α2A-Adrenergic Receptor Activation Decreases Parabrachial Nucleus Excitatory Drive onto BNST CRF Neurons and Reduces Their Activity In Vivo

Abstract

Stress contributes to numerous psychiatric disorders. Corticotropin releasing factor (CRF) signaling and CRF neurons in the bed nucleus of the stria terminalis (BNST) drive negative affective behaviors, thus agents that decrease activity of these cells may be of therapeutic interest. Here, we show that acute restraint stress increases cFos expression in CRF neurons in the mouse dorsal BNST, consistent with a role for these neurons in stress-related behaviors. We find that activation of α_2A-adrenergic receptors (ARs) by the agonist guanfacine reduced cFos expression in these neurons both in stressed and unstressed conditions. Further, we find that α- and β-ARs differentially regulate excitatory drive onto these neurons. Pharmacological and channelrhodopsin-assisted mapping experiments suggest that α_2A-ARs specifically reduce excitatory drive from parabrachial nucleus (PBN) afferents onto CRF neurons. Given that the α_2A-AR is a G_i-linked GPCR, we assessed the impact of activating the G_i-coupled DREADD hM4Di in the PBN on restraint stress regulation of BNST CRF neurons. CNO activation of PBN hM4Di reduced stress-induced Fos in BNST Crh neurons. Further, using Prkcd as an additional marker of BNST neuronal identity, we uncovered a female-specific upregulation of the coexpression of Prkcd/Crh in BNST neurons following stress, which was prevented by ovariectomy. These findings show that stress activates BNST CRF neurons, and that α_2A-AR activation suppresses the in vivo activity of these cells, at least in part by suppressing excitatory drive from PBN inputs onto CRF neurons.SIGNIFICANCE STATEMENT Stress is a major variable contributing to mood disorders. Here, we show that stress increases activation of BNST CRF neurons that drive negative affective behavior. We find that the clinically well tolerated α_2A-AR agonist guanfacine reduces activity of these cells in vivo, and reduces excitatory PBN inputs onto these cells ex vivo Additionally, we uncover a novel sex-dependent coexpression of Prkcd with Crh in female BNST neurons after stress, an effect abolished by ovariectomy. These results demonstrate input-specific interactions between norepinephrine and CRF, and point to an action by which guanfacine may reduce negative affective responses.

Keywords: BNST; CRF; norepinephrine; parabrachial; stress.

Figure 1.

Restraint stress induces transient cFos expression in BNST CRF neurons in an α_2A-AR sensitive manner. a, Schematic showing timeline of animal injection, restraint stress, and in situ hybridization/immunohistochemistry assays, along with image of coronal section with the dBNST highlighted (purple) to denote region used for analysis throughout. b, Representative images of cFos labeling in the dBNST showing control, 30 min, 1 h, 2 h, and 4 h ± SEM time points after restraint stress. Scale bar, 100 μm. c, Summary data showing the mean ± SEM number of cFos-labeled cells in dBNST sections from control conditions or at various time points following a 1 h restraint stress. n = 3 male mice/group. *Indicates significant difference from control conditions (***p < 0.001, *p < 0.05). d, Summary bar graph showing the percentage of Crh neurons that express Fos 30 min after restraint stress in male and female mice. Males: no stress n = 5, stress n = 5; Females: no stress n = 5, stress n = 4. *Indicates significant difference between stress conditions (*p < 0.05, **p < 0.01). e, Example image of RNA in situ assay (gray, DAPI-labeled nuclei; magenta, Crh transcripts; cyan, Fos transcripts). Boxes provide magnified examples of cells expressing one or both transcripts (magenta, Crh; brown, Crh/Fos; cyan, Fos). f, Representative fluorescent immunohistochemical images of merged cFos (green) and CRF (red) labeling in the dBNST 30 min after restraint stress for no stress/saline, no stress/guanfacine, stress/saline, and stress/guanfacine conditions. Scale bar, 100 μm. g, Summary bar graph showing the mean ± SEM percentage of cFos+ CRF neurons 30 min after restraint stress for the following conditions: no stress/saline (n = 9; 6 male/3 female), no stress/guanfacine (n = 6; 4 male/2 female), stress/saline (n = 8; 5 male/3 female), stress/guanfacine (n = 9; 4 male/5 female). *Indicates significant difference compared with each other group (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Figure 2.

NE regulation of excitatory drive onto CRF neurons is mediated by α-AR signaling and can be blocked by the of α_2A-AR antagonist atipamezole. a–d, Bar graphs summarizing the effects of AR agonists on EPSC amplitude (left) and PPR (right) in BNST CRF neurons. a, Effects of NE (n = 5 cells, N = 3 male mice). b, Effects of α₁-AR agonist methoxamine (Methox; n = 8 cells, 4 male mice). c, Effects of α_2A-AR agonist guanfacine (Guan; n = 5 cells, N = 3 male mice). d, Effects of β-AR agonist isoproterenol (Iso; n = 6 cells, N = 3 male mice) *Indicates significant difference compared with baseline (*p < 0.05). e, Representative EPSC traces before (black) and after (red) AR agonist application. f, Time course showing effects of pretreatment with α_2A-AR antagonist atipamezole on NE modulation of EPSC amplitude in BNST CRF neurons, graphed as a ±SEM. g, Bar graph comparing EPSC amplitude during atipamezole pretreatment before and after NE application (n = 7 cells, N = 1 male, 3 female mice).

Figure 3.

