ELEVATED tph2 mRNA EXPRESSION IN A RAT MODEL OF CHRONIC ANXIETY

Abstract

Background Allelic variations in TPH2, the gene encoding tryptophan hydroxylase 2, the rate-limiting enzyme for brain serotonin (5-HT) biosynthesis, may be genetic predictors of panic disorder and panic responses to panicogenic challenges in healthy volunteers. To test the hypothesis that tph2 mRNA is altered in chronic anxiety states, we measured tph2 expression in an established rat model of panic disorder. Methods We implanted 16 adult, male rats with bilateral guide cannulae and then primed them with daily injections of the corticotropin-releasing factor (CRF) receptor agonist, urocortin 1 (UCN1, 6 fmoles/100 nl per side, n = 8) or vehicle (n = 8) into the basolateral amygdaloid complex (BL) for 5 consecutive days. Anxiety-like behavior was assessed, 24 hr prior to and 48 hr following priming, in the social interaction (SI) test. A third group (n = 7) served as undisturbed home cage controls. All rats were killed 3 days after the last intra-BL injection to analyze tph2 and slc6a4 (gene encoding the serotonin transporter, SERT) mRNA expression in the dorsal raphe nucleus (DR), the main source of serotonergic projections to anxiety-related brain regions, using in situ hybridization histochemistry. Results UCN1 priming increased anxiety-related behavior in the SI test compared to vehicle-injected controls and elevated tph2, but not slc6a4, mRNA expression in DR subregions, including the ventrolateral DR/ventrolateral periaqueductal gray (DRVL/VLPAG), a subregion previously implicated in control of panic-related physiologic responses. Tph2 mRNA expression in the DRVL/VLPAG was correlated with increased anxiety-related behavior. Conclusion Our data support the hypothesis that chronic anxiety states are associated with dysregulated tph2 expression.

Development×environment interactions control tph2 mRNA expression

“…No. 74104, Qiagen, Valencia, USA), and used in an ISHH assay as described previously (Donner and Handa, 2009; Donner et al, 2012). To detect slc6a4 mRNA, a synthetic 50-base antisense oligonucleotide (5′-ACT GCA GAG TAC CCA TTG GAT ATT TGG CTA GGC TCT GCC CTG TCC GCT GT-3′, Integrated DNA Technologies, Coralville, IA, USA), complementary to bases 207–256 of rat slc6a4 mRNA (previously reported as bases 77–126 Fujita et al, 1993; Hansen and Mikkelsen, 1998), based on the published sequence of the rat serotonin transporter cDNA clone (Blakely et al, 1991) of slc6a4 mRNA ( Rattus norvegicus solute carrier family 6 (neurotransmitter transporter, serotonin), member 4, mRNA; GenBank Accession no., NM_013034.3) was used as previously described (Gardner et al, 2009a).…”

The Deakin/Graeff hypothesis: Focus on serotonergic inhibition of panic

“…First, we have shown that amygdala priming (using 5 × daily bilateral infusions of 6 fmol urocortin 1 into the BL) in male rats, which results in a chronic anxiety-like state as measured in the social interaction test, resulted in increased expression of tph2 , encoding Tph2, the rate-limiting enzyme in biosynthesis of serotonin, selectively in the DRVL/VLPAG serotonergic neurons and the caudal DRV serotonergic neurons (which have also been shown to project to the DPAG (Stezhka and Lovick, 1994;Vertes, 1991)), but not in any other subdivision of the DR (Donner et al, 2012). In addition, tph2 mRNA expression was highly correlated with anxiety-like behavior in the social interaction test ( r = 0.729; p = 0.001) (Donner et al, 2012). Second, we have shown that social defeat increases tph2 mRNA expression, but only in the DRVL/VLPAG serotonergic neurons, and only in rats previously exposed to maternal separation (Gardner et al, 2009).…”

“…Priming of the BLA, a structure critical for processing fear-related stimuli and gating output of the CE (Adolphs, 2013;LeDoux et al, 1988), by repeated urocortin 1 (UCN1) injections increases anxiety-like behavior and elevates tph2 mRNA in the DRVL/VLPAG (Donner et al, 2012). Microinjection of urocortin 2 (UCN2), a neuropeptide in the CRH family and a selective CRHR2 agonist, directly into the DR activates serotonergic neurons and increases extracellular 5-HT in DR projection regions like the BLA (Amat et al, 2004).…”

“…We and others have found evidence for the following systems: 1) Panic inhibition system , a serotonergic system in the ventrolateral part of the dorsal raphe nucleus (DRVL)/ventrolateral periaqueductal gray (VLPAG), projecting to the DPAG, that a) is activated by panicogenic agents, including sodium lactate (Johnson et al, 2008) and hypercapnia (elevated atmospheric CO2; Johnson et al, 2005), b) selectively responds with increased tph2 mRNA expression following amygdala priming (Donner et al, 2012), a model of vulnerability to sodium lactate-induced panic attacks (Johnson et al, 2013;Sajdyk et al, 1999), and c) is selectively dysregulated in an animal model of vulnerability to lactate-induced panic-like responses (Johnson et al, 2004;Johnson et al, 2007). 2) Conflict anxiety facilitation system , a serotonergic system in the midline dorsal and caudal parts of the dorsal raphe nucleus (DRD/DRC), projecting to the basolateral nucleus of the amygdala (BLA; Abrams et al, 2005), that is activated by anxiogenic drugs (Abrams et al, 2005), anxiety-related neuropeptides (Staub et al, 2005;Staub et al, 2006), and anxiety-provoking stimuli (Spannuth et al, 2011); this system is sensitized following inescapable shock in a model of learned helplessness (Rozeske et al, 2011).…”

Opposite-sex housing reactivates the declining GnRH system in aged transgenic male mice with FGF signaling deficiency

“…After dehydration and coverslipping, sections were photographed under identical camera settings. The outlines of two evenly spaced sections from each animal were manually traced, and their optical density (OD) was measured using NIH ImageJ while control-ling for background color and threshold levels (15,16). OD measurements of two sections from a single animal were averaged to obtain a value for that animal.…”