Figures and data
Chronic hM3Dq activation chronically alters the activity of SNc dopamine neurons.
(A) Graphical illustration summarizing experimental design. Recombinant AAV encoding a conditional allele of the hM3Dq(DREADD)-mCherry was injected bilaterally into the ventral midbrain of 4-5 month-old DATIRESCre mice. CNO (300 mg/L) or vehicle (2% sucrose in water) was administered ad libitum via drinking water for two weeks and the animals perfused the next day. Changes in locomotion were assessed with running wheels. The two days preceding start of treatment were used as a measure for baseline locomotion.
(B) Representative traces of wheel usage for animals given control vehicle water (top) or CNO water (bottom). Arrows denote start of treatment. Grey background shading indicates dark cycle hours.
(C) Mean wheel usage for selected days during the experiment, segregated by light (left) or dark (right) cycles. n=10 animals/group from 2 independent experiments. *p≤0.05, **p<0.01, ***p<0.001 by two-way ANOVA and Holm-Sidak post hoc test.
(D) Spontaneous firing rate was measured during the first 2 min of whole cell recordings. **p ý 0.01, ***p ý 0.005 by t-test or permutation (non-parametric) analysis.
(E) Time course of responses to bath application of 1 μM CNO ex vivo measured in current clamp in neurons from vehicle-treated mice: 4.9 +/-2.9 mV (n = 5), in neurons from CNO-treated mice: -0.5 +/-1.0 mV (n = 9); p = 0.05, t test.
Chronic activation with AAV-hM3Dq-DREADDs is preferentially toxic to nigrostriatal axons.
DATIRESCre mice expressing hM3Dq(DREADD)-mCherry (A-C, E-H), or no virus injection (D) in DA neurons. Example images of TH (A,D,E) and mCherry (B,F) immunoreactivity in striatal sections of mice treated for two or four weeks with vehicle (left) or CNO (right) via drinking water. DA neuron projection areas in dorsal and ventral striatum are indicated with dotted lines. Quantifications for TH (A,D,E) and mCherry (B,F) optical density at 2 and 4 weeks show preferential loss in CPu. n = 8-9 animals/group, 3-5 sections/animal, from two independent experiments. (C,G,H) Images of TH and mCherry (4 weeks only) immunoreactivity in midbrain of vehicle (top) or CNO (bottom) treated mice at 2 or 4 weeks. SN and VTA regions are indicated with dotted lines. (C,G,H) Stereology estimating the number of TH+ or mCherry+ DA neurons. Chronic CNO treatment of hM3Dq(DREADD)-expressing mice shows a significant decrease in both TH and mCherry immunoreactivity and DA neuron number. n = 4-5 (A,C, E-H) or 3 (D) animals/group. Scale bars indicate 100μm in the midbrain and 200μm in the striatum. Error bars indicate mean ± SEM. *p<0.05, **p<0.01, ***p < 0.001 by two-way ANOVA and Holm-Sidak post hoc test. SN: substantia nigra, VTA: ventral tegmental area, CPu: caudate putamen, NAc-C: nucleus accumbens core, NAc-Sh: nucleus accumbens shell, OT: olfactory tubercule.
Chronic hM3Dq activation increases baseline calcium in parallel with axonal degeneration.
(A) Transgenic mice expressing Ca2+-reporter GCaMP6 specifically in DA neurons were injected bilaterally with AAV-DIO-hM3Dq-mCherry and implanted with an optical probe for baseline Ca2+ measurements during a 14-day chronic chemogenetic activation.
(B) Representative image of photometry probe placement in mouse midbrain to record from dopamine neurons co-expressing hM3Dq-mCherry (magenta) and GCaMP6 (cyan). Scale bar is 200 μm.
(C) Representative high-magnification images of GCaMP6 (cyan), mCherry reporter (magenta) and co-expression. Scale bar is 10 μm.
(D) Representative raw traces of baseline Ca2+ fluorescence in mice treated with vehicle vs CNO.
(E) Representative isosbestic corrected traces in mice treated with vehicle vs CNO.
(F) Baseline Ca2+ fluorescence levels of DA neurons in mice treated with vehicle or CNO for 14 days (gray shaded area), and following wash. Error bars indicate mean ± SEM. *, p<0.05 by two-way ANOVA, n = 7 mice/group from 2 independent experiments.
Spatial transcriptomics reveals midbrain DA and striatal target DEGs altered by chronic DA neuron hyperactivity
(A) DATIRESCre animals that received CNO (CNO only, n=2 mice) or were injected with AAV-hM3Dq-mCherry and received vehicle (GqVeh, n=3 mice) or CNO (GqCNO, n=3 mice) were administered CNO for one week before brains were flash frozen for spatial transcriptomic analysis.
(B) Image of midbrain and striatal sections stained with TH (green), NeuN (purple), and DAPI (blue) shows discs assigned to regions of interest. Inset shows a disc containing two TH+ cell bodies. Scale bars indicate 500µm.
(C) Expression of dopaminergic and striatal genes is confined to expected spatial regions. Expression of genes involved in DA metabolism decrease with chronic CNO. 2-49 discs were compiled per VTA and 1-7 discs were compiled per SN. 357-560 capture areas were compiled per CP. The thalamus was selected as a midbrain control region, while white matter tracts (white) and the lateral septal complex (LSX) were used as striatal controls. *p<0.05, **p<0.01, ***p<0.001 by one-way ANOVA followed by Holm-Sidak post hoc test in the SN, VTA, and CP.
(D) Principal components analysis of midbrain regions (left) and the caudate putamen (right) for GqCNO, GqVeh, and CNO only groups.
(E) Hits were used for Enrichr pathway analysis if significant in both GqCNO vs CNO alone and GqCNO vs GqVeh comparisons. Gene rankings for hit analysis were established using fold change score (FCS) and signal-to-noise score (SNS).
(F) Volcano plots comparing GqCNO vs GqVeh in the SN, VTA, and CP. Genes highlighted are also significantly altered when comparing GqCNO vs CNO only.
