12Through the corpus callosum, interhemispheric communication is mediated by callosal projection (CP) 13 neurons. Using retrograde labeling, we identified a population of layer 6 (L6) excitatory neurons as the 14 main conveyer of transcallosal information in the monocular zone of the mouse primary visual cortex (V1). 15 Distinct from L6 corticothalamic (CT) population, V1 L6 CP neurons contribute to an extensive reciprocal 16 network across multiple sensory cortices over two hemispheres. Receiving both local and long-range 17 cortical inputs, they encode orientation, direction, and receptive field information, while are also highly 18 spontaneous active. The spontaneous activity of L6 CP neurons exhibits complex relationships with brain 19 states and stimulus presentation, distinct from the spontaneous activity patterns of the CT population. The 20 anatomical and functional properties of these L6 CP neurons enable them to broadcast visual and nonvisual 21 information across two hemispheres, and thus play a major role in regulating and coordinating brain-wide 22 activity events. 23 24 25 26 27 28 29
Results
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CP neurons in mouse V1 are dominantly located in L6
62Traditionally, CP neurons were labeled with intra-parenchymal injection of retrograde tracers, such as 63 horseradish peroxidase or Fluoro-Gold (FG), with varying efficacy 18 . To label CP neurons with high 64 efficacy, we took advantage of a recently developed recombinant AAV variant (rAAV2-retro) that permits 65 efficient retrograde access to projection neurons 19 . We injected rAAV2-retro carrying GFP (rAAV2-66 retro.CAG.GFP) into the monocular zone of right V1 of a transgenic mouse line with L5 excitatory neurons 67 labeled with H2B-mCherry (Rbp4-Cre × Rosa26 LSL CAG H2B mCherry 20 ). We observed brightly labeled 68 CP neurons in the left monocular V1 predominantly located in the cortical layer below L5, with few cells 69 in the supragranular layer (L2/3) ( Figure 1A). More superficial CP neurons were observed at the border of 70 V1 and V2L, consistent with earlier studies 2, 8, 9 . Immunostaining with anti-GABA antibody indicated that 71 these neurons were not GABAergic (Supplementary Figure 1A and 1B). Therefore, CP neurons in mouse 72 V1 are dominantly excitatory neurons located in L6.
73L6 CP neurons and NTSR1-positive CT neurons are distinct populations 74 Having identified L6 neurons as the main callosal-projecting neurons in mouse monocular V1, we then 75 asked whether these CP neurons were distinct from the thalamus-projecting L6 CT neurons. To this end, 76 we utilized a Cre-recombinase transgenic mouse line NTSR1-Cre (NTSR1-cre GN220) that selectively 77 labels L6 CT neurons in V1 21, 22, 23 . Crossed with the Cre reporter line Ai14 24 , the resulting mice had L6 CT 78 neurons expressing red fluorescent protein tdTomato. Injecting rAAV2-retro.syn.GFP in the right V1 and 79 labeling CP neurons in the left hemisphere, we similarly observed that CP neurons were mostly located in 80 L6 (Figure 1B), with high-resolution image stacks ...