Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 12 (11)

Quantitative Whole-Brain 3D Imaging of Tyrosine Hydroxylase-Labeled Neuron Architecture in the Mouse MPTP Model of Parkinson's Disease

Affiliations

Quantitative Whole-Brain 3D Imaging of Tyrosine Hydroxylase-Labeled Neuron Architecture in the Mouse MPTP Model of Parkinson's Disease

Urmas Roostalu et al. Dis Model Mech.

Abstract

Parkinson's disease (PD) is a basal ganglia movement disorder characterized by progressive degeneration of the nigrostriatal dopaminergic system. Immunohistochemical methods have been widely used for characterization of dopaminergic neuronal injury in animal models of PD, including the MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) mouse model. However, conventional immunohistochemical techniques applied to tissue sections have inherent limitations with respect to loss of 3D resolution, yielding insufficient information on the architecture of the dopaminergic system. To provide a more comprehensive and non-biased map of MPTP-induced changes in central dopaminergic pathways, we used iDISCO immunolabeling, light-sheet fluorescence microscopy (LSFM) and deep-learning computational methods for whole-brain three-dimensional visualization and automated quantitation of tyrosine hydroxylase (TH)-positive neurons in the adult mouse brain. Mice terminated 7 days after acute MPTP administration demonstrated widespread alterations in TH expression. Compared to vehicle controls, MPTP-dosed mice showed a significant loss of TH-positive neurons in the substantia nigra pars compacta and ventral tegmental area. Also, MPTP dosing reduced overall TH signal intensity in basal ganglia nuclei, i.e. the substantia nigra, caudate-putamen, globus pallidus and subthalamic nucleus. In contrast, increased TH signal intensity was predominantly observed in limbic regions, including several subdivisions of the amygdala and hypothalamus. In conclusion, mouse whole-brain 3D imaging is ideal for unbiased automated counting and densitometric analysis of TH-positive cells. The LSFM-deep learning pipeline tracked brain-wide changes in catecholaminergic pathways in the MPTP mouse model of PD, and may be applied for preclinical characterization of compounds targeting dopaminergic neurotransmission.

Keywords: Imaging; Light-sheet fluorescence microscopy; Neurotoxicity model; Parkinson's disease; Tyrosine hydroxylase; iDISCO.

Conflict of interest statement

Competing interestsU.R., C.B.G.S., D.D.T., J.L.S., K.F., P.B., H.H.H. and J.H.-S. are employed by Gubra; L.M.J., V.I.J. and L.B.K. are employed by Novo Nordisk; N.V. and J.J. are owners of Gubra.

