A key role for orexin in panic anxiety

Abstract

Panic disorder is a severe anxiety disorder with recurrent, debilitating panic attacks. In individuals with panic disorder there is evidence of decreased central gamma-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.

Figure 1

a) Left: Mean no. of c-Fos/ORX-A-ir neurons in the DMH/PeF and LH of rats challenged with either sodium lactate (Lac) or saline (Sal). Bars with black lines = no. of c-Fos/ORX-A-ir neurons; grey, open bars = total no. of ORX-A-ir neurons. Middle: photomicrographs representing c-Fos (blue nuclei) and ORX-A-ir neurons (brown cytoplasmic) in the DMH with arrows indicating c-Fos/ORX-A-ir neurons. Scale bars = 50 μm, 125 μm for inset. Right: Mean no. of c-Fos/ORX-A-ir neurons in the DMH/PeF correlated with changes in social interaction (SI). Effects of prior (48h) injections of small interfering (si) RNA targeting prepro-orexin mRNA (siORX), but not control siRNA (siCON), into the DMH/PeF of panic-prone rats (l-AG treated) on: b) anxiety-like responses (SI duration, * and † indicate p<0.05); c) general locomotor activity; d) heart rate (HR); and e) mean arterial blood pressure (MAP). For locomotor and cardiovascular data, *, a, and b indicate p<0.05. f) Coronal illustration of unilateral l-AG infusions, bilateral siORX or siCON injections, and micropunches taken for mRNA assays; g) Effects of siORX into the DMH/PeF of control rats on concentrations of local prepro-ORX (ppORX) mRNA in the combined DMH/LH. Effects of bilateral injections of siORX or siCON into the DMH/PeF of panic-prone rats challenged with Lac or saline on local h) ppORX, i) pro-dynorphin (pDyn) and j) proopiomelanocortin (POMC) mRNA. The last bar in figures 1h-j represents the concentration of mRNA in the DMH/LH of untreated homecage control rats. For Fig. 1h-j, * and # respectively indicate p<0.05 compared to siCON/Sal or siORX+Sal groups. All mRNA levels are expressed relative to beta-actin mRNA levels. Bars and lines represent means; error bars represent SEM. Abbreviations: contra, contralateral; DA, dorsal hypothalamic area; DMH, dorsomedial hypothalamus; ipsi, ipsilateral; LH, lateral hypothalamus.

Figure 2

a) Effect of systemically injecting the ORX1 receptor antagonist (SB334867) or benzodiazepine (alprazolam) into panic-prone rats prior to NaLac challenges on a) anxiety-like responses [social interaction (SI)], general locomotor activity, heart rate (HR), and mean arterial blood pressure (MAP). For anxiety tests in Fig. 2a, and 2b, * and + indicate significant differences between groups using a Tukey's HSD tests with p<0.05. For locomotor and cardiovascular data in Fig 2a–c * indicates significantly different from baseline using a Dunnet's test and † indicate significant differences between groups using a Tukey's HSD or paired t-test with p<0.05. b) Effects of systemically injecting SB334867 into panic-prone rats prior to NaLac challenges on anxiety-like responses (decreased time spent in center of open field), general locomotor activity, HR, and MAP. c) Effects of systemically injecting a 2^nd ORX1 receptor antagonist (SB408124) into panic-prone rats prior to NaLac on SI duration, general locomotor activity, HR and MAP. See Suppl. Fig. 2a–2c for respective probe-placement verification. Bars and lines in graphs represent the mean, and error bars represent the standard error of the mean.

Figure 3

Assessment of defensive shock associated behaviors in panic-prone rats (l-AG treated) challenged with isotonic saline (Sal) or hypertonic sodium lactate with a prior intraperitoneal injection of either a vehicle (Veh) or an orexin 1 receptor antagonist (30mg/kg SB334868: SB33). Duration of: a) freezing, near probe (see gray shaded area in defensive shock cage illustration in inset in figure 3a) grooming, burying; and b) number of line crossings (see dashed line in illustration inset in figure 3a). * indicates significant differences between all other groups using a Tukey's HSD tests with p<0.05. c) Effects of unilateral injections of the ORX1 receptor antagonist SB334867 (300pmoles/100nl) into the bed nucleus of the stria terminalis (BNST) of l-AG treated rats, prior to a sodium lactate challenge, on the duration of SI (compared to SI duration following vehicle injection). Bars in graphs represent the mean, and error bars represent the standard error of the mean. * indicates significant differences between groups using a paired t-test with p<0.05. See Supplemental Figure 1d for l-AG infusion placement verification in DMH/PeF and injections sites into the BNST.

Figure 4

Results of the human translational study: bar graph illustrates ORX concentrations in cerebrospinal fluid (CSF: obtained by lumbar puncture) samples from all subjects, which were assayed using a radioimmunoassay. A cohort of subjects who presented with acute suicidal behavior was systematically assessed for psychiatric symptoms utilizing the comprehensive psychopathological rating scale (CPRS), where item 3 (inner tension) assesses panic anxiety. A threshold cut off at 1.5 on this item was used to define a subject as having significant panic symptoms. All subjects with substance abuse and traces of antidepressive, neuroleptic or anxiolytic medication in the blood were excluded from the analysis. A total of 53 medication-free subjects were included comprising three groups: subjects with panic anxiety without major depressive disorder (MDD) (n=12); subjects with both panic and co-morbid MDD (n=13); and subjects without panic, without MDD (n=28). * indicates significant differences from other groups, using Kruskall Wallis ANOVA (P=0.004); and two-tailed Mann-Whitney U-test (subjects with panic and MDD, p= 0.002; subjects without panic, p=0.01). Bars in graph represent the mean, and error bars represent standard deviation of the mean. Supplemental Table 2 describes details of subject characteristics. Age and gender did not have any impact on CSF ORX levels (Pearsons R and Mann-Whitney U-tests, P>0.1).