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. Author manuscript; available in PMC: 2022 Nov 14.
Published in final edited form as: Nat Neurosci. 2021 Feb 22;24(3):401–411. doi: 10.1038/s41593-021-00797-6

Extended Data Fig. 6 |. Anatomical features of deep and superficial cortical ID2/Nkx2.1 interneurons.

Extended Data Fig. 6 |

a, Depth distribution of ID2 + /Nkx2.1+ neurons (tdTomato+ or GFP+) in the medial prefrontal cortex (mPFC), PPC and anterior and posterior regions of the primary visual cortex (V1). b, Photographs showing distribution of GFP+ (or tdTomato+) neurons in the mPFC of an ID2/Nkx2.1::Ai80 mouse (Ai65) (top and middle images, reverse contrast epifluorescence; n = 47 neurons in 9 samples from 3 mice). Most neurons were found in layer 6. c, A layer 6 tdTomato+ ID2/Nkx2.1 neuron (red) at two different depth levels. Note the putative PV+ (cyan) puncta in close apposition to the tdTomato+ cell (arrows). Top, 0.38 μm-thick single optical section. Bottom, 0.76 μm-thick maximum intensity z-projection (representative micrograph, tested in 3 mice). d, Top: Z projections (confocal maximum-intensity projection) of two intracellularly-filled tdTomato+ lD2/Nkx2.1 neuron from two ID2/NKx2.1::Ai65 mouse. Bottom: reconstruction of an intracellularly-filled tdTomato+ lD2/Nkx2.1 neuron (from a 223.2 μm thick confocal stack, maximum-intensity projection as inset) of an ID2/NKx2.1::Ai65 mouse. The two reconstructions of the ID2/Nkx2.1 neurons showed similarities to neurogliaform cells in L2/324,29 and L126. Total dendritic length: 7968.5 μm and 11641 μm for the cell in Fig. 5d and the cell in Extended Data Fig. 6, respectively; total axonal length: 77873 μm and 53669 μm; membrane surface in dendrites and soma: 1.02 × 105 μm2 and 2.81 × 105 μm2; membrane surface in axons: 9.21 × 105 μm2 and 1.14 × 106 μm2; number of dendritic branches: 117 and 105; axonal branches: 747 and 314. e, ID2+/Nkx2.1+ neuron (red) in L6b weakly immunoreactive for both NPY and nNos (arrow; n = 64 tdTomato+ ID2/Nkx2.1 neuron from 3 mice were tested against NPY). Note also strongly immunoreactive NPY+ nNOS+ neuron lacking tdTomato expression (arrowhead on top). f, A tdTomato+ ID2/Nkx2.1 neuron in L5 of PPC immunoreactive for calretinin (white arrowheads) and lacking expression of Calbindin (widefield epifluorescence; n = 54 tdTomato+ ID2/Nkx2.1 neuron from 3 mice were tested, 2 of which were positive for calretinin). g, PV+ tdTomato+ neuron in L2 of V1 (representative micrograph, tested in 3 mice). h, Left: a layer 2 tdTomato+ ID2/Nkx2.1 neuron in layer 2 (widefield epifluorescence, reverse contrast). right, high magnification of the marked rectangle area shows the tdTomato+ ID2/Nkx2.1 neuron axon distribution (‘cartridges’, some of them marked by arrows) typical of axo-axonic neurons (widefield epifluorescence, reverse contrast; representative micrograph, tested in 3 mice). The ID2/Nkx2.1 cells labeled here are identical to the Lamp5/Lhx6 type described in ref. 21,22. ID2 and Lamp5 are both broadly expressed in putative neurogliaform interneurons21, and Lhx6 is expressed downstream of Nkx2.1 in cortical interneurons. The proportion of the Lamp5/Lhx6 subtype has been reported to be 1.4 ± 0.2% of total interneurons22, with the majority located in L5/6.