BNST CRF neurons receive kinetically distinct excitatory input from both the insular cortex and PBN, but the PBN input can be regulated by both NE and guanfacine. a, Illustrated mouse indicating that for Figure 3, CRF-tdtomato reporter mice were stereotaxically injected with AAV5-CaMKIIα-ChR2:YFP, along with example stereo microscope images of injection sites for insular cortex and PBN. b, Summary of percentage of BNST CRF cells that respond to insula or PBN activation. c, Representative image of CGRP labeling (purple; marking PBN input) around BNST CRF neurons (red) demonstrating CRF cells surrounded by CGRP (white arrow; white box: magnified image of cell) and CRF cells without PBN input (yellow arrow; yellow box: magnified image of cell). Scale bar, 50 μm. d, Bar graphs comparing oEPSC kinetics between PBN (n = 6 cells, female mice) and insula (n = 10 cells, female mice) stimulation. Top left, Latency to oEPSC peak (ms). Top right, Representative oEPSC traces from insula (red) and PBN (black). Bottom left, Rise time (10–90%) of oEPSC (ms). Bottom right, Decay time (90–10%) of oEPSC (ms). *Indicates significant difference using unpaired t test (*p < 0.05). e, f, Summary graphs showing effect of NE on oEPSC amplitude, graphed as percentage of baseline, for PBN stimulation (d; n = 4, N = 3 female mice) and insula stimulation (e; n = 4, N = 4 female mice). Inset, Representative oEPSC traces before (black) and after (red) NE application. g, Summary graph showing effect of guanfacine on oEPSC amplitude, graphed as percentage of baseline, for PBN stimulation (n = 5, 2 male mice, N = 2 female mice). Inset, Representative oEPSC traces before (black) and after (red) guanfacine application. h–j, Bar graphs comparing average oEPSC amplitude, graphed as percentage of baseline, 5 min before and after drug wash-on. *Indicates significant difference using paired t test (*p < 0.05). All data are represented as mean ± SEM.

Figure 4.

Activation of PBN-expressed Gi-coupled DREADD hM4Di blunts stress-induced Fos activation in Crh cells. a, Schematic showing timeline of animal injection, restraint stress, and RNA in situ hybridization assay. b, Summary bar graph showing effect of CNO injection on percentage of Crh cells that express Fos 30 min after restraint stress (n = 10 mice/group, 5 male/5 female; exception: no stress/saline n = 9). *Indicates significant difference between treatment groups (**p < 0.01, ****p < 0.0001). c, Bar graph summarizing results of control immunohistochemistry experiment showing effect of CNO injection on male C57 mice (saline: n = 4; CNO: n = 3) with no hM4Di DREADD expression. d, Example confocal image showing AAV5-hSyn-hM4D(Gi)-mCherry PBN injection site. Scale bar, 100 μm. scp, Superior cerebellar peduncle; CB, cerebellum. e, Time course of CNO application on EPSC amplitude in BNST CRF cells, graphed as percentage of baseline, comparing control mice (n = 8 cells, N = 2 male, 2 female mice) and PBN hM4Di-expressing mice (n = 7 cells, N = 1 male, 1 female mouse). f, Representative EPSC traces before (black) and after (red) CNO application in mice expressing PBN-hM4Di. g, Summary bar graph showing average EPSC amplitude as a percentage of baseline during last 5 min of CNO application. *Indicates significant difference between control and hM4Di groups using unpaired t test (**p < 0.01). All data are represented as mean ± SEM.

Figure 5.

RNA in situ assay reveals a female-specific stress-induced increase in Prkcd/Crh coexpression that can be altered by ovariectomy. a, Summary bar graph showing the percentage of Crh neurons that express Fos 30 min after restraint stress in male and female mice. *Indicates significant difference compared with each other group (**p < 0.01, ***p < 0.001). b, Example image of RNA in situ assay (gray, DAPI-labeled nuclei; magenta, Crh transcripts; yellow, Prkcd transcripts; cyan, Fos transcripts). Boxes provide magnified examples of cells expressing multiple transcripts (maroon, Crh/Fos; orange, Crh/Prkcd; green, Prkcd/Fos; blue, Crh/Prkcd/Fos). c, Summary bar graph showing the number of Crh-expressing cells 30 min after restraint stress in male and female mice. d, Summary bar graph showing the number of Prkcd-expressing cells 30 min after restraint stress in male and female mice. *Indicates significant difference between stress conditions (*p < 0.05). e, Summary bar graph showing the percentage of colocalized Crh/Prkcd cells that express Fos 30 min after restraint stress in male and female mice. *Indicates significant difference between stress conditions (*p < 0.05). f, Summary bar graph showing the percentage of Crh cells (lacking Prkcd) that express Fos 30 min after restraint stress in male and female mice. *Indicates significant difference between stress conditions (**p < 0.01, ***p < 0.001). a–f, Males: no stress n = 5, stress n = 5; Females: no stress n = 5, stress n = 4. g, Summary bar graph showing effect of CNO injection on percentage of Crh cells that express Prkcd 30 min after restraint stress in male and female mice (n = 5/mice per group; exception: female, no stress/saline n = 4). h, i, Summary bar graphs showing effect of ovariectomy on percentage of Crh cells that express Fos (h) or Prkcd (i) 30 min after restraint stress in female mice (n = 5 mice/group). *Indicates significant difference between stress conditions (**p < 0.01, *p < 0.05). All data are represented as mean ± SEM.