Acute engagement of Gq-mediated signaling in the bed nucleus of the stria terminalis induces anxiety-like behavior

Abstract

The bed nucleus of the stria terminalis (BNST) is a brain region important for regulating anxiety-related behavior in both humans and rodents. Here we used a chemogenetic strategy to investigate how engagement of G protein-coupled receptor (GPCR) signaling cascades in genetically defined GABAergic BNST neurons modulates anxiety-related behavior and downstream circuit function. We saw that stimulation of vesicular γ-aminobutyric acid (GABA) transporter (VGAT)-expressing BNST neurons using hM3Dq, but neither hM4Di nor rM3Ds designer receptors exclusively activated by a designer drug (DREADD), promotes anxiety-like behavior. Further, we identified that activation of hM3Dq receptors in BNST VGAT neurons can induce a long-term depression-like state of glutamatergic synaptic transmission, indicating DREADD-induced changes in synaptic plasticity. Further, we used DREADD-assisted metabolic mapping to profile brain-wide network activity following activation of G_q-mediated signaling in BNST VGAT neurons and saw increased activity within ventral midbrain structures, including the ventral tegmental area and hindbrain structures such as the locus coeruleus and parabrachial nucleus. These results highlight that G_q-mediated signaling in BNST VGAT neurons can drive downstream network activity that correlates with anxiety-like behavior and points to the importance of identifying endogenous GPCRs within genetically defined cell populations. We next used a microfluidics approach to profile the receptorome of single BNST VGAT neurons. This approach yielded multiple G_q-coupled receptors that are associated with anxiety-like behavior and several potential novel candidates for regulation of anxiety-like behavior. From this, we identified that stimulation of the G_q-coupled receptor 5-HT_2CR in the BNST is sufficient to elevate anxiety-like behavior in an acoustic startle task. Together, these results provide a novel profile of receptors within genetically defined BNST VGAT neurons that may serve as therapeutic targets for regulating anxiety states and provide a blueprint for examining how G-protein-mediated signaling in a genetically defined cell type can be used to assess behavior and brain-wide circuit function.

Figure 1

Select modulation of BNST VGAT neurons using DREADDs. (a) Stereotaxic delivery of viruses encoding a Cre-inducible mCherry or DREADD (hM3Dq or hM4Di) into the BNST of VGAT-Cre mice. (b) Representative sections showing expression of a Cre-inducible mCherry (left), hM3Dq (middle), or hM4Di (right). Scale bars indicate 500 µm. (c) Schematic of ex vivo slice electrophysiology in hM3Dq-mCherry-expressing BNST neurons in the presence of CNO. (d) Representative traces from an hM3Dq-expressing BNST VGAT neuron depolarized by bath application of 10 µM CNO in the presence of TTX, but not in the presence of the PLC inhibitor U73122. (e) Average change in resting membrane potential during the last 2 minutes of CNO application in ACSF + TTX with and without U73122 (right). N: ACSF, 6 cells from 4 mice; ACSF + U73122, 4 cells from two mice. (f) Time course of hM3Dq-induced reduction in evoked EPSCs is blocked by CB₁R antagonist SR 141716A. **, p<0.01, Mann Whitney test. Error bars indicate SEM. (g) Representative superimposed average evoked responses of the five minutes before CNO bath application (solid trace) and 20 to 25 minutes of washout (dotted line). (h) Mean evoked EPSC amplitude during minutes 30–35 (20–25 minutes of washout). F_(2,10)=10.36, p=0.0037. *p<0.05, Tukey’s multiple comparison test; **p<0.01 Tukey’s multiple comparison test. (i) Schematic of ex vivo slice electrophysiology in hM4Di-mCherry-expressing BNST neurons in the presence of CNO. (j) Representative tracing showing hyperpolarization of hM4Di-mCherry-expressing BNST neuron in the presence of 10 µM CNO. (k) Mean hyperpolarization induced by 10 µM CNO. **p<0.01, one-sample t-test. N: 10 cells from 6 mice. (l) Mean change in rheobase following bath CNO application. **p<0.01, one-sample t-test. N: 5 cells from 4 mice.

