Whole-brain input mapping of the lateral versus medial anterodorsal bed nucleus of the stria terminalis in the mouse - PubMed (original) (raw)
Whole-brain input mapping of the lateral versus medial anterodorsal bed nucleus of the stria terminalis in the mouse
Yanjun Sun et al. Neurobiol Stress. 2023.
Abstract
The anterior portion of the bed nucleus of the stria terminalis (BNST) modulates fear and stress responses. The anterodorsal BNST (adBNST) can be anatomically subdivided further into the lateral and medial divisions. Although output projections of BNST subregions have been studied, the local and global input connections to these subregions remain poorly understood. To further understand BNST-centered circuit operations, we have applied new viral-genetic tracing and functional circuit mapping to determine detailed synaptic circuit inputs to lateral and medial subregions of adBNST in the mouse. Monosynaptic canine adenovirus type 2 (CAV2) and rabies virus-based retrograde tracers were injected in the adBNST subregions. The amygdalar complex, hypothalamus and hippocampal formation account for the majority of overall inputs to adBNST. However, lateral versus medial adBNST subregions have distinct patterns of long-range cortical and limbic brain inputs. The lateral adBNST has more input connections from prefrontal (prelimbic, infralimbic, cingulate) and insular cortices, anterior thalamus and ectorhinal/perirhinal cortices. In contrast, the medial adBNST received biased inputs from the medial amygdala, lateral septum, hypothalamus nuclei and ventral subiculum. We confirmed long-range functional inputs from the amydalohippocampal area and basolateral amygdala to the adBNST using ChR2-assisted circuit mapping. Selected novel BNST inputs are also validated with the AAV axonal tracing data from the Allen Institute Mouse Brain Connectivity Atlas. Together, these results provide a comprehensive map of the differential afferent inputs to lateral and medial adBNST subregions, and offer new insight into the functional operations of BNST circuitry for stress and anxiety-related behaviors.
Keywords: BNST; CAV2; Monosynaptic inputs; Rabies; Retrograde.
© 2023 The Authors.
Conflict of interest statement
The authors declare no conflict of interest.
Figures
Fig. 1
CAV2-Cre injections into subregions of the BNST in the Cre reporter Ai9 mice. (A) Top left: schematic illustration of the CAV2-Cre virus injection into the alBNST of an Ai9 mouse. CAV2-Cre-infected neurons near the injection site express tdTomato red fluorescent protein. Top right: injection site images from two representative mice targeting alBNST. The panels on the right are zoom-in views of the boxed regions in the panels on the left. The white dotted line shows the contours of the entire adBNST. Scale bar, 1 mm and 200 μm respectively and apply to similar panels in B and C. Bottom: example anterior to posterior images of the alBNST injection site from a third representative case. Scale bar: 200 μm. (B) Panels organized similar to A, but for targeting the amBNST. (C) Organized similar to A, but for targeting the entire adBNST. (D) Cre-dependent AAV injections targeting the oval region of the BNST (BNSTov) using the PKC-δ-Cre mouse revealed axonal projections to the nearby amBNST and ventral BNST. Scale bar: 200 μm. Data images were obtained and modified from Allen Mouse Brain Connectivity Atlas:
connectivity. brain-map.org/projection/experiment/522078446
. Image credit: Allen Institute for Brain Science (Oh et al., 2014). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Fig. 2
Limbic and cortical regions provide strong afferent input to the alBNST. Schematic of E1-deleted CAV2 viral vector expressing the Cre recombinase and of CAV2-Cre-mediated retrograde tracing in the alBNST of Ai9 mice. The alBNST injection sites are shown in Fig. 1A. (A) An example of tdTomato-labeled neurons in the prelimbic cortex (PrL) providing monosynaptic afferent input into the alBNST. Scale bar, 1 mm. (B) High-magnification view of the PrL neurons showing the boxed region in panel A. Scale bar, 200 μm. (C–D) Labeled neurons in the cingulate cortex (Cg1) and PrL. (E) Labeled neurons in the angular insular (AI) and piriform (Pir) cortices. (F) Labeled neurons in the ectorhinal (ECT) and perirhinal (PRh) cortices. (G-H) Labeled neurons in the anteromedial thalamic nucleus (AM). (I-J) Labeled neurons in the thalamus, basolateral amygdaloid nucleus (BLA), lateral (CeL) and medial (CeM) subdivisions of the central amygdaloid nucleus (CeA). (K-L) Labeled neurons in the BLA and CeA. (M-N) Labeled neurons in the posterior part of the basolateral amygdaloid nucleus (BLP), amygdalohippocampal area (AHiA) and posteromedial cortical amygdaloid nucleus (PMCo). (O–P) Labeled neurons in the ventral subiculum/prosubiculum, APir, and PMCo. (Q–R) Labeled neurons in the lateral entorhinal cortex (LEnt), medial entorhinal cortex (MEnt), dorsal raphe (DR), and caudal linear nucleus of the raphe (CLi).
