Fig 2: nNOS is mostly expressed in GABAergic, non-dopaminergic neurons in the PBP part of the VTA and SNc, and mostly in non-GABAergic, non-dopaminergic (putatively glutamatergic) neurons in the VTAR and RLi. A–D, Representative images of triple immunolabelling for nNOS, HA, and TH, in the SNc and sub-regions of the VTA. Yellow arrows indicate nNOS+ neurons. E, Graphs show the mean (±SEM; n = 3 mice, 1469 cells) and individual data points for percentage of nNOS+ cells localized in each region, percentage of nNOS+ cells that colocalized with HA, and the percentage of HA+ cells that were nNOS+. F, Schematic illustrating the localization of nNOS+/GABAergic neurons and nNOS+/glutamatergic neurons in the VTA and SNc. Yellow arrows indicate exemplar neurons; *p < 0.05.

Fig 3: Comparison of three different anti-nNOS antibodies (for details, see Tables 1, 2) in the midbrain of wild-type and nNOS-deficient mice. Representative images of double immunolabelling for nNOS (magenta) and TH (green). A, Anti-nNOS (Sigma Aldrich; N7155; AB_260795) exhibited non-specific immunolabelling that was also seen in tissue from nNOS-deficient mice. B, Anti-nNOS (Cell Signaling; 4234; AB_10694499) also exhibited somewhat non-specific immunolabelling, that was only partially absent in tissue from nNOS-deficient mice. C, Anti-nNOS (Sigma Aldrich; N2280; AB_260754) exhibited specific immunolabelling that was absent in tissue from nNOS-deficient mice. In wild-type tissue, nNOS+ cells were observed in the IPN, RLi, PBP, SNc, SNr, and substantia nigra pars lateralis (SNL). There was no colocalization between nNOS and TH.

Fig 4: Cell body expression of ChR2-mCherry was mostly colocalised with either nNOS or TH in the SNc. Representative images of triple immunolabelling for ChR2-mCherry, nNOS, TH in the SNc, showing exemplar cells exhibiting either: A, colocalisation of nNOS and ChR2-mCherry, but not TH. B, expression of ChR2-mCherry, but neither nNOS nor TH. or C, co-localisation of ChR2-mCherry and TH, but not nNOS. D, Graph shows the mean (+SEM) and individual data points for percentage of ChR2-mCherry that were colocalised with nNOS and/or TH (129 ChR2-mCherry+ cells, five mice). Most ChR2-mCherry+ neurons were either nNOS+ or TH+. Yellow arrows indicate exemplar neurons.

Fig 5: Absence of immunoreactivity for various conventional amacrine markers in EYFP-positive INL cells. Vertical sections prepared from ChAT-ChR2-EYFP retinas were co-immunolabeled with antibodies probing EYFP and TH (dopaminergic AC marker, A1-A3), VIP (VIP-expressing AC marker, B1-B3), bNOS (bNOS-expressing AC marker, C1-C3), VGluT3 (glutamatergic AC marker, D1-D3), and VGluT1 (glutamatergic neuronal marker, E1-E3). Note that virtually no EYFP-positive INL cells are co-labeled by any of the markers listed above. In all the panels, arrows point to EYFP-positive cells, and all these cells were negative to markers listed. TH, tyrosine hydroxylase; VIP, vasoactive intestinal polypeptide; bNOS, nitric oxide synthase, brain (251–270); VGluT3, vesicular glutamate transporter 3; VGluT1, vesicular glutamate transporter 1. Scale bars = 40 µm in A3 (applies to A1-A3) and 20 µm in E3 (applies to B1-E3).