frequently co-localized with tryptophan hydroxylase, a marker of serotonergic neurons. This study provides evidence that CRF₂ receptor protein is expressed in the DR, and that CRF₂ receptors are expressed in topographically organized subpopulations of cells in the DR, including serotonergic neurons. Furthermore, these data are consistent with the hypothesis that CRF₂ receptors play an important role in the regulation of stress-related physiology and behavior through actions on serotonergic and non-serotonergic neurons within the DR.

The Role of the Medial Prefrontal Cortex in Regulating Social Familiarity-Induced Anxiolysis

Overcoming specific fears and subsequent anxiety can be greatly enhanced by the presence of familiar social partners, but the neural circuitry that controls this phenomenon remains unclear. To overcome this, the social interaction (SI) habituation test was developed in this lab to systematically investigate the effects of social familiarity on anxiety-like behavior in rats. Here, we show that social familiarity selectively reduced anxiety-like behaviors induced by an ethological anxiogenic stimulus. The anxiolytic effect of social familiarity could be elicited over multiple training sessions and was specific to both the presence of the anxiogenic stimulus and the familiar social partner. In addition, socially familiar conspecifics served as a safety signal, as anxiety-like responses returned in the absence of the familiar partner. The expression of the social familiarity-induced anxiolysis (SFiA) appears dependent on the prefrontal cortex (PFC), an area associated with cortical regulation of fear and anxiety behaviors. Inhibition of the PFC, with bilateral injections of the GABAA agonist muscimol, selectively blocked the expression of SFiA while having no effect on SI with a novel partner. Finally, the effect of D-cycloserine, a cognitive enhancer that clinically enhances behavioral treatments for anxiety, was investigated with SFiA. D-cycloserine, when paired with familiarity training sessions, selectively enhanced the rate at which SFiA was acquired. Collectively, these outcomes suggest that the PFC has a pivotal role in SFiA, a complex behavior involving the integration of social cues of familiarity with contextual and emotional information to regulate anxiety-like behavior.