Chronic stress increases pituitary adenylate cyclase-activating peptide (PACAP) and brain-derived neurotrophic factor (BDNF) mRNA expression in the bed nucleus of the stria terminalis (BNST): roles for PACAP in anxiety-like behavior

Abstract

Exposure to chronic stress has been argued to produce maladaptive anxiety-like behavioral states, and many of the brain regions associated with stressor responding also mediate anxiety-like behavior. Pituitary adenylate cyclase activating polypeptide (PACAP) and its specific G protein-coupled PAC(1) receptor have been associated with many of these stress- and anxiety-associated brain regions, and signaling via this peptidergic system may facilitate the neuroplasticity associated with pathological affective states. Here we investigated whether chronic stress increased transcript expression for PACAP, PAC(1) receptor, brain-derived neurotrophic factor (BDNF), and tyrosine receptor kinase B (TrkB) in several nuclei. In rats exposed to a 7 days chronic variate stress paradigm, chronic stress enhanced baseline startle responding induced by handling and exposure to bright lights. Following chronic stress, quantitative transcript assessments of brain regions demonstrated dramatic increases in PACAP and PAC(1) receptor, BDNF, and TrkB receptor mRNA expression selectively in the dorsal aspect of the anterolateral bed nucleus of the stria terminalis (dBNST). Related vasoactive intestinal peptide (VIP) and VPAC receptor, and other stress peptide transcript levels were not altered compared to controls. Moreover, acute PACAP38 infusion into the dBNST resulted in a robust dose-dependent anxiogenic response on baseline startle responding that persisted for 7 days. PACAP/PAC(1) receptor signaling has established trophic functions and its coordinate effects with chronic stress-induced dBNST BDNF and TrkB transcript expression may underlie the maladaptive BNST remodeling and plasticity associated with anxiety-like behavior.

Figure 1. Chronic variate stress is anxiogenic

The startle responses of chronically stressed and control rats were measured in a dark environment (Phase 1). Half of the stressed and control animals were retested in the dark (Dark-Dark), while half were tested in bright light (Dark-Light). Stressed rats exhibited startle enhancement from phase 1 to phase 2 regardless of whether phase 2 testing was in the dark or in the light, indicative of a stress-induced enhancement of the anxiogenic effects of handling on baseline startle amplitude, which has been shown to be mediated by the BNST. * Two-tailed t-test p < 0.05, ** One-tailed t-test p < 0.05

Figure 2. Location of tissue samples harvested for quantitative PCR analysis

Adult male rats were exposed to chronic variate stress for 7 days as described in the text and the indicated brain regions were harvested for quantitative PCR analyses. Coordinates represent mm from Bregma. A. medial prefrontal cortex; B. lateral septum; C. dorsal aspect of the anterolateral BNST; D. ventral aspect of the anterolateral BNST; E. hippocampus; F. habenula; G. central amygdala; H. PVN; I. basolateral amygdala; J. dorsal raphe nucleus; K. locus coeruleus.

Figure 3. Chronic variate stress increases PACAP, PAC₁ receptor and neurotrophin-related transcripts in the BNST

Adult male rats were exposed to chronic variate stress for 7 days as described in the text and the indicated brain regions were harvested for quantitative PCR analyses. All tissue samples from each region were reverse transcribed at the same time with random hexamers to allow quantitation and normalization across samples against 18S RNA. n = 6 for each group; data represent mean fold-change ± SEM against tissue from control unstressed animals. Asterisk, significantly different at p < 0.05. mPFC, medial prefrontal cortex; dBNST, dorsolateral BNST; vBNST, ventrolateral BNST; LS, lateral septum; DR, dorsal raphe nucleus; LC, locus coeruleus; BLA, basolateral amygdala; CEA, central amygdala; PVN, hypothalamic paraventricular nucleus; Hi, hippocampus; hb, habenula; SCG superior cervical ganglion. ND, not determined.

Figure 4. Experiment 3 BNST PACAP infusion sites

Cannulae placements for rats that received 0, 0.1, 0.5 or 1 μg PACAP38 (0.5 μl/side) infusions into the BNST. Each circle represents the center of one injection. Coordinates represent mm from Bregma.

Figure 5. PACAP infusion into the BNST is anxiogenic

The BNST of adult male rats were cannulated bilaterally and the animals were tested for baseline startle for 2 days before PACAP38 injections (see Methods). Twenty-four hours later, the animals were baseline tested again before intra-BNST peptide infusion (0, 0.1 μg, 0.5 μg, 1 μg in 0.5 μl saline vehicle/side) and retesting. BNST PACAP38 dose-dependently increased startle amplitude. Data represent the mean response ± SEM to the last 9 startle stimuli. * Significantly different at p < 0.05.

Figure 6. Experiment 4 BNST PACAP infusion sites

Cannulae placements for rats that received 1 μg PACAP38 (0.5 μl/side) infusions into the BNST. Each circle represents the center of one injection. Coordinates represent mm from Bregma.

Figure 7. The anxiogenic effect of BNST PACAP infusion persists for 7 days

The BNST of adult male rats were cannulated bilaterally and the animals were tested for baseline startle as described in Experiment 3. Rats were tested for baseline startle immediately after intra-BNST infusion with either 1 μg PACAP38 or vehicle (0.5 μl/side), and subsequently returned to their home cages. 7 days later, rats were returned to the startle chambers and tested again. Data represent startle amplitude after injections into the BNST, as a percent of pre-injection day 1 baseline. A. BNST PACAP38 increased startle amplitude immediately after infusion. Blocks represent the mean response to the last 9 startle stimuli. B. The increase in startle amplitude after a single PACAP38 injection was maintained 7 days later, suggesting plasticity changes within the BNST. Data represent mean ± SEM. * Significantly different at p < 0.05.