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, three-way MM ANOVAs), regardless of virus type (F1,18 = 104.4, p < .001). ***p < .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 fibre optic tip is outlined in red. Atlas image adapted from the Allen Brain Atlas (Goldowitz, 2010). (B) Tissue images were obtained from 6-OHDA ChR2 animals around bregma −1.22 mm, and (C) a 6-OHDA eYFP animal more caudally around bregma −1.46 mm. Images show the distribution of DAPI (blue), eYFP (green), c-Fos (yellow), and TH (magenta). Landmarks are outlined in white (3V: third ventricle; mtt: mammillothalamic tract), 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 (D) and a 6-OHDA eYFP animal (E). Scale bars are set to 350 μm. 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. Scale bars are set to 50 μm.

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 (1 min) with unilateral photoactivation of the A13 region. (B-E) 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 the ChR2 groups: (B) distance travelled (ChR2 vs. eYFP: p = 0.005), (C) locomotor bouts (ChR2 vs. eYFP: p = 0.005), (D) duration of locomotion in the open field (ChR2 vs. eYFP: p = 0.005), and (E) animal movement speed (ChR2 vs. eYFP: p < 0.001). (F-I) Group averaged instantaneous velocity graphs showing no increase in a sham eYFP (F) or 6-OHDA eYFP mouse (H), with increases in velocity during stimulation in a sham ChR2 (G) and 6-OHDA ChR2 (I) mouse. (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. The time for release is taken as the experimenter removes their hand from the animal’s tail. (L, M) Graphs showing the response of animals to photoactivation of the A13 region while performing the pole test. (L) A13 region photoactivation also led to shorter total descent time in ChR2 compared to eYFP mice (ChR2 vs. eYFP: p = 0.004), and (M) 6-OHDA ChR2 mice showed a greater reduction in descent time compared to sham ChR2 (6-OHDA ChR2 vs. sham ChR2: p = 0.012; 6-OHDA ChR2: n = 5; sham ChR2: n = 7). ***p < .001, **p < .01, *p < .05. Bonferroni’s post hoc comparisons between 6-OHDA ChR2 and sham eYFP, sham ChR2, and 6-OHDA eYFP at stim time point as a, b, and c respectively. Error bars indicate SEMs.

Unilateral nigrostriatal degeneration leads to large-scale changes in the organization of the A13 region afferent and efferent distributions across the neuraxis.

We used correlation matrices to summarize any observable patterns in the distribution patterns of inputs and outputs of the A13 region. A correlation matrix was calculated by correlating the proportion of input from one brain region to another in a pairwise manner across 251 brain regions delimited by registration with Allen Brain Atlas. If two brain regions among mice (eg. brain regions A and B) contribute a similar input, they are highly correlated (A). Using the color legend showing various correlation strengths, the intersecting box in the matrix in this example will be colored dark red (B). If no relationship is found between contributions from two brain regions, the intersecting box will be colored yellow. If the contribution from one brain region was negatively correlated with another brain region among mice, then the intersecting box will be colored blue. The afferent distribution pattern in the sham displayed a higher level of inter-regional correlation between brain regions (C) than 6-OHDA injected mice (D). Indeed, two distinct bands of anti-correlated afferent regions were identified in the 6-OHDA injected mice (see black boxes in D). These two bands arose from the cortical plate subregions (motor, sensory, visual, and prefrontal), and striatal and pallidal subregions showing distinct inputs compared to the rest of the neuraxis. In contrast, the projection patterns of A13 efferents displayed a higher level of inter-regional correlation between brain regions following a unilateral nigrostriatal degeneration (F) compared to sham (E). In sham, proportions of A13-cortical/ striatal efferents were negatively correlated to A13-pallidal/ thalamic/ hypothalamic/ midbrain efferents (see black boxes in E). However, these distinct projection patterns disappeared following nigrostriatal degeneration, suggesting A13 efferent distributions becoming more distributed across the neuraxis.

Differential remodeling of A13 region connectome following a unilateral nigrostriatal degeneration.

The distributions of the A13 connectome in sham served as a basis for an in-depth comparison against 6-OHDA mouse models. Example registered slices (using WholeBrain software 64 with light-sheet data, 2X objective, 4X optical zoom) at rostral areas show changes in sham (A) afferents compared to 6-OHDA lesioned animals (B). Graph showing major brain regions contributing afferents to A13 in sham mice (C). The graph illustrates the change in the proportion of afferents in 6-OHDA compared to sham mice (D). Representative registered slices showing sham proportions of efferents in sham (E) compared to 6-OHDA mice (F). The magnified black box section displays an example of mCherry+ fibers (left) segmented using Ilastik and ImageJ software (right). Graph showing major brain regions receiving efferents from the A13 in sham mice (G). The graph illustrates the change in the proportion of efferents in 6-OHDA compared to sham mice (H). Error bars represent SEMs. Anterograde and retrograde viruses were injected into the ipsilesional A13 (see methods). Abbreviations from Allen Brain. Atlas: CTXpl (cortical plate), CTXsp (cortical subplate), STR (striatum), PAL (palladium), TH (thalamus), HYP (hypothalamus), P (pons), MB (midbrain), MY (Medulla), and CB (cerebellum).

Comparing descending dopamine pathways for locomotor control.

Simplified connectivity map for the 3 dopamine pathways. The first pathway is the classical VTA/SNc projection to the striatum, and the SNr/GPi projects to the MLR. The VTA/SNc also directly projects to the MLR (Ryczko et al., 2016). The mZI/A13 region projects dopaminergic projections to the MLR (Sharma et al., 2018). Canonical pathways are in black, while non-canonical pathways are in red.

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

Protocol for Whole Brain Clearing.

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

(A-B) 30-minute open field experiment group averages for sham (A) and 6-OHDA (B) animals with photoactivation plotted as 1-minute bins of distance travelled. Blue bars indicate 1-minute trials with photoactivation. (C) Locomotion distance travelled for the six sham ChR2 animals at baseline and at the five pre-timepoints compared using a 1-way RM ANOVA (F5,25 = 0.486, P = .783). 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. (B) The average time for the ChR2 group animals to begin locomotion after the onset of photoactivation. Means plotted with error bars indicating ± SEM. Asterisks indicate significant comparisons using the Wilcoxon signed-rank test: ** P ≤ 0.05.

Preservation of TH+ A13 cells in Parkinsonian mouse models.

Representative slices of SNc (AP: −3.08 mm, A) and A13 region (AP: −1.355 mm, D) following registration with WholeBrain software 64. 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. Scale bars are 50 μm. 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; A13 region vs. SNc: P = 0.029). 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. 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 consistency of afferent and efferent proportions across mice was 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.