Orexin 1 receptors are a novel target to modulate panic responses and the panic brain network

Abstract

Background: Although the hypothalamic orexin system is known to regulate appetitive behaviors and promote wakefulness and arousal (Sakurai, 2007 [56]), this system may also be important in adaptive and pathological anxiety/stress responses (Suzuki et al., 2005 [4]). In a recent study, we demonstrated that CSF orexin levels were significantly higher in patients experiencing panic attacks compared to non-panicking depressed subjects (Johnson et al., 2010 [9]). Furthermore, genetically silencing orexin synthesis or blocking orexin 1 receptors attenuated lactate-induced panic in an animal model of panic disorder. Therefore, in the present study, we tested if orexin (ORX) modulates panic responses and brain pathways activated by two different panicogenic drugs.

Methods: We conducted a series of pharmacological, behavioral, physiological and immunohistochemical experiments to study the modulation by the orexinergic inputs of anxiety behaviors, autonomic responses, and activation of brain pathways elicited by systemic injections of anxiogenic/panicogenic drugs in rats.

Results: We show that systemic injections of two different anxiogenic/panicogenic drugs (FG-7142, an inverse agonist at the benzodiazepine site of the GABA(A) receptor, and caffeine, a nonselective competitive adenosine receptor antagonist) increased c-Fos induction in a specific subset of orexin neurons located in the dorsomedial/perifornical (DMH/PeF) but not the lateral hypothalamus. Pretreating rats with an orexin 1 receptor antagonist attenuated the FG-7142-induced anxiety-like behaviors, increased heart rate, and neuronal activation in key panic pathways, including subregions of the central nucleus of the amygdala, bed nucleus of the stria terminalis, periaqueductal gray and in the rostroventrolateral medulla.

Conclusion: Overall, the data here suggest that the ORX neurons in the DMH/PeF region are critical to eliciting coordinated panic responses and that ORX1 receptor antagonists constitute a potential novel treatment strategy for panic and related anxiety disorders. The neural pathways through which ORX1 receptor antagonists attenuate panic responses involve the extended amygdala, periaqueductal gray, and medullary autonomic centers.

Figure 1

Effects of anxiogenic drugs on c-Fos expression in orexin (ORX) neurons. a) Graph illustrating the effects of caffeine (Caff) and FG-7142 (FG) on c-Fos expression in the dorsomedial/perifornical hypothalamic (DMH/PeF) region (left) and lateral hypothalamus (right). Black-lined bars in graph illustrate the numbers of ORX neurons from each treatment group that expressed c-Fos and gray-lined bars represent the total number of ORX neurons present. Error bars indicate the standard error of the mean (SEM) and * indicates significance compared to the vehicle group (Fisher’s LSD test). b) photomicrographs of a coronal section of the DMH/PeF region depicting ORX-immunoreactive (ir) neurons (brown cytoplasmic stain) with or without nuclear blue/black c-Fos-immunostained nuclei. Black arrows in Fig. 1b indicate double labelling whereas gray arrow indicates a c-Fos-ir/ORX-immunonegative cell. Scale bar, 10 μm.

Figure 2

Effects of FG-7142 and the ORX 1 receptor antagonist SB334867 (SB33) on behavior in the open-field test. a) Illustration of an open-field test box with outer, middle and center regions. A decrease in time spent exploring the center and middle regions indicates an increase in anxiety-associated behavior. b) Bar graphs illustrate the duration of time each treatment group spent in regions of the open-field test (i.e., from left to right the graphs indicate the time spent in the outer and middle perimeter and center of the open-field during the 5-min test). Treatment groups were as follows: vehicle pre-treatment + vehicle; vehicle pre-treatment + FG-7142; SB334867 pre-treatment + FG-7142. Bars represent the means and error bars represent the SEMs. Abbreviations: Veh, vehicle; SB33, SB334667 (orexin 1 receptor antagonist).

Figure 3

Effects of FG-7142 and the ORX 1 receptor antagonist SB334867 (SB33) on behavior in the social interaction (SI) test. a) Illustration of an SI test box where a decrease in the time spent initiating social contacts with a partner rat indicates an increase in anxiety-associated behavior. b) Bar graph illustrates the duration of time each treatment group spent initiating social contacts during a 5-min test. The baseline bar indicates the duration of social interaction from all rats at 5 days prior to treatments. Treatment groups were as follows: vehicle pre-treatment + vehicle; vehicle pre-treatment + FG-7142; SB334867 pre-treatment + FG-7142. Bars represent the means and error bars represent the SEMs. * p < 0.05, different from all other treatment groups (Fisher’s SD test). Abbreviations: Veh, vehicle; SB33, SB334667 (orexin 1 receptor antagonist).

