α(2A)-adrenergic receptors filter parabrachial inputs to the bed nucleus of the stria terminalis

Abstract

α2-adrenergic receptors (AR) within the bed nucleus of the stria terminalis (BNST) reduce stress-reward interactions in rodent models. In addition to their roles as autoreceptors, BNST α(2A)-ARs suppress glutamatergic transmission. One prominent glutamatergic input to the BNST originates from the parabrachial nucleus (PBN) and consists of asymmetric axosomatic synapses containing calcitonin gene-related peptide (CGRP) and vGluT2. Here we provide immunoelectron microscopic data showing that many asymmetric axosomatic synapses in the BNST contain α(2A)-ARs. Further, we examined optically evoked glutamate release ex vivo in BNST from mice with virally delivered channelrhodopsin2 (ChR2) expression in PBN. In BNST from these animals, ChR2 partially colocalized with CGRP, and activation generated EPSCs in dorsal anterolateral BNST neurons that elicited two cell-type-specific outcomes: (1) feedforward inhibition or (2) an EPSP that elicited firing. We found that the α(2A)-AR agonist guanfacine selectively inhibited this PBN input to the BNST, preferentially reducing the excitatory response in ex vivo mouse brain slices. To begin to assess the overall impact of α(2A)-AR control of this PBN input on BNST excitatory transmission, we used a Thy1-COP4 mouse line with little postsynaptic ChR2 expression nor colocalization of ChR2 with CGRP in the BNST. In slices from these mice, we found that guanfacine enhanced, rather than suppressed, optogenetically initiated excitatory drive in BNST. Thus, our study reveals distinct actions of PBN afferents within the BNST and suggests that α(2A)-AR agonists may filter excitatory transmission in the BNST by inhibiting a component of the PBN input while enhancing the actions of other inputs.

Keywords: adrenergic receptors; bed nucleus of the stria terminalis; excitatory transmission; extended amygdala; norephinephrine; optogenetics.

Figure 1.

Optogenetic targeting of the parabrachial input to the BNST. A, Illustrated mouse indicating that the experiments done in Figure 1A–E used a C57BL/6J mouse that was injected with AAV5-CaMKIIα-ChR2:YFP at least 5 weeks prior. B, Atlas image of BNST slice indicating that we are recording within the dorsal BNST while optically stimulating PBN terminals in the BNST (slice image adapted from the Franklin and Paxinos Mouse Brain Atlas). C, Representative image of ImageJ (Fiji) image analysis done of a BNST cell and its surrounding neuropil after injection of AAV5-CaMKIIα-ChR2:YFP into the PBN of a C57BL/6J mouse 5 weeks prior. Colocalization of the gray value of CGRP (purple) with the gray value of ChR2 (green) is observed. D, Image (63×) characterizing expression of ChR2-YFP in the BNST (D1, left) and at the PBN injection site (D2, right) 5 weeks after microinjection into the PBN. Left, Colocalization (yellow, indicated by white arrow) of CGRP (purple) with ChR2-YFP (green) in the dorsal BNST (D1). Right, ChR2-YFP expression (yellow) at the PBN injection site (D2). E, Representative traces of the dual component oEPSC generated by stimulation of the PBN input to the BNST. F, CGRP staining (green) can be seen in the dorsal anterolateral BNST of wild-type mice (F1, left) and is absent in CGRP KO mice (F2, right). GAD67 staining is shown in the dorsal BNST (red). G, Colocalization of VGluT2 (green) with CGRP staining (red) in the dorsal anterolateral BNST.

Figure 2.

Parabrachial inputs to the BNST innervate at least two types of neurons. A, Illustrated mouse indicating that the experiments done in Figure 2 used C57BL/6J mice that were injected with AAV5-CaMKIIα-ChR2:YFP at least 5 weeks prior. B, A representative trace of a PBN-activated cell firing action potentials is shown (B1). A representative trace of an IPSP recorded from a PBN-inhibited cell is shown (B2). A pie chart showing the relative prevalence of each cell type is shown. PBN-activated cells comprise 52.9% of the 34 cells recorded from, and PBN-inhibited cells comprise 47.1% (B3). C, Stepwise current injection into a PBN-activated cell; I_h current can be seen at hyperpolarized potentials (C1). Stepwise current injection into a PBN-inhibited cell; no I_h current is seen at hyperpolarized potentials (C2). Overlapping action potentials showing that action potentials generated in PBN-activated cells (top) have a faster rise time than PBN-inhibited cells (bottom) (C3). D, Kynurenic acid (4 mm) blocks the feedforward IPSP generated by PBN-inhibited cells. Representative traces of an IPSP before drug application (D1) and after drug application (D2) are shown. E, Time course showing the block of feedforward IPSPs generated in PBN-inhibited cells by kynurenic acid (4 mm) (p < 0.01, n = 5).

