Role of Basolateral Amygdalar Somatostatin 2 Receptors in a Rat Model of Chronic Anxiety.

Repeated exposure to stress has been implicated in inducing chronic anxiety states. Stress related increases in anxiety responses are likely mediated by activation of corticotropin-releasing factor receptors (CRFR) in the amygdala, particularly the basolateral amygdala (BLA). Within the BLA, acute injections of the CRFR agonist urocortin 1 (Ucn1) leads to acute anxiety, whereas repeated daily injections of subthreshold-doses of Ucn1 produces a long-lasting, persistent anxiety-like phenotype, a phenomenon referred to as Ucn1-priming. Relative gene expressions from the BLA of vehicle and Ucn1-primed rats were analyzed with quantitative RT-PCR using a predesigned panel of 82 neuroscience-related genes. Compared to vehicle-primed rats, only expression of the somatostatin receptor 2 gene (Sstr2) was significantly reduced in the BLA of Ucn1-primed rats. The contribution of Sstr2 on an anxiety phenotype was tested by injecting a Sstr2 antagonist into the BLA in un-primed rats. The Sstr2 antagonist increased anxiety-like behavior. Notably, pretreatment with Sstr2 agonist injected into the BLA blocked anxiety-inducing effects of acute Ucn1 BLA-injections and delayed anxiety expression during Ucn1-priming. However, concomitant Sstr2 agonist pretreatment during Ucn-1 priming did not prevent either the development of a chronic anxiety state or a reduction of BLA Sstr2 expression induced by priming. The data demonstrate that the persistent anxiety-like phenotype observed with Ucn1-priming in the BLA is associated with a selective reduction of Sstr2 gene expression. Although Sstr2 activation in the BLA blocks acute anxiogenic effects of stress and down-regulation of BLA Sstr2, it does not suppress the long-term consequences of prolonged exposure to stress-related challenges.

Psychosocial impairment following mild blast-induced traumatic brain injury in rats.

Traumatic brain injury (TBI) is associated with increased risk for mental health disorders, impacting post-injury quality of life and societal reintegration. TBI is also associated with deficits in psychosocial processing, defined as the cognitive integration of social and emotional behaviors, however little is known about how these deficits manifest and their contributions to post-TBI mental health. In this pre-clinical investigation using rats, a single mild blast TBI (mbTBI) induced impairment of psychosocial processing in the absence of confounding physical polytrauma, post-injury motor deficits, affective abnormalities, or deficits in non-social behavior. Impairment severity correlated with acute upregulations of a known oxidative stress metabolite, 3-hydroxypropylmercapturic acid (3-HPMA), in urine. Resting state fMRI alterations in the acute post-injury period implicated key brain regions known to regulate psychosocial behavior, including orbitofrontal cortex (OFC), which is congruent with our previous report of elevated acrolein, a marker of neurotrauma and 3-HPMA precursor, in this region following mbTBI. OFC of mbTBI-exposed rats demonstrated elevated mRNA expression of metabotropic glutamate receptors 1 and 5 (mGluR1/5) and injection of mGluR1/5-selective agonist in OFC of uninjured rats approximated mbTBI-induced psychosocial processing impairment, demonstrating a novel role for OFC in this psychosocial behavior. Furthermore, OFC may serve as a hotspot for TBI-induced disruption of psychosocial processing and subsequent mental health disorders.

From anxiety to autism: spectrum of abnormal social behaviors modeled by progressive disruption of inhibitory neuronal function in the basolateral amygdala in Wistar rats.

RATIONALE: Social behaviors are disrupted in several psychiatric disorders. The amygdala is a key brain region involved in social behaviors, and amygdala pathology has been implicated in disease states ranging from social anxiety disorder to autism. OBJECTIVE: To test the effects of progressive disruption of the inhibitory function within the basolateral nucleus of the amygdala (BLA) on conspecific social interaction in rats and investigate functional networks from the ventral medial prefrontal cortex (mPFCv) to the BLA. MATERIALS AND METHODS: BLA inhibitory tone was disrupted by priming it with the stress-peptide corticotrophin releasing factor (CRF) receptor agonist urocortin 1 (Ucn 1, 6 fmol), or by selective lesioning of a subset of BLA-GABAergic interneurons containing neurokinin 1 receptors using the targeted toxin SSP-Saporin. The effects of the disruption of GABAergic tone in the BLA were examined using a repeated exposure and habituation paradigm of social interaction (SI/h). Lesions and selectivity of lesions were confirmed postmortem. Additionally, effects of stimulating mPFCv on cFos activity in interneurons of the BLA were examined. RESULTS: Rats primed with Ucn 1 showed persistent social inhibition, which could be overcome with habituation, putatively modeling social anxiety. Rats with a selective lesioning of a subset of GABAergic interneurons in the BLA exhibited persistent social inhibition that was not reversed by SI/h paradigm. We also demonstrate selective functional inputs to this subset of interneurons when mPFCv was activated. CONCLUSIONS: These models with different gradations of disrupted BLA inhibition could help to study social dysfunction in disorders ranging from social anxiety to autism spectrum disorders.

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.

Interaction between glycogenin and glycogen synthase.

Glycogen synthase plays a key role in regulating glycogen metabolism. In a search for regulators of glycogen synthase, a yeast two-hybrid study was performed. Two glycogen synthase-interacting proteins were identified in human skeletal muscle, glycogenin-1, and nebulin. The interaction with glycogenin was found to be mediated by the region of glycogenin which contains the 33 COOH-terminal amino acid residues. The regions in glycogen synthase containing both NH2- and COOH-terminal phosphorylation sites are not involved in the interaction. The core segment of glycogen synthase from Glu21 to Gly503 does not bind COOH-terminal fragment of glycogenin. However, this region of glycogen synthase binds full-length glycogenin indicating that glycogenin contains at least one additional interacting site for glycogen synthase besides the COOH-terminus. We demonstrate that the COOH-terminal fragment of glycogenin can be used as an effective high affinity reagent for the purification of glycogen synthase from skeletal muscle and liver.

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, Ser(640) (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.

GNIP, a novel protein that binds and activates glycogenin, the self-glucosylating initiator of glycogen biosynthesis.

Glycogenin is a self-glucosylating protein involved in the initiation of glycogen biosynthesis. Self-glucosylation leads to the formation of an oligosaccharide chain, which, when long enough, supports the action of glycogen synthase to elongate it and form a mature glycogen molecule. To identify possible regulators of glycogenin, the yeast two-hybrid strategy was employed. By using rabbit skeletal muscle glycogenin as a bait, cDNAs encoding three different proteins were isolated from the human skeletal muscle cDNA library. Two of the cDNAs encoded glycogenin and glycogen synthase, respectively, proteins known to be interactors. The third cDNA encoded a polypeptide of unknown function and was designated GNIP (glycogenin interacting protein). Northern blot analysis revealed that GNIP mRNA is highly expressed in skeletal muscle. The gene for GNIP generates at least four isoforms by alternative splicing. The largest isoform GNIP1 contains, from NH(2)- to COOH-terminal, a RING finger, a B box, a putative coiled-coil region, and a B30.2-like motif. The previously identified protein TRIM7 (tripartite motif containing protein 7) is also derived from the GNIP gene and is composed of the RING finger, B box, and coiled-coil regions. The GNIP2 and GNIP3 isoforms consist of the coiled-coil region and B30.2-like domain. Physical interaction between GNIP2 and glycogenin was confirmed by co-immunoprecipitation, and in addition GNIP2 was shown to stimulate glycogenin self-glucosylation 3-4-fold. GNIPs may represent a novel participant in the initiation of glycogen synthesis.