Figure 4

Effects of FG-7142 and the ORX 1 receptor antagonist SB334867 (SB) on c-Fos expression in anxiety- and stress-related brain regions. a) Diagrammatic illustrations of brain areas where subregional analyses of the numbers of c-Fos-ir cells were conducted in coronal brain sections as illustrated in a standard stereotaxic atlas of the rat brain [16]. The anterior-posterior coordinate (in mm from bregma) is indicated below each section. Studies by Singewald and colleagues where they observed significant increases in c-Fos-ir cells in specific brain regions following FG-7142 treatment was referenced in selecting regions to assess c-Fos responses [14, 15]. Open circles indicate region with no significant differences between groups; black-filled circles indicate regions where only a FG-7142 effect was observed; and gray-filled circles indicate regions where there was an effect with FG-7142 that was attenuated with the orexin 1 receptor antagonist (i.e., SB334867). Small circles indicate regions counted at 600X magnification and large circles indicate regions counted at 400× magnification. A 1 mm scale bar is located on the right side of the figure. Abbreviations: prelimbic cortex (PRL) and infralimbic cortex (IL) at +2.70 mm bregma; bed nucleus of the stria terminalis (BNST) divisions that included the anterior part of BNST (aBNST) at +0.20 mm bregma; the intermediate (LSI) and ventral (LSV) part of lateral septum at +0.20 and −0.30 mm bregma; the hypothalamic paraventricular nucleus (PVN) at −1.80 mm bregma; the amygdala subdivisions [central amygdaloid nucleus (CeA); basolateral amygdaloid nucleus, anterior part (BLA); lateral amygdaloid nucleus (LA); and the medial amygdaloid nucleus (MeA)] at −2.56 mm bregma; the dorsomedial (DMH) and perifornical (PeF) hypothalamus at −3.14 mm bregma; the dorsal (DPAG), dorsolateral (DLPAG) and ventrolateral VLPAG) periaqueductal gray and dorsal raphe nucleus (DRN) at −7.80 mm bregma;, the lateral (lPBN) and medial (mPBN) part of the parabrachial nucleus at −9.30 mm bregma; the locus coeruleus (LC) at −10.04 mm bregma; and rostroventrolateral medulla (RVLM) at −11.60 mm bregma. b) Graphs illustrating the effects of SB334867 pretreatment on the FG-7142-induced increaseas in numbers of c-Fos-immunoreactive cells in specific forebrain and brainstem regions. Treatment groups were as follows: vehicle pre-treatment + vehicle; vehicle pre-treatment + FG-7142; SB334867 pre-treatment + FG-7142. Bars represent means and errors bars indicated S.E.M.s *, p < 0.05 (Fisher’s Protected LSD). c) The first column in this table indicates brain regions where there was no significant effect of either FG-7142 or SB334867 interaction on the no. of c-Fos-ir cells. Subsequent columns indicate the mean of the no. of c-Fos-ir cells +/- SEM from each treatment group in each brain region. Abbreviations: Veh, vehicle, FG, FG-7142; SB33, SB334667 (orexin 1 receptor antagonist).

Figure 5

Effects of FG-7142 and the ORX 1 receptor antagonist SB334867 (SB33) on heart rate (HR) and locomotor activity. a, b) Line graphs illustrate changes in a) heart rate [HR in beats/min (BPM)] comparing 5 min baseline values (pre-injection) to values measured post-injection, and b) general activity counts between rats systemically injected with vehicle or FG-7142. c, d) Line graphs illustrate changes in c) heart rate [HR in beats/min (BPM)] comparing 5 min baseline values (pre-injection) to values measured post-injection, and d) general activity counts between rats systemically pre-injected with vehicle or SB334867 30 min prior to a systemic FG-7142 injection. *, p < 0.05, between-subjects effects (2-tailed paired t-test). #, p < 0.05, within-subjects effects over time (Dunnett’s test). There were no significant baseline HR or activity differences between groups (see results section). Values of lines represent means, whereas error bars represent S.E.M.s.

Figure 6

Hypothetical model depicting mechanisms through which FG-7142, a beta-carboline, negatively modulates GABA-GABA_A receptor-mediated chloride influx [55] on orexin neurons that leads to depolarization and subsequent release of orexin at postsynaptic targets in the brain and spinal cord that express orexin 1 receptors to increase anxiety-associated behavior and cardioexcitation. Abbreviations: BNST, bed nucleus of the stria terminalis; CeA, central amygdala, DLPAG, dorsolateral periaqueductal gray; ORX, orexin; ORX1-R, orexin 1 receptor; RVLM, rostroventrolateral medulla.