Figure 3.

Immunolabeling for HA-tagged α_2A-AR in mouse BNST. A, Illustrated mouse indicating that the experiments done in Figure 3 used HA-α_2A-AR-knockin mice (A1). Immunolabeling directed against the HA tag produced some patches of intracellular labeling in neuronal elements (arrow); however, the bulk of labeling observed appeared to be extracellular (arrowhead), appearing to fill spaces between elements of the neuropil, producing the effect of “outlining” them with reaction product (A2). B–E, When animals were treated with clonidine before death, the “outlining” was less frequent and instead reaction product was observed inside neuronal elements. Dendrites (B, arrow) and preterminal axons (C, arrowheads) were commonly observed. Labeled dendritic spines (C, arrow) and axon terminals (D, E, arrows) were also seen. The labeled axon terminals sometimes made asymmetric synaptic contacts (E). Scale bar, 500 nm. F, Bar graph showing relative abundance of α_2A-ARs in each synapse type in both the dorsal and ventral BNST.

Figure 4.

Guanfacine depresses excitatory transmission from the parabrachial nucleus projection to the BNST. A, Illustrated mouse indicating that the experiments done in Figure 4 used C57BL/6J mice that were injected with AAV5-CaMKIIα-ChR2:YFP at least 5 weeks prior. B, Individual experiment showing decrease in excitatory transmission of the PBN input to the BNST by guanfacine that is reversed by atipamezole (1 μm). Example traces of each phase of drug application are shown (insets). C, Individual experiments showing that excitatory transmission is significantly decreased from baseline by guanfacine (1 μm) (p < 0.01, n = 7). Subsequent application of atipamezole reverses this depression such that amplitudes of oEPSCS are no longer significantly different from baseline (not significant, n = 7). *, Significant; n.s., not significant. D, Guanfacine (1 μm) was applied to oEPSCs recorded from stimulation of the PBN afferents in the BNST. Guanfacine was allowed to washout for 30 min. With prolonged washout, the depression of the excitatory PBN input to the BNST did not reverse to baseline levels (p < 0.05, n = 3). E, Guanfacine (1 μm) has no apparent effect on excitatory transmission from the BLA to the BNST in normal ACSF (white circles) (p < 0.01, n = 5). Preincubation of ex vivo BNST slices with atipamezole (1 μm) does not alter the effect of guanfacine on excitatory transmission from the BLA to the BNST (black circles) (p < 0.01, n = 4). F, Baclofen (10 μm) depresses excitatory transmission from the BLA to the BNST (p < 0.01, n = 9). Image of the BLA injection site 5 weeks after injection of the AAV5-CaMKII α-ChR2:YFP into the BLA (inset, right). Expression of ChR2-YFP in the dorsal BNST 5 weeks after injection of the viral vector into the BLA (inset, left).

Figure 5.

Guanfacine reduces EPSPs of PBN-activated cells in the BNST. A, Illustrated mouse indicating that the experiments done in Figure 5 used C57BL/6J mice that were injected with AAV5-CaMKIIα-ChR2:YFP at least 5 weeks prior. B, Guanfacine (1 μm) significantly decreases the size of EPSPs recorded from PBN-activated cells in the BNST (p < 0.05, n = 5). C, Guanfacine (1 μm) has variable effects on IPSPs recorded from the PBN-inhibited cells in the BNST (not significant, p = 11). D, Individual experiments showing the effect of guanfacine (1 μm) on EPSPs recorded from PBN-activated cells in the BNST. EPSPs are reduced by guanfacine across all experiments. E, Individual experiments showing the effect of guanfacine (1 μm) on IPSPs recorded from PBN-activated cells in the BNST. The effect of guanfacine on IPSPs is more variable with IPSPs being reduced in some experiments and unaltered in others.

Figure 6.

Guanfacine increases field potentials in the dBNST of Thy1-COP4 transgenic mice. A, Illustrated mouse indicating that the experiments done in Figure 6 used Thy1-COP4 transgenic mice. B, Image of dorsal BNST (63×) showing lack of colocalization of CGRP (red) with ChR2 (green) in Thy1-COP4 mice. NeuN staining is shown in blue (B1). Representative image of ImageJ (Fiji) analysis done of a BNST cell and its surrounding neuropil in a Thy1-COP4 transgenic mouse. ImageJ (Fiji) image analysis does not show colocalization of CGRP (red) with the ChR2 (green). There is also no observed colocalization of CGRP or ChR2 with NeuN (blue) (B2). C, Optical field potential (oN) size is increased with guanfacine (1 μm) application in Thy1-COP4 mice (p < 0.05, n = 7). Representative trace of oN is shown (inset). D, Preincubation of ex vivo BNST slices with atipamezole (1 μm) blocks the increase in size of the optical field potentials with subsequent guanfacine (1 μm) application (not significant, n = 5).