Fig. 3
Amygdalar regions provide robust input to the amBNST. Schematic of E1-deleted CAV2 viral vector expressing the Cre recombinase and of CAV2-Cre-mediated retrograde tracing in the amBNST of Ai9 mice. The amBNST injection site is shown in Fig. 1B. (A–B) Example of sparse labeling in the PrL and infralimbic cortex (IL). Scale bar, 1 mm in panel A and 200 μm in panel B. (C–D) Sparse labeling in the Cg1 and PrL. (E–F) Labeled neurons in the lateral hypothalamus (LH). (G–H) Labeled neurons in the anterior basomedial amygdaloid nucleus (BMA), the anterodorsal (CeMAD), anteroventral (CeMAV), and capsular (CeC) subdivisions of the central amygdaloid nucleus (CeA). Labeling of the BLA was sparse. (I–J) Labeled neurons in the CeA and the anterior part of the BMA. (K–L) Labeled neurons in the posterior part of the BLP and the AHiA. (M–N) Labeled neurons in the APir and PMCo. (O–P) Labeled neurons in the LEnt, APir, and ventral subiculum/prosubiculum. (Q) Weak labeling in the DpG and PAG. (R) Strong labeling in the posterior BNST.
Fig. 4
Input areas to the entire adBNST overlap with areas providing input to the alBNST and amBNST. Schematic of E1-deleted CAV2 viral vector expressing the Cre recombinase and of CAV2-Cre-mediated retrograde tracing in the adBNST of Ai9 mice. The adBNST injection site is shown in Fig. 1C. (A–B) Labeled neurons in the PrL and Cg1. Scale bar, 1 mm in panel A and 200 μm in panel B. (C–D) Labeled neurons in the PrL. (E–F) Labeled neurons in the thalamus and LH. (G–H) Labeled neurons in the BLA, CeMAV, and BMA. (I–J) Labeled neurons in the BLA, BMP, BMA, and CeA. (K–N) Labeled neurons in the BLP, BMP, AHiA, and PMCo. (O–P) Labeled neurons in the Sub, APir, PMCo, and AHiA. (Q–R) Labeled neurons in the LEnt.
Fig. 5
Quantification of afferent inputs to the alBNST versus amBNST. The graphs show the anatomical distribution (% of total inputs) of structures providing afferent input to the alBNST (black) and amBNST (red). The contribution of afferents from each anatomical brain region on the x-axis is shown as a percentage of the total number of labeled neurons providing afferent inputs into the BNST subregions. See Table 1, Table 2, Table 3 for detailed quantification and statistical comparisons. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Fig. 6
Retrograde tracing of afferent inputs to the alBNST using a modified rabies virus. Schematic of SADΔG-EGFP virus injection into the alBNST of wild-type C57BL/6 mice. A modified rabies virus expressing EGFP was co-injected with red fluorescent microbeads for unambiguous identification of the injected area. The gray dotted line shows the contours of the entire adBNST. (A–B) A representative image of an injection site into the alBNST. Scale bar, 1 mm in panel A and 200 μm in panel B. (C–D) Retrogradely-labeled neurons expressing EGFP in the Cg1 and PrL. (E–F) Labeled neurons in the mediodorsal (MD), anteromedial (AM), and central medial (CM) thalamic nuclei. (G–H) Labeled neurons in the BLA and CeA. (I–J) Labeled neurons in the BLP and AHiA. (K–L) Labeled neurons in the APir, BLP, and AHiA. (M–N) Labeled neurons in the DR and CLi. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Fig. 7
Retrograde tracing of afferent inputs to the amBNST using a modified rabies virus. Schematic of SADΔG-EGFP virus injection into the amBNST of wild-type C57BL/6 mice. A modified rabies virus expressing EGFP was co-injected with red fluorescent microbeads for unambiguous identification of the injected area. The gray dotted line shows the contours of the entire adBNST. (A–B) A representative image of an injection site into the amBNST. Scale bar, 1 mm in panel A and 200 μm in panel B. (C–E) Labeled neurons in the septohypothalamic nucleus (SHy), ventromedial preoptic nucleus (VMPO), and lateral septal nucleus (LS). (F–G) Labeled neurons in the hypothalamus. (H–I) Labeled neurons in the BLA, CeA, and BMA. (J–K) Labeled neurons in the BLP and AHiA. (L–M) Labeled neurons in the posterior part of the BLP. (N–P) Labeled neurons in hippocampus area CA1, Sub/ProS, LEnt, and PMCo. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Fig. 8
Verification of functional synaptic input from amydalohippocampal transition area (AHiA) and basolateral amygdala (BLA) to the adBNST. (A) Bright field image showing AAV expression of ChR2-Venus at the injection site in the AHiA. (B) The BNST (dotted white line) superimposed on ChR2-Venus-expressing axons (green) originating from the viral injection shown in panel A. The red circle indicates the recorded cell location in the BNST. (C) Example of excitatory postsynaptic current (EPSC) responses recorded from an amBNST neuron after repeated optogenetic stimulation. (D) Bright field image showing AAV expression of ChR2-Venus at the injection site in the BLA. (E) The BNST (dotted white line) superimposed on ChR2-Venus-expressing axons (green) originating from the viral injection shown in panel D. The red circle indicates the recorded cell location in the BNST. (F) Example inhibitory postsynaptic current (IPSC) responses recorded from a BNST neuron in response to repeated optogenetic stimulation. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Fig. 9
Verification of novel projections to the BNST using anterograde AAV tracing. (A) Top: the injection site of AAV-EGFP in the prelimbic cortex (PrL) in a wild type C57BL/6 mouse. The green color represents the signal from the AAV expression, while the maroon color represents the structure of the brain section. Scale bar: 1 mm and applies to all other panels on the top. Middle: the corresponding axonal projections in the BNST. Scale bar: 200 μm and applies to all other middle and bottom panels. Bottom: same as the middle panel, but segmented EGFP signals. Data images were obtained and modified from Allen Mouse Brain Connectivity Atlas:
connectivity. brain-map.org/projection/experiment/157711748
. (B) Same as A, but for an injection in the anteromedial thalamic nucleus (AM). Data images were obtained and modified from Allen Mouse Brain Connectivity Atlas:
connectivity. brain-map.org/projection/experiment/158840459
. (C) Same as A, but for an injection in the piriform cortex (Pir). Data images were obtained and modified from Allen Mouse Brain Connectivity Atlas:
connectivity. brain-map.org/projection/experiment/146857301
. (D) Same as A, but for an injection in the ectorhinal and perirhinal cortices (ECT/PRh). Data images were obtained and modified from Allen Mouse Brain Connectivity Atlas:
connectivity. brain-map.org/projection/experiment/180435652
. Image credit for this figure: Allen Institute for Brain Science (Oh et al., 2014). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
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