Experimental design and confirmation of unilateral TH+ depletion in the SNc via 6-OHDA lesion.

(A) Illustration of experimental timeline. (B) Dual ipsilateral stereotaxic injection into the mFB and A13 region. (C) TH+ cells in SNc of sham (top) compared to 6-OHDA injected mouse (bottom). Magnified areas outlined by yellow squares are shown on the right. (D) Unilateral injection of 6-OHDA (6-OHDA ChR2: n = 5, 6-OHDA eYFP: n = 5) into the MFB resulted in greater percentage of TH+ loss compared to sham in the SNc (sham ChR2: n = 7, sham eYFP: n = 5, 2-way ANOVA), regardless of virus type (F1,18 = 104.4, p < 0.001). ***p < 0.001. Error bars indicate SEMs.

Post hoc c-Fos expression and targeting of the mZI and A13.

(A) Diagram showing the A13 DAergic nucleus in dark magenta encapsulated by the ZI in light magenta. The fiber optic tip is outlined in red. Atlas image adapted from the Allen Brain Atlas (Goldowitz, 2010). (B) Tissue images were obtained from a 6-OHDA ChR2 animal and (C) a 6-OHDA eYFP animal. Images show the distribution of DAPI (blue), eYFP (green), c-Fos (yellow), and TH (magenta). Landmarks are outlined in white (3V: third ventricle; A13 and mZI as shown in A), and the optic cannula tip is shown in red. Higher magnification images of the A13 DAergic nucleus are outlined by the yellow boxes in a 6-OHDA ChR2 animal (Di-Dvi) and a 6-OHDA eYFP animal (Ei-Evi). Images show isolated channels in the top rows of the respective groups: eYFP (i), TH (ii), and c-Fos (iii). Merged channels for eYFP and c-Fos (iv), TH and c-Fos (v), and a merge of all four channels (vi) are presented in the bottom rows of their respective groups. White arrowheads in the merged images highlight overlap in merged markers. Red arrows show triple colocalization of eYFP, c-Fos and TH. (Dvi) contains a magnified example of triple-labelled neurons, as highlighted in the yellow box. (F) Graph shows increase in the intensity of c-Fos fluorescence after photoactivation in 6-OHDA ChR2 mice (p = 0.05).

Ispilesional photoactivation of the A13 region in a unilateral 6-OHDA mouse model rescues motor deficits.

(A) Schematic of open field experiment design and example traces for open field testing (each testing box is 1 min - total test is 4 min) with unilateral photoactivation of the A13 region. (B-E) Group averaged instantaneous velocity graphs showing no increase in a sham eYFP (B) or 6-OHDA eYFP mouse (C), with increases in velocity during stimulation in a sham ChR2 (D) and 6-OHDA ChR2 (E) mouse. (F-I) Effects of photoactivation on open field metrics for sham eYFP (n = 5), sham ChR2 (n = 6), 6-OHDA eYFP (n = 5), and 6-OHDA ChR2 (n = 5) groups (three-way MM ANOVAs, post hoc Bonferroni pairwise). Photoactivation increased in both sham and 6-OHDA ChR2 groups: (F) distance travelled (ChR2 vs. eYFP: p < 0.01), (G) locomotor bouts (ChR2 vs. eYFP: p < 0.01), (H) animal movement speed (ChR2 vs. eYFP: p < 0.001) and (I) duration of locomotion in the open field (ChR2 vs. eYFP: p < 0.001). (J) The graph presents animal rotational bias using the turn angle sum. There was a significant increase in 6-OHDA ChR2 rotational bias during A13 region photoactivation (6-OHDA ChR2 vs. 6-OHDA eYFP: p < 0.001). (K) Diagram depicting the pole test. A mouse is placed on a vertical pole facing upwards time for release is derived from time the experimenter removes their hand from the animal’s tail to when it touches the ground. (L, M) Photoactivation of the A13 region led to shorter descent times in 6-OHDA ChR2 mice compared to 6-OHDA eYFP mice (p < 0.01). A pre-op baseline was performed, followed by post-op three weeks later. On experiment day, performance with no stim (Exp - NS) was compared with photostimulation (Exp - Stim). (M) 6-OHDA ChR2 mice showed a greater reduction in descent time compared to 6-OHDA eYFP mice (6-OHDA ChR2 vs. 6-OHDA eYFP: p < 0.001). ***p < 0.001, **p < 0.01. Error bars indicate SEMs.

Nigrostriatal degeneration causes widespread changes in A13 region input and output connections.