Figure 2

Chemogenetic activation of BNST VGAT neurons increases anxiety-like behavior. (a) Behavioral assay design. VGAT-Cre mice expressing DIO-mCherry, DIO-hM3Dq, or DIO-hM4Di in the BNST were injected with 3.0 mg/kg CNO 30 minutes before being tested in exploratory assays. (b) Elevated plus maze (EPM). (c) Distance traveled (left; F_(2,23)=1.413, p=0.26), time in open arms (middle; F_(2,23)=3.894, p<0.05, One-Way ANOVA and Tukey’s post-hoc test) and probability of an open arm entry (right; F_(2,23)=6.305, p<0.01, One-Way ANOVA, p<0.05, Tukey’s post-hoc test) during a 5 minute EPM session. N: 8 mcherry, 9 hM3Dq, 9 hM4Di). (d) Averaged heat maps showing time spent in open and closed arms for mCherry (left), hM3Dq (center), and hM4Di (right) expressing mice. (e) Open field. Distance traveled (left; F_(2,18)=0.2503, p=0.78, One-Way ANOVA. N: 7 mCherry, 6 hM3Dq, 8 hM4Di), time in the center (middle; F_(2,18)=0.5538, p=0.58. N: 7 mCherry, 6 hM3Dq, 8 hM4Di), and latency to enter the center (right; F_(2,22) = 0.3674, p=0.6967. N: 8 mCherry, 9 hM3Dq, 8 hM4Di) during a 30 minute open field session. (f) Light-dark box. Time in (left; F_(2,23)=5.266, p<0.05, One-Way ANOVA, p<0.05, Tukey’s post-hoc test) and entrances to (right; F_(2,23)=3.629, p<0.05), One-Way ANOVA, p<0.05, Tukey’s post-hoc test) the light compartment during a 15 minute session. N: 8 mCherry, 9 hM3Dq, 9 hM4Di. *p <0.05 relative to mCherry, # p<0.05 relative to hM4Di, Tukey’s post hoc test. Error bars indicate SEM.

Figure 3

Metabolic mapping of downstream activity following CNO-induced activation of hM3Dq in BNST VGAT neurons. (a) Injection of a DIO-mCherry to the BNST of a VGAT-Cre to label projection fibers. (b) mCherry fluorescence observed in the BNST injection site (left) and fluorescent fibers in the VTA (middle) and PB/LC (right). Scale bars: 1 mm. (c) µPET imaging timeline. Mice were injected with vehicle or CNO and five minutes later injected with FDG. 25 minutes later mice were anesthetized with isoflurane and placed on the scanning bed for a 20 minute scanning session. (d) Representative image of a DIO-hM3Dq-mCherry BNST injection site. Scale bar: 500 µm. (e) Increased FDG uptake in areas corresponding to the BNST (left) VTA (middle) and PB/LC (right) following activation of hM3Dq in BNST VGAT neurons. (f) Representative injection of a DIO-hM3Dq-mCherry to a Cre-negative control mouse. (g) No change in FDG uptake in the BNST (left), VTA (middle), or PB/LC (right) from pooled adjacent controls. N: 4 mice per group. A.c.: anterior commissure.

Figure 4

Single-cell qPCR analysis reveals G_q-coupled GPCRs in BNST VGAT cells. (a) BNST tissue was dissected from a VGAT-Cre×L10-EGFP mouse and dissociated to create a single-cell suspension. Following FACS, individual EGFP-positive cells were captured on a C1 chip and used for single-cell qPCR with probes targeting G_q-coupled receptors. (b) Heat map depicting delta Ct values of individual probes from 149 cells positive for Slc32a1, Map2, and Gad1 and/or Gad2. Values are normalized to RN18S. (c) Percent of cells expressing transcripts for each G_q-coupled GPCR. (d–h) Representative fluorescent in situ hybridization sections for assessing colocalization of VGAT mRNA and mRNA for mGluR5 (d), M₁mAchR (e), 5-HT_2CR (f), α1-AR (g), α1-BR (h). Scale bar: 100 µm. (i) Percent of VGAT mRNA positive cells expressing various GPCR transcripts. Error bars indicate SEM. Gene (Protein): Grm5 (MGLUR5), Ntsr2 (NTSR2), Chrm1 (CHRM1), Htr2c (HTR2C), Grm1 (mGluR1), Adra1a (α1-AR), Tacr1 (TACR1), Htr2a (5-HT_2AR), Chrm3 (M₃ mAChR), Calcr (CT), Oxtr (OXTR), Chrm5 (M₅ mAChR), Adra1b (α1-BR), Ntsr1 (NTSR1), Adra1d (α1-DR), Npsr1 (NPSR1), Gpr171(GPR171), Cckar (CCKAR), Htr2b (5-HT_2BR), Avpr1b (AVPR1B).

Figure 5

Local infusion of mCPP to the BNST increases acoustic startle. (a) Mice with cannulae inserted over the BNST received an infusion of 1 µg mCPP and were immediately placed in the acoustic startle chamber. Following a 5 minute habituation period, mice were presented with 30 startle stimuli. (b) Mice treated with mCPP showed an exacerbated acoustic startle response (t(14) = 3.015, p = 0.0093). N: 7 vehicle, 9 mCPP. **p<0.01, unpaired t-test. Error bars indicate SEM.