Fig 1.
SNc DA neurons have a longer axonal arborization than VTA DA neurons in vivo.
Axonal arborization was estimated by performing AAV injections in adult DAT-Cre mice to conditionally express eYFP in either VTA or SNc DA neurons and visualized by immunofluorescent labelling for eYFP and TH (A). The extent of the axonal arborization was then observed in the striatum (B) and measured thought-out the ventral and dorsal striatum for VTA (C) and SNc (D) targeted injections. Direct (E) and relative (F) comparisons of the total striatal axonal arborization size was performed. N = 3 brains/group, mean ± SEM, *p>0.05. Brain schematics modified from The Mouse Brain in Stereotaxic Coordinates 3rd Edition by George Paxinos and Keith B.J. Franklin [73].
Fig 2.
Increase in DAT but not TH striatal expression in D2-cKO mice, without a change in the number of DA neurons.
TH (A) and DAT (B) immunofluorescence levels were measured in the ventral and dorsal striatum in controls and D2-cKO mice. Scale bar = 50 μm. % area covered (C,F), mean signal intensity (D,G) and integrated signal (E,H) were quantified for TH and DAT signals as well as the number of striosomes (I) and their size (J) using DAT signal. N = 15–22 brains/group, mean ± SEM, *p>0.05. The number of DA neurons in the SNc, VTA and RRF (K) was measured using stereological counting. N = 4–5 brains/group, mean ± SEM.
Fig 3.
Increased axonal arborization size of SNc but not VTA DA neurons in D2-cKO mice.
The axonal arborization of SNc or VTA DA neurons was selectively visualized using AAV injections in adult DATIRES-Cre/+;DRD2LOX/LOX mice to express eYFP in either SNc or VTA DA neurons. The extent of the axonal arborization was observed in the striatum and measured thoughout the ventral and dorsal striatum for SNc (A) and VTA (B) targeted injections. N = 6–10 brains/group, mean ± SEM, **p>0.01. Colocalization of YFP-positive SNc dopaminergic axonal processes (C) with TH and DAT, and the colocalization of TH and DAT were measured with Mander’s coefficients (M1 and M2) (D). Colocalization of YFP-positive SNc dopaminergic axonal processes (E) with VMAT2 and colocalization of VMAT2 and DAT were measured with Mander’s coefficients (F). M1 = proportion of signal 1 that colocalize with signal 2, M2 = proportion of signal 2 that colocalize with signal 1. Scale bar = 80 μm. N = 24–35 images/group, mean ± SEM, *p>0.05, ***p>0.001, ****p>0.0001.
Fig 4.
Decrease in striatal DA release in D2-cKO mice without changes in DA reuptake kinetics or in striatal surface DAT levels.
Cyclic voltammetry was used to measure the amount of DA released during single electrical stimuli on acute brain slices (A) in the ventral and dorsal striatum (B). Single stimulations were also used in the presence of the DAT antagonist nomifensine (C). DA reuptake kinetics were extracted from recordings obtained in response to single pulse stimulation by fitting an exponential curve based on the Michaelis-Menten equation for the dorsal (D) and ventral (E) striatum. N = 8–9 brains/group, mean ± SEM, *p>0.05, ***p>0.001. Surface biotinylation assay was performed on the striatum of a separate cohort of control and D2-cKO mice (F). Biotinylated surface DAT band intensities were normalized to Na+/K+-ATPase band intensities using ImageJ gel analysis software. N = 10 brains/group, mean ± SEM.
Fig 5.
SNc DA neurons from D2-cKO are not more vulnerable to α-synuclein overexpression.
α-synuclein viral overexpression in the mesencephalon was used to evaluate the impact of the increased axonal arborization size of D2-cKO SNc DA neurons on their vulnerability. In the mesencephalon (A), SNc TH+ (B), RRF TH+ (C), SNc TH- (D) and VTA TH+ neurons (E) were counted using stereological methods. N = 9 brains/group, mean ± SEM, ** p>0.01, **** p>0.0001. Scale bars = 1mm and 50 μm. In the dorsal and ventral striatum (F), TH signal area (G), mean signal intensity (H) and integrated signal (I) were measured. N = 9–12 brains/group, mean ± SEM, ** p>0.01, *** p>0.001. Scale bars = 1mm and 50 μm.
Fig 6.
SNc DA neurons from D2-cKO mice are more vulnerable to 6-OHDA.
The 6-OHDA partial lesion model was used to evaluate the impact of the increased axonal arborization size of D2-cKO SNc DA neurons on their vulnerability. (A) TH immunohistochemistry illustrating the density of dopaminergic innervation at different coordinates along the rostro-caudal axis of the striatum in a CTL mouse. Scale bar = 1 mm. (B) TH immunohistochemistry was used to localize and count the number of DA neurons in the SNc, RRF and VTA. Quantification of the survival of TH+ SNc (C), RRF (D), VTA (E) and TH- SNc neurons (F) was performed using stereological counting methods. N = 10–11 brains/group, mean ± SEM, *p>0.05, *** p>0.001, **** p>0.0001. The signal area (J), total area (H) and signal intensity (I) of TH (G)was measured in the dorsal and ventral striatum. DAT was also quantified in the dorsal striatum (J, K, L). N = 6–8 brains/group, mean ± SEM, **** p>0.0001.
Fig 7.
Behavioral changes following 6-OHDA injection.
Paw preference (A) and total number of steps (B) were measured during a stepping test. Rotational behaviour was assessed at basal state (C) and after amphetamine administration (D). Total number of rotations were also measured (E). N = 4–11 brains/group, mean ± SEM, ** p>0.01, **** p>0.0001.