Correlation matrices were used to visualize input and output patterns of the A13 region. Here, we focus on motor-related pathways. (A) Brain regions with similar input patterns show high correlation. (B) Correlation strength is shown by cell color in the matrix: yellow represents strong positive correlations, magenta indicates no correlation, and black represents strong negative correlations. (C, D) Sham animals displayed stronger interregional correlations of inputs from motor-related regions across the neuroaxis to the A13 region compared to 6-OHDA-lesioned mice. This suggests a broader distribution of inputs among motor related cortical, subcortical, and brainstem regions in sham condition. (D) In 6-OHDA-lesioned mice, inputs to the A13 region from the STN, PAG, and PPN became negatively correlated, unlike inputs from other motor-related regions. In contrast, inputs from motor-related pallidal and incertohypothalamic areas showed stronger positive correlations with cortical inputs, suggesting these regions had a relatively greater influence on A13 activity. (E, F) In contrast, output patterns from the A13 region showed strong interregional correlation among cortical and brainstem motor related regions in 6-OHDA-lesioned mice compared to sham animals. (E) In sham animals, A13 outputs to cortical regions were negatively correlated with outputs to thalamic, hypothalamic, and midbrain regions. This pattern disappeared after nigrostriatal degeneration, suggesting a more widespread distribution of A13 outputs. MOp (primary motor cortex), MOs (secondary motor cortex), SSp (primary somatosensory area), PALd (pallidum, dorsal), VM (ventral medial thalamic nucleus), LHA (lateral hypothalamus), STN (subthalamic nucleus), ZI (zona incerta), SNr (substantia nigra pars reticular), MRN (midbrain reticular nucleus), SCm (superior colliculus, motor), PAG (periaqueductal gray), CUN (cuneiform), RN (red nucleus), SNc (substantia nigra pars compacta), PPN (pedunculopontine nucleus), TRN (tegmental reticular nucleus), and PRNr (pontine reticular nucleus).

Unilateral nigrostriatal degeneration causes distinct changes in A13 connectivity.

(A) A13 input and output connections to motor-related brain areas. Anterograde and retrograde viruses were injected into the A13 region on the lesioned side (see methods). The graph shows the relative change in input (afferents) and output (efferents) connections in 6-OHDA-lesioned mice compared to sham controls. (B, C) Brain regions showing changes in A13 input connections in sham (B) and 6-OHDA-lesioned (C) mice. (D, E) Brain regions showing changes in A13 output connections in sham (D) and 6-OHDA-lesioned (E) mice. Error bars represent SEMs. 6-OHDA: n = 3; sham: n = 2. Brain region abbreviations are from the Allen Brain Atlas. MOp (primary motor cortex), MOs (secondary motor cortex), SSp (primary somatosensory area), LHA (lateral hypothalamus), STN (subthalamic nucleus), ZI (zona incerta), SNr (substantia nigra pars reticulata), MRN (midbrain reticular nucleus), SCm (superior colliculus, motor), PAG (periaqueductal gray), CUN (cuneiform), RN (red nucleus), SNc (substantia nigra pars compacta), PPN (pedunculopontine nucleus), and TRN (tegmental reticular nucleus).

List of antibodies used for immunohistochemical staining of the A13 and SNc regions, as well as the whole brain.

Protocol for Whole Brain Clearing.

Quantification of channelrhodopsin viral spread in the rostral-caudal direction from the injection site in 6-OHDA-treated and sham animals.

The left panel shows a representative image of viral spread, including the optic fibre track, visualized using the “fire” lookup table in FIJI/ImageJ software, with targeting precision confirmed by coronal overlays from the Mouse Allen Brain Atlas to the A13/Mzi region. The right panel displays a graph illustrating the percentage of viral spread across anterior-posterior coordinates for all ChR2-infected mice, calculated as the ratio of viral expression area to total tissue section area. Data were obtained from mouse brain sections injected with AAVDJ-CaMKIIα-ChR2 into the A13/Mzi region, sectioned at 50 µm, and analyzed using the VS120 Virtual Slide Scanner. Quantification was performed by blinded analysts using FIJI/ImageJ, with defined regions of interest (ROIs).

Time course of open field locomotion distance travelled over 30 minutes.

Locomotion distance travelled for the six sham ChR2 animals at baseline and at the five pre-stimulation timepoints compared using a 1-way RM ANOVA (F5,25 = 0.49, p = 0.78). Data indicate mean ± SEM bars.

Characterization of A13 region photoactivation temporal dynamics on locomotion initiation.

