nNOS-Expressing Neurons in the Ventral Tegmental Area and Substantia Nigra Pars Compacta

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.

AAV injection into the VTA and SNc of NOS1cre± mice lead to expression of ChR2-mCherry in cell bodies in distinct regions depending on injection volume/position. A, Representative images of ChR2-mCherry (magenta) and TH (green) in cell bodies for each injected group. Mice were grouped based on the distribution of ChR2-mCherry expressing cell bodies (yellow arrows indicate sub-regions where robust cell body expression was observed). Group 1 exhibited expression in the VTAR, RLi, PBP and SNc, group 2 exhibited expression in the dorso-lateral VTAR, PBP, and SNc, and group 3 exhibited expression that was restricted to the PBP and SNc (Table 3). B, Higher magnification, representative images illustrate the robust expression of ChR2-mCherry (magenta) in cell bodies and dendrites intermingled with TH (green)-expressing neurons. Right-hand images show higher magnification images of ChR2-mCherry-expressing neurons.

When ChR2-mCherry expression was restricted to cell bodies in the PBP part of the VTA and the SNc, no axonal projections were found outside of the VTA and SNc. Representative images of axon-expressed ChR2-mCherry for each group. group 1 exhibited extensive projections (for full summary, see Table 3) to multiple regions. Images are shown for the VP, PO, MD, LH, IPN, and PMnR/MnR, where the most extensive axonal expression was observed (pink tick indicates robust axonal expression). Group 2 exhibited sparse projections that were limited to the LH. Group 3 (which had cell body labeling restricted to the PBP and SNc) did not exhibit any axonal expression outside of the VTA and SNc.

Cell body expression of ChR2-mCherry was colocalized with nNOS immunolabelling in the VTA. A, Representative images of triple immunolabelling for ChR2-mCherry, nNOS, TH in the PBP. Graph shows the mean (±SEM) and individual data points for percentage of ChR2-mCherry that were colocalized with nNOS and/or TH (230 ChR2-mCherry+ cells, five mice). Almost all ChR2-mCherry+ neurons were nNOS+ and TH-. B, Representative images of triple immunolabelling for ChR2-mCherry, nNOS, TH in the VTAR. Graph shows the mean (±SEM) and individual data points for percentage of ChR2-mCherry that were colocalized with nNOS and/or TH (40 ChR2-mCherry+ cells, four mice). Almost all ChR2-mCherry+ neurons were nNOS+ and TH-. C, Representative images of triple immunolabelling for ChR2-mCherry, nNOS, TH in the RLi part of the VTA. Graph shows the mean (±SEM) and individual data points for percentage of ChR2-mCherry that were colocalized with nNOS and/or TH (155 ChR2-mCherry+ cells, three mice). Almost all ChR2-mCherry+ neurons were nNOS+ and TH-; *p < 0.05.

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.

mCherry-expressing neurons that were also TH+, co-expressed AADC and DAT. Representative images of triple immunolabelling for mCherry, TH and AADC or mCherry, TH, and DAT. mCherry+ neurons that were TH+ were also AADC+ and DAT+. Yellow arrows indicate exemplar cell bodies, exhibiting triple colocalization.

Axonal expression of ChR2-mCherry+ was colocalized with GABAergic synaptic boutons in the VTA and SNc. A, Representative images of immunolabelling for ChR2-mCherry, VGAT, and TH in the PBP. ChR2-mCherry colocalizes with VGAT puncta in a single z-plane image suggesting the presence of GABAergic synapses. B, Representative images of immunolabelling for ChR2-mCherry, VGAT, and TH in the SNc. ChR2-mCherry colocalizes with VGAT puncta in a single z-plane image suggesting the presence of GABAergic synapses. These puncta are also often TH+. Yellow arrows indicate exemplar puncta.

Axonal expression of ChR2-mCherry was colocalized with glutamatergic synaptic boutons in the VP. A, Representative images of immunolabelling for ChR2-mCherry and substance P (which is highly expressed in the VP). Extensive innervation was observed in the VP compared to the neighboring parts of the NAc and septum. B, High-magnification representative images of immunolabelling for ChR2-mCherry, substance P, and VGluT2 in the VP. Colocalization between ChR2-mCherry andVGluT2 puncta can be seen in single z-plane images, suggesting that these projections are glutamatergic. C, Representative images of immunolabelling for ChR2-mCherry, VGluT2, and TH, in the RLi, occasionally also revealed cell bodies that expressed VGluT2. D, High-magnification representative images of immunolabelling for ChR2-mCherry, substance P, and VGAT in the VP. On some occasions, colocalization between ChR2-mCherry and VGAT puncta was observed in single z-plane images, suggesting that some these projections are also be GABAergic.