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 the SNc of a sham animal (top) compared to a 6-OHDA–injected mouse (bottom). Magnified areas, outlined by yellow squares, are shown at right. (D) Unilateral injection of 6-OHDA into the mFB (6-OHDA ChR2: n = 5; 6-OHDA eYFP: n = 5) resulted in a significantly greater percentage loss of TH⁺ cells in the SNc compared to sham animals (sham ChR2: n = 7; sham eYFP: n = 5), regardless of virus type (two-way ANOVA: 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 dopaminergic (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 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 each group: (i) eYFP, (ii) TH, and (iii) c-Fos. Merged channels are shown in the bottom rows: (iv) eYFP/ChR2 + c-Fos, (v) TH + c-Fos, (vi) all three channels merged. White arrowheads in the merged images highlight areas of marker overlap. Red arrows indicate triple colocalization of ChR2, c-Fos, and TH. (Dvi) contains a magnified example of triple-labeled neurons, highlighted in the yellow box. (F) Graph shows an increase in c-Fos fluorescence intensity after photoactivation in 6-OHDA ChR2 mice (p = 0.05).

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

A. A representative image of viral spread, including the optic fiber track, visualized using the “fire” lookup table in FIJI/ImageJ software. Targeting precision was confirmed by coronal overlays from the Mouse Allen Brain Atlas onto the A1xxxx3 region. B, C. Graphs illustrating the percentage of viral spread across anterior–posterior coordinates for ChR2-transfected Sham (B), and 6-OHDA (C) mice, calculated as the ratio of viral expression area to total tissue section area. Data were obtained from brain sections of mice injected with AAVDJ-CaMKIIα-ChR2 into the A13 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).

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

(A) Schematic of the open-field experiment design and example traces from open-field testing. Each testing bin represents 1 min (total duration: 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), and increased 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.Statistical comparisons used three-way mixed-model ANOVAs with post hoc Bonferroni pairwise tests. Photoactivation significantly increased locomotor activity in both sham and 6-OHDA ChR2 groups in the following metrics: (F) distance travelled (ChR2 vs. eYFP: p = 0.005), (G) locomotor bouts (ChR2 vs. eYFP: p = 0.005), (H) 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. A significant increase in rotational bias was observed in 6-OHDA ChR2 mice during A13 region photoactivation (6-OHDA ChR2 vs. 6-OHDA eYFP: p < 0.001). (K) Diagram of the pole test. A mouse is placed facing upward on a vertical pole; “time to release” is defined as the interval from the experimenter removing their hand from the animal’s tail to when the animal touches the ground. (L, M) Photoactivation of the A13 region decreased the time required to navigate to the base in 6-OHDA ChR2 mice compared to 6-OHDA eYFP mice (p = 0.004). A pre-op baseline was performed, followed by post-op testing three weeks later. On the experiment day, performance with no stimulation (Exp – NS) was compared to photoactivation (Exp – Stim). (M) 6-OHDA ChR2 mice showed a further reduction in time to reach the base 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.

Time course of open-field locomotion distance traveled over 30 minutes.

Locomotion distance traveled for the six sham ChR2 animals at baseline and across five pre-stimulation timepoints was compared using a one-way repeated-measures ANOVA (F₅,₂₅ = 0.49, p = 0.78). Data are presented as mean ± SEM.

Characterization of A13 region photoactivation temporal dynamics on locomotion initiation.

(A) Percentage of trials in which at least one bout of locomotion was observed. Data are plotted as box-and-whisker plots, with the horizontal line within the box indicating the group median, the interquartile range represented by the box edges, and the whiskers denoting group minimum and maximum. Asterisks indicate significant comparisons using the Wilcoxon signed-rank test: *p = 0.71. (B) The average latency to initiate locomotion after photoactivation onset in ChR2-group animals was not significantly different from that of sham controls (p = 0.953). Means are 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. A There was a loss 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. Additionally, TH+ A13 cells were present on the ipsilesional side 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 and brain region (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 the VTA and A13 region (VTA vs. SNc: p = 0.005; A13region vs. SNc: p = 0.03). In contrast, sham showed no significant difference in TH+ cell loss across SNc, VTA and A13 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.

