Figure 2. Anatomical organization of a Tacr1-defined spinoparabrachial circuit.

(A) Spinal^Tacr1 neurons were transduced to express hM3Dq-mCherry (cartoon, left); typical confocal image of a coronal section of the PBN showing mCherry-positive Spinal^Tacr1 projections (right panel, red) in two regions of the PBN (PBN-EL and PBN-SL). Application of CNO resulted in Fos induction throughout the PBN (green). The nuclear stain DAPI (blue) highlights the overall anatomy of the region. Scale = 100 μm. (B) Multichannel in situ hybridization shows Tacr1 transcript (red) is localized to the PBN-SL. Co-staining for the glutamate vesicular transporter Slc17a6 (Vglut2; cyan) and GABA vesicular transporter SLC32A1 (Vgat; green) reveals most Tacr1-positive PBN-SL neurons are glutamatergic and hence excitatory. (C) Quantification of the percentage of Tacr1-positive cells co-expressing Tac1 (left plot) and Vglut2 (right plot). (D) A cartoon depicting the strategy used to label spinal^Tacr1 presynaptic specializations and PBN-SL^Tacr1 postsynaptic specializations in the same animal. Cre-dependent viral vectors were injected in the lumbar spinal cord (AAV-DIO-SypYFP to label presynaptic termini) and PBN (AAV-PSD95tagRFP to label postsynaptic densities) in Tacr1^Cre mice. (E) Example confocal image of a coronal section from the PBN showing a low-magnification view of the organization of Spinal^Tacr1 presynaptic specializations (green) and PBN-SL^Tacr1 postsynaptic specializations (red). Scale = 100 μm.(F) Super-resolution imaging (Airyscan) of sections from three different mice showing close apposition of SypYFP and PSD95tagRFP puncta indicative of synaptic connections. Scale = 1 μm. (G) Quantification of number of SypYFP puncta with PSD95tagRFP puncta in close apposition (left graph) and vice versa (right graph) demonstrate that the majority of spinal projection neurons target PBN-SL^Tacr1 neurons; n = 7 sections from n = 3 mice. (H) A cartoon depicting the viral strategy for anterograde tracing of PBN-SL^Tacr1 neuron projections using injection of AAV-DIO-tdTomato into the PBN of Tacr1^Cre mice. (I) Confocal image of coronal section showing tdTomato labeling (red) of cell bodies of PBN-SL^Tacr1 neurons. (J) Dense projections were found in two major brain regions, (left image; boxed regions): the medial thalamus (MTh) and a region encompassing part of the lateral hypothalamus (LH) and the parasubthalamic nucleus (PSTN). Right images show larger magnification views of boxed regions. Scale = 1 mm.

Figure 2—figure supplement 1. Tac1 and Tacr1 are differentially expressed in the PBN.

Typical confocal image of two color in situ hybridization showing Tacr1 expression (red) is enriched in the PBN-SL and is largely non-overlapping with Tac1 (green). Right side is an enlargement of the PBN-SL showing densely clustered Tacr1 neurons. Scale = 100 μm.

Figure 2—figure supplement 2. Viral-assisted visualization of PBN-SL^Tacr1 projection terminals.

The presynaptic terminals of PBN-SL^Tacr1 neurons were labeled by transducing Tacr1^cre mice with a viral vector encoding a Cre-dependent synaptophysin-GFP fusion construct (AAV-DIO-SypYFP). Confocal imaging of coronal sections of the Medial Thalamus (MTh) and Lateral Hypothalamus/Parasubthalamic nucleus (LH/PSTN) reveal densities of GFP-positive puncta in both regions indicating that PBN-SL^Tacr1 neurons make presynaptic terminals in both regions. Scale = 100 μm.