Article Figures & Data
Figures
- Figure 1.
Use of FoxP2 to target-specific clusters of ITCs. The gene FoxP2 is expressed in ITCs and can be used to define different clusters. a, Immunofluorescence for FoxP2 expression in different regions of the amygdala. Number in lower left corner indicates location of coronal section relative to bregma in the anterior-posterior axis. Scale bar represents ∼500 μm. The clusters are labeled according to prior descriptions. The main (mITC), lateral (lITC), and caudal (cITC) nuclei were targeted for input-output mapping circled in yellow and marked by yellow arrow. b, c, FoxP2-cre mice were injected with a cre-dependent mCherry viral vector and tissue was assayed for mCherry and FoxP2 expression. Representative image of the amygdala region where the mITC were targeted with viral vector, see Extended Data Figure 1-1 for targeting other ITC clusters. FoxP2 represented in green, and mCherry in red, in the merged image (left) and individual grayscale images (right). Scale bar is 200 μm. c, Zoomed in image taken from the white box in b presenting the merged image and individual grayscale channels as in b. Scale bar is 25 μm. d, Schematic of experimental strategy for anterograde and retrograde tracing approach performed in FoxP2-cre mice.
- Figure 2.
Anterograde tracing of the main ITC cluster. FoxP2-cre Mice were injected with AAV-phSyn1(s)-FLEX-tdTomato-T2A-SypEGFP into the mITC cluster. a, Injection site targeting the main ITCs, showing the cytosolic td-Tomato. Scale bar represents ∼0.5 mm. Number in lower left corner indicates location of coronal section relative to bregma in the anterior-posterior axis. The white box depicts a region shown at higher magnification (right), image is ∼200 × 200 μm. b, Representative images of projection targets identified by synaptophysin-eGFP expression. c, Corrected Total Fluorescence data for regions targeted by the mITC (n = 3). Error bars show SEM. d, Pie charts depicting the category of regions targeted by the mITC cluster.
- Figure 3.
Retrograde tracing of inputs to the main ITC cluster. Monosynaptic rabies vectors, AAV-hSyn-FLEX-TVA-P2A-GFP-2A-oG and EnvA-G-Deleted Rabies-mCherry, were injected into FoxP2-cre mice to identify the inputs to the mITC cluster. a, Representative coronal section showing targeting of the helper virus (green) and rabies (red) in the mITCs. Scale bar represents ∼0.5 mm. White box in merged image outlines the region shown in b of starter cells in the mITC cluster, image is ∼200 × 200 μm. c, Representative images show regions identified to have presynaptic neurons identified by mCherry expression. Number in lower left corner indicates location of coronal section relative to bregma in the anterior-posterior axis. d, bar graphs show corrected total fluorescence quantification of the presynaptic regions (n = 3). Error bars show SEM. e, The pie chart depicts the category of regions that project to the mITC cluster.
- Figure 4.
Anterograde tracing of the caudal ITC cluster. FoxP2-cre Mice were injected with AAV-phSyn1(s)-FLEX-tdTomato-T2A-SypEGFP into the cITC cluster. a, Injection site targeting the cITCs, showing the cytosolic td-Tomato. Scale bar represents ∼0.5 mm. The white box depicts a region shown at higher magnification (right), scale bar is ∼50 μm. b, Representative images of projection targets identified by expression of synaptophysin-eGFP. Number in lower left corner indicates location of coronal section relative to bregma in the anterior-posterior axis. c, Corrected total fluorescence data for regions targeted by the cITC (n = 3). Error bars show SEM. d, Pie charts depicting the category of regions targeted by the cITC cluster.
- Figure 5.
Retrograde tracing of inputs to the caudal ITC cluster. Monosynaptic rabies vectors were used to trace the inputs to the cITC cluster. a, Representative coronal section showing targeting of the helper virus (green) and rabies (red) in the cITCs. Scale bar represents ∼0.5 mm. White box in merged image outlines the region shown in b of starter cells in the mITC cluster, image is ∼200 × 200 μm. c, Representative images show regions identified to have presynaptic neurons identified by mCherry expression. Number in lower left corner indicates location of coronal section relative to bregma in the anterior-posterior axis. d, bar graphs show corrected total fluorescence quantification of the presynaptic regions (n = 3). Error bars show SEM. e, The pie charts depict the category of regions that project to the cITC cluster.
- Figure 6.
Anterograde tracing of the lateral ITC cluster. FoxP2-cre mice were injected with AAV-phSyn1(s)-FLEX-tdTomato-T2A-SypEGFP into the lITC cluster. a, Injection site targeting the lITCs, showing the cytosolic td-Tomato. Scale bar represents ∼0.5 mm. The white box depicts a region shown at higher magnification (right), scale bar is ∼50 μm. b, Representative images of projection targets identified by synaptophysin-eGFP expression. Number in lower left corner indicates location of coronal section relative to bregma in the anterior-posterior axis. c, Corrected total fluorescence data for regions targeted by the lITC (n = 3). Error bars show SEM. d, Pie charts depicting the category of regions targeted by the lITC cluster.
- Figure 7.
Retrograde tracing of inputs to the lateral ITC cluster. Monosynaptic rabies vectors were used to trace the inputs to the lITC cluster. a, Representative coronal sections showing targeting of the helper virus (green) and rabies (red) in the lITCs. Scale bar represents ∼0.5 mm. White box in merged image outlines the region shown in b of starter cells in the mITC cluster, image is ∼200 × 200 μm. c, Representative images show regions identified to have presynaptic neurons identified by mCherry expression. Number in lower left corner indicates location of coronal section relative to bregma in the anterior-posterior axis. d, bar graphs show corrected total fluorescence quantification of the presynaptic regions (n = 3). Error bars show SEM. e, The pie charts depict the category of regions that project to the lITC cluster.
- Figure 8.
Schematic representation of the relative strength of afferent and efferent connections with the three ITC clusters. A summary diagram representing the inputs and outputs of the different ITC clusters. Line thickness represents the relative anatomic connection strength, measured by fluorescence signal, between presynaptic inputs (left column of regions) and projection targets (right hand column of regions) of the three ITC nuclei evaluated.
Tables
AA Anterior amygdala nucleus aCeA Anterior central amygdala Aco Anterior cortical amygdala AHiAL Amygdalohippocampal area aITC Anterior intercalated cell nucleus APir Amygdalopiriform transition area AUV Secondary auditory cortex BLA Basolateral amygdaloid nucleus, anterior part BLP Basolateral amygdaloid nucleus, posterior part BLV Basolateral amygdaloid nucleus, ventral part BMA Basomedial amygdaloid nucleus, anterior part BMP Basomedial amygdaloid nucleus, posterior part BNST Bed nucleus of the stria terminalis CA1py Hippocampal CA1, pyramidal cell layer cITC Caudal intercalated cell nucleus DLEnt Dorsolateral entorhinal cortex DLO Dorsolateral orbital cortex EA Extended amygdala HDB Horizontal limb of the diagonal band InsularCtx Insular cortex IPAC Interstitial nucleus of the posterior limb of the anterior commissure ITC Intercalated cells LA Lateral amygdaloid nucleus lITC Lateral intercalated cell nucleus MCPO Magnocellular preoptic nucleus mdITC Medial intercalated cell nucleus MeAD Medial amygdaloid nucleus, anterodorsal MeAV Medial amygdaloid nucleus, anteroventral MePD Medial amygdaloid nucleus, posterodorsal MePV Medial amygdaloid nucleus, posteroventral mITC Main intercalated cell nucleus pCeA Posterior central amygdaloid nucleus PF Parafascicular thalamic nucleus PIL Posterior intralaminar thalamic nucleus Piri Piriform cortex PLCo Posterolateral cortical amygdaloid area PMCO Posteromedial cortical amygdaloid area PMV Premammillary nucleus, ventral part PO Posterior thalamic nuclear group PP Peripeduncular nucleus PRh Perirhinal cortex PSTH Parasubthalamic nucleus PVA Paraventricular thalamic nucleus RAPir Rostral amygdalopiriform area SIB Substantia innominata, basal part TeA Temporal association area VMH Ventromedial hypothalamic nucleus VP Ventral pallidum ZIC Zona incerta, caudal part
Extended Data Figure 1-1
FoxP2-cre line labels FoxP2 ITCs. A cre-dependent reporter (AAV-FLEX-mCherry) was targeted to different ITC clusters in FoxP2-cre mice. The brain tissue was stained with a FoxP2 antibody to analyze the overlap in reporter and FoxP2 expression. a, Individual channels and merged images for each of the three ITC clusters. Scale bar is 100 μm. b, Zoomed in image from the white boxes in a showing the merged image (left) and individual channel (right). Download Figure 1-1, TIF file.
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