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. Author manuscript; available in PMC: 2020 Aug 21.
Published in final edited form as: Neuron. 2019 Jul 1;103(4):583–597.e8. doi: 10.1016/j.neuron.2019.05.047

Figure 5. Neural Circuit-based HiUGE Labeling.

Figure 5.

(A) Illustration of cortico-striatal circuit-selective C-term labeling of ßIII-tubulin by injection of AAV2-retro mouse Tubb3 GS-gRNA into the striatum and 2 lateral injections of AAV2/9 HA and Myc-epitope donors in the motor cortex. (B) Representative image showing GFP labeling in the motor cortex, indicating retrogradely accessed Cre-dependent Cas9– 2A-GFP expression in projection neurons. (C) Immunolabeling of HA (arrows) and Myc-epitope (arrowheads) tagged ßIII-tubulin, imaged from the boxed area in (B). (D) Enlarged images from the boxed areas in (C), showing cells positive for (i) HA or (ii) Myc-epitope. (E) GFP signal from the AAV2-retro injected striatum. (F) Zoomed image of the boxed area in (E), showing GFP-positive axon bundles that contain fibers positive for HA or Myc-epitope. (G, H) Enlarged images showing fibers positive for (i) HA or (ii) Myc-epitope within GFP-positive axon bundles from boxed areas in (F). (I) Illustration of thalamo-cortical circuit-selective C-term labeling of ßIII-tubulin by injection of AAV2- retro mouse Tubb3 GS-gRNA in the somatosensory cortex and injection of AAV2/9 HA-epitope donor in the thalamus. (J) Representative image showing retrogradely activated Cas9-2A-GFP expression within the thalamus (boxed area) and local cortical networks (mostly cells within layer II/III and layer VI). (K) Zoomed image of the boxed area in (J), showing retrogradely accessed and HiUGE edited thalamic neurons positive for HA-epitope (arrows) and Cas9-2A-GFP. Scale bar is indicated in each panel.