Injection core in a sham brain showing viral tracer spread in the A13 region.

Viral tracers (AAV8-CamKII-mCherry and AAVrg-CAG-GFP) were mixed 1:1. Light-sheet images around the injection site were acquired using a 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 and visualized in IMARIS 9.8 (Belfast, United Kingdom). Images from the 2008 Allen Reference Atlas were overlaid on 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 region at each coordinate (rows ‘i’) are displayed below for each fluorophore (rows ‘ii’). Scale bars: 200 µm for rows ‘i’ and 100 µm for rows ‘ii’.

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

Correlation matrices were used to visualize the input and output patterns of the A13 region, focusing on motor-related pathways.

(A) Brain regions with similar input patterns exhibit strong correlations. (B) Correlation strength is represented by cell color in the matrix: yellow indicates strong positive correlations, magenta denotes no correlation, and black indicates strong negative correlations. (C, D) Sham animals displayed stronger interregional correlations among inputs from motor-related regions across the neuraxis 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 animals. (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 may exert greater influence on A13 activity. (E, F) In contrast, output patterns from the A13 region showed stronger interregional correlations 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 was lost following nigrostriatal degeneration, suggesting a more distributed pattern 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 reticulata), MRN (midbrain reticular nucleus), SCm (superior colliculus, motor), PAG (periaqueductal gray), CUN (cuneiform nucleus), RN (red nucleus), SNc (substantia nigra pars compacta), PPN (pedunculopontine nucleus), TRN (tegmental reticular nucleus), PRNr (pontine reticular nucleus).

Ipsilateral (A–F) and contralateral (G–L) afferent and efferent proportions in sham (blue) and 6-OHDA (orange) mice.

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.

Unilateral nigrostriatal degeneration causes distinct changes in A13 connectivity.

(A) Relative changes in A13 afferent (input) connections in 6-OHDA-lesioned mice compared to sham controls. (B, C) Brain regions showing A13 input connections in sham (B) and 6-OHDA–lesioned (C) mice. (D) Relative changes in A13 efferent (output) connections in 6-OHDA-lesioned mice compared to sham controls. (E, F) Brain regions showing A13 output connections in sham (E) and 6-OHDA–lesioned (F) mice. 6-OHDA: n = 3; sham: n = 2. Brain region abbreviations follow 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 nucleus), RN (red nucleus), SNc (substantia nigra pars compacta), PPN (pedunculopontine nucleus), TRN (tegmental reticular nucleus).

Examples of retrogradely labeled GFP-positive fibers and cells from selected regions illustrating projections to the A13 region.

Cell bodies projecting to A13 were visualized through whole-brain imaging. GFP expression was detected using light-sheet microscopy with a 2× objective, 6.3× optical zoom, and a z-step size of 2 µm (xyz resolution: 0.477 µm × 0.477 µm × 2 µm). Brain regions were delimited by registration with the Allen Brain Atlas (see Methods) and cropped from selected 90 µm image stacks using ImageJ software. A background filter (Gaussian smoothing with a rolling ball radius of 20 pixels) and a minimum filter (radius = 1 pixel) were applied in ImageJ. Scale bar = 50 µm.

Examples of anterogradely labeled mCherry-positive fibers from selected regions illustrating projections to the A13 region.

mCherry expression was detected using light-sheet microscopy with a 2× objective, 6.3× optical zoom, and a z-step size of 2 µm (xyz resolution: 0.477 µm × 0.477 µm × 2 µm). Brain regions were delineated by registration with the Allen Brain Atlas (see Methods) and cropped from selected 90 µm image stacks using ImageJ software. A background filter (Gaussian smoothing with a rolling ball radius of 5 pixels) and a minimum filter (radius = 1 pixel) were applied in ImageJ. Scale bar = 50 µm.