(A) Percent of trials where there was at least one bout of locomotion. Data are plotted as box and whiskers with the horizontal line through the box indicating the group median, interquartile range indicated by the limits of the box, and group minimum and maximum indicated by the whiskers. Asterisks indicate significant comparisons using the Wilcoxon signed-rank test: * p< 0.05. (B) The average time for the ChR2 group animals to begin locomotion after the onset of photoactivation was not different from sham mice (p = 0.953). Means plotted with error bars indicating ± SEM.

Preservation of TH+ A13 cells in Parkinsonian mouse models.

Representative slices of SNc (AP: -3.08mm, A) and A13 region (AP: -1.355mm, D) following registration with WholeBrain software. Full 3D brain is available (see Movie S4). There was a lack of TH+ SNc cells following 6-OHDA injections at the MFB (A). (B, C) Zoomed sections (90 μm thickness) of red boxes in panel A in left to right order. Meanwhile, TH+ VTA cells were preserved bilaterally. In addition, TH+ A13 cells were present ipsilesional to 6-OHDA injections (D). (E, F) Zoomed sections (90 μm thickness) of red boxes in panel D in left to right order. When calculating the percentage of TH+ cell loss normalized to the intact side, there was a significant interaction between the condition group and brain regions (repeated measures two-way ANOVA with post hoc Bonferroni pairwise, sham: n = 3, 6-OHDA: n = 6) (G) 6-OHDA-treated mice showed a significantly greater percentage of TH+ cell loss in SNc compared to VTA and A13 region (VTA vs. SNc: p = 0.005; A13region vs. SNc: p < 0.05). In contrast, sham showed no significant difference in TH+ cell loss across SNc, VTA and A13 region regions (p > 0.05). *p < 0.05, and **p < 0.01. (H) Dual ipsilateral stereotaxic injection into the MFB and A13 region. Scale bars are 50 μm unless otherwise indicated.

Example of the injection core in a sham brain for viral tracers and the rostral and caudal spread to the injection site (A13).

Viral tracers (AAV8-CamKII-mCherry and AAVrg-CAG-GFP) were mixed 50:50. Light-sheet images around the injection site were obtained with 2x objective, 6.3x optical zoom, and a z-step size of 2 µm (xyz resolution = 0.477 µm x 0.477 µm x 2 µm). Background filtering (median value of 20 pixels and Gaussian smoothing with a sigma value of 10) was performed in ImageJ software 1 and visualized in IMARIS 9.8 (Belfast, United Kingdom). 2008 Allen reference atlas images were overlaid on top of 90 µm maximum intensity projections taken from IMARIS 9.8 (Belfast, United Kingdom): -1.26 mm (A), -1.36 mm (B), and -1.46 mm (C). Zoomed in sections of each white rectangular area at each coordinate (rows ‘i’) are displayed below for each fluorophore (rows ‘ii’). Scale bars for rows ‘i’ are 200 µm and for rows ‘ii are 100 µm.

The ipsilateral (A-F) and contralateral (G-L) afferent and efferent proportions across Sham (blue) and 6-OHDA (orange) mice were consistent when compared in a pairwise manner. An experimental variation on the total labeling of neurons and fibers was minimized by dividing the afferent cell counts or efferent fiber areas in each brain region by the total number found in a brain to obtain the proportion of total inputs and outputs. Using Spearman’s correlation analysis, we found afferent and efferent proportions across animals to be consistent among each other with an average correlation of 0.91 (SEM = 0.02). M1 = mouse #1, M2 = mouse #2, M3 = mouse #3.

Examples of retrogradely labelled GFP positive fibers and cells from selected regions illustrating cell bodies from whole brain imaging that project to the A13 region. Expressions of GFP detected using light-sheet microscopy with 2x objective, 6.3x optical zoom, and a z-step size of 2 µm (xyz resolution = 0.477 µm x 0.477 µm x 2 µm). Brain regions were delimited by registration with Allen Brain Atlas (see Methods) and cropped out of selected 90 µm image stack in ImageJ software 1. A background filter (Gaussian smoothing with a rolling ball radius of 20 pixels) and a minimum filter with radius of 1 pixel were performed in ImageJ software 1. Scale bar = 50 µm.

Examples of anterogradely labelled mCherry positive fibers from selected regions illustrating cell bodies from whole brain imaging that project to the A13 region. Expressions of mCherry detected using light-sheet microscopy with 2x objective, 6.3x optical zoom, and a z-step size of 2 µm (xyz resolution = 0.477 µm x 0.477 µm x 2 µm). Brain regions were delimited by registration with Allen Brain Atlas (see Methods) and cropped out of selected 90 µm image stack in ImageJ software 1. A background filter (Gaussian smoothing with a rolling ball radius of 5 pixels) and a minimum filter with a radius of 1 pixel were applied in ImageJ software 1. Scale bar = 50 µm.