Predator Stress-Induced CRF Release Causes Enduring Sensitization of Basolateral Amygdala Norepinephrine Systems that Promote PTSD-Like Startle Abnormalities

Effects on %PPI of repeated (three presentations) ferret exposure or of various intra-BLA treatments and subsequent subthreshold challenge injections. A, Repeated ferret exposure (termed 1st Ferret, 2nd Ferret, and 3rd Ferret) and subsequent intra-BLA challenges with NE (0.3 μg; which took place 4, 11, and 18 d after the termination of ferret exposure) or systemic yohimbine (1 mg/kg) with accompanying control mock injections (mock2 to mock4). Note that the final challenge (yohimbine) took place 28 d after ferret exposures had ended. B, Repeated intra-BLA CRF infusions (termed first CRF, second CRF, and third CRF; all were 200 ng) with subsequent intra-BLA NE challenge injection (0.3 μg) 4 d after repeated CRF infusions ended. C, Repeated intra-BLA NE infusions (termed first NE, second NE, and third NE; all were 20 μg) with subsequent intra-BLA CRF challenge injection (200 ng) 4 d after repeated NE infusions ended. D, Repeated intra-BLA vehicle (termed first vehicle, second vehicle, and third vehicle) with subsequent intra-BLA NE challenge injection (0.3 μg) 4 d after repeated vehicle infusions ended. Values are reported as the mean ± SEM. *p < 0.05, **p < 0.01, compared with corresponding mock infusion; +p < 0.06, $p < 0.05, compared with the first NE infusion.

Effects on %PPI of CRF1-R antagonist (NBI27914, 1 μg) and repeated ferret exposure (three exposures total in each group: 1st Ferret, 2nd Ferret, and 3rd Ferret). A–C, CRF1-R antagonist was injected into the BLA immediately before (A) or 30 min after (B) each ferret exposure, or immediately before the intra-BLA NE challenge (C; 0.3 μg of NE). Values are reported as the mean ± SEM. NE challenges took place 4 d after the termination of the repeated ferret exposures. NBI, NBI27914. *p < 0.05, compared with mock2.

Effects on %PPI of intra-BLA infusion of the α1 noradrenergic receptor agonist PHEN or the β-receptor agonist ISO. PHEN (30 μg) or ISO (30 μg) were tested 4 d after the termination of the repeated ferret exposures (three exposures total in each group: 1st Ferret, 2nd Ferret, and 3rd Ferret). Values are reported as the mean ± SEM. *p < 0.05, compared with mock2.

A–D, Representative injector tip locations within the CeA (A) and BLA (C) of the amygdala, which are indicated by arrows; chartings depicting the location of infusions of CRF (squares), NE (triangles), or vehicle (circles) into CeA (B) and BLA (D). VM, Ventromedial thalamus; opt, optic tract; Pe, periventricular hypothalamus.

A high degree of colocalization of CRF1 and α1 receptors is seen in BLA neurons. A–A3, Immunofluorescence labeling of CRF1 receptors (A), α1 receptors (A1), DAPI-labeled cells (A2), and their merge (A3) showing the coexpression of red, green, and blue in the same individual cells. A4 shows a high-magnification image of the cell identified with an arrow. B–B2, Double-immunofluorescence labeling in BLA showing α1 receptor (B), NeuN (B1), and their merge (B2). Arrows indicate individual neurons.

BLA neurons that express α1 NE receptors are not GABAergic presumptive interneurons. A, A1, Double-immunofluorescence labeling of GAD67 (A) and α1 NE receptors (A1) from the same section/slice in BLA. B, B1, Immunoperoxidase labeling of GAD67 (brown) and α1 NE receptors (blue) in BLA. Arrows indicate individual cells.

Retrogradely transported microsphere beads injected into nucleus accumbens (NAcc) are deposited in basolateral amygdala (BLA) neurons that express CRF1 receptors, indicating that repeated ferret exposure changes the functional sensitivity of BLA output projections neurons that innervate the NAcc. A–E, Line drawing of NAcc area for microsphere infusion (A); overlaid fluorescent image (B) and close-up (C) showing restricted deposition of retrograde tracer in NAcc; immunofluorescence labeling of CRF1 receptors in the BLA (D), BLA cells with fluorescence-labeled microspheres (E), and the merged image (F) showing CRF1 receptors colabeled with cells that are filled with the microspheres. Arrows indicated representative cells.

Schematic of the working model for predator stress-induced effects in the basolateral amygdala. A, Before our studies, it was known only that α1 () and CRF1 () receptors were both present in BLA, but their localization relative to each other and on different types of BLA cells (glia, orange; GABAergic inhibitory interneurons, blue; excitatory output neurons, green) was not known. B, We completed immunohistochemical and tract-tracing studies (Experiment 6; Figs. 6, 7, 8) to address these gaps in knowledge. First, we found that α1 and CRF1 receptors are colocalized on the same neurons and not on glial cells. Second, we failed to find these receptors on GABAergic cells. Third, we found that these receptors are present on BLA glutamatergic projection neurons that innervate the NAcc. Finally, we found that these output projection neurons that express CRF1 and α1 receptors are innervated by GABAergic neurons (presumptive inhibitory interneurons). C, Working model for behavioral sensitization induced by repeated predator exposure. Repeated exposure to predator stress sensitizes α1 receptors on BLA output neurons via initial activation of CRF1 receptors. Sensitized α1 receptors are hence abnormally responsive to low levels of NE, leading to increased glutamate release in downstream targets such as the NAcc, and thereby disrupting PPI. This putative mechanism of stress-induced BLA NE α1 receptor sensitization could thus contribute to startle abnormalities (exaggerated startle and disrupted PPI) that are seen in post-traumatic stress disorder.

Preabsorption with α1 peptide and CRF1 peptide eliminates receptor-like labeling in the BLA, shown with parallel fluorescence and immunoperoxidase methods. A–A2, α1 Preabsorption and secondary alone controls with fluorescence labeling. A3–A5, Bright-field images of the preabsorption and secondary-alone controls for α1 receptor using an immunoperoxidase staining method. B–B2, CRF1 preabsorption and secondary-alone controls with fluorescence labeling. B3–B5, bright-field images of the preabsorption and secondary alone controls for CRF1 receptor, using an immunoperoxidase method. With the CRF1 receptor antibody preabsorption, we noticed some residual labeling resembling blood vessels (see B1), which is consistent with the presence of CRF2-R (CRF2-R is also recognized to some degree by the sc-1757 antibody; Lukkes et al., 2011). However, receptor-like, “ring-shaped” structures were eliminated by primary antibody preabsorption with CRFR1 antigen. Asterisks denote presumptive receptors; arrows denote presumptive blood vessels.