Figures

Fig. 1.
Fig. 1.
Generation of brain-wide tyrosine hydroxylase expression map in the mouse. (A) Light-sheet fluorescence brain imaging of tyrosine hydroxylase (TH) expression in representative vehicle-dosed control mouse. (B) Further magnification (5×) of boxed midbrain area in panel A. (C) Mean fluorescence intensity TH expression pattern, generated from seven individual mouse brains. (D) Map of average TH expression in major catecholaminergic brain regions (colour-coded for easier visualization). (E,F) Virtual cross-section through the midbrain region of the mean fluorescence intensity image of the mouse brain, depicting conspicuous TH expression in the VTA, SNc and SNr. ACB, nucleus accumbens; ARH, arcuate nucleus; CP, caudate–putamen; LRN, lateral reticular nucleus; LC, locus coeruleus; ME, median eminence; NTS, nucleus of the solitary tract; OT, olfactory tubercle; PGRNl, paragigantocellular nucleus; PRN, pontine reticular formation; PVH, paraventricular nucleus; RR, retrorubral field; SNc, substantia nigra pars compacta; SNr, substantia nigra pars reticulata; SCH, suprachiasmatic nucleus; SOC, superior olivary complex; VTA, ventral tegmental area; ZI, zona incerta. Scale bars: 1 mm.
Fig. 2.
Fig. 2.
Automated whole-brain imaging of MPTP-induced changes in tyrosine hydroxylase TH expression in the mouse. (A) Mean fluorescence intensity signature of TH expression in vehicle control (left panel) and MPTP-dosed (right panel) mice. TH expression from each scanned brain was transferred to an average brain 3D coordinate mesh with mean expression calculated for each voxel. (B) Visualization of mean change in TH expression in MPTP-dosed mice (n=10) compared to vehicle-dosed control mice (n=7). Brain regions with altered average TH signal intensity are delineated in blue (downregulation) or red (upregulation). (C) Fold change (log2 scale) in TH expression for every brain region of MPTP-dosed mice compared to vehicle controls. Only regions that show a statistically significant change between the groups are indicated (P<0.01 and P<0.001; unpaired two-tailed t-test). False discovery rate correction was applied. Brain regions are ranked by statistical significance from left to right. ADP, anterodorsal preoptic nucleus; ASO, accessory supraoptic group; BMA, basomedial amygdala nucleus; BST, bed nucleus of stria terminalis; CP, caudate–putamen; CEA, central amygdala nucleus (i.e. both CEAl and CEAm); CEAl, central amygdala nucleus, lateral part; CEAm, central amygdala nucleus (medial part); FS, fundus of striatum; DMH, dorsomedial nucleus of the hypothalamus; GPi, globus pallidus (internal segment); IA, intercalated amygdala nucleus; IF, interfascicular nucleus raphe; LGv, lateral geniculate complex (ventral part); LPO, lateral preoptic area; LS, lateral septal nucleus; MDRNd, medullary reticular nucleus (dorsal part); MEA, medial amygdala nucleus; NLOT, nucleus of the lateral olfactory tract; PD, posterodorsal preoptic nucleus; PH, posterior hypothalamic nucleus; PS, parastrial nucleus; SNr, substantia nigra pars reticulata; STN, subthalamic nucleus. Scale bars: 1 mm.
Fig. 3.
Fig. 3.
Automated voxel-based whole-brain quantitative analysis of changes in tyrosine hydroxylase expression in MPTP-dosed mice. (A) Virtual coronal sections (20 µm) from 3D reconstructed average MPTP mouse whole brain (see also Fig. 2B). MPTP-dosed mouse brain regions with significantly altered mean TH signal intensity are delineated in blue (downregulation) or red (upregulation). Virtual coronal sections sampled at the level of forebrain, midbrain and brainstem are shown. (B) Voxel-based statistical analysis performed on 3D-imaged brains. Brain regions in MPTP-dosed mice with significant regulation of TH expression are indicated, as compared to vehicle controls (P<0.01 and P<0.001; unpaired two-tailed t-test; NS, not significant). (C) Representative light-sheet fluorescence images from vehicle control mouse. (D) Representative light-sheet fluorescence images from MPTP-dosed mouse. CEA, central amygdala nucleus; CP, caudate-putamen; BST, bed nucleus of stria terminalis; MDRNd, medullary reticular nucleus (dorsal part); STN, subthalamic nucleus; SNr, substantia nigra pars reticulata. Scale bars: 1 mm in A,B; 500 µm in C,D.
Fig. 4.
Fig. 4.
Development of deep learning-based method for counting of tyrosine hydroxylase-positive cells in the mouse midbrain. (A) Cell nuclei distinguishable in high-resolution scan of TH-stained mouse brain. (B) Deep-learning computational model applied to high-resolution midbrain scans for automated identification and registration of nuclei in TH-expressing cells. Boxed area is magnified in upper right corner. (C,D) Automated detection of TH-positive cells in midbrain whole 3D-image stack from vehicle-dosed mouse. Panel C, dorsal view; panel D, corresponding map of TH-positive cells detected in the sample. SNc, substantia nigra pars compacta; VTA, ventral tegmental area. Scale bars: 500 µm.
Fig. 5.
Fig. 5.
3D cell quantification of tyrosine hydroxylase-positive cells in the mouse midbrain. (A,B) Visualization of 3D coordinates of midbrain dopaminergic areas in mouse brain. False-colored region in A is magnified in B. (C,D) Coronal midbrain sections constructed from the whole 3D-image stack in representative vehicle (C)- and MPTP (D)-dosed mice, respectively. (E) Automated deep learning-based counting of TH-positive neurons in the SNc and VTA. **P<0.01; ***P<0.001 (one-way ANOVA, compared to vehicle controls). (F) TH signal intensity in the caudate–putamen plotted against corresponding number of TH-positive cells in the SNc. CP, caudate–putamen; SNc, substantia nigra pars compacta; SNr, substantia nigra pars reticulata; VTA, ventral tegmental area. Scale bars: 100 µm.
Fig. 6.
Fig. 6.
Correlation between tyrosine hydroxylase-positive cells and motor coordination skills. (A) Time spent on rotarod over three consecutive tests separated by 30 min (performed on study day 6). *P<0.05; ***P<0.001 (Dunnett's test on two-factor linear regression model with interaction, compared to vehicle controls). (B) Number of TH-positive cells in the substantia nigra pars compacta (SNc) plotted against mean rotarod test performance in vehicle (n=7)- and MPTP (n=9)-dosed mice (r=0.77). Impaired rotarod performance was observed at ≥50% depletion of TH-positive SNc cells compared to mean level in vehicle control mice. (C) MPTP-dosed mice show impaired performance compared to vehicle-dosed mice in a composite motor behavioral test. **P<0.01 (unpaired t-test).

Similar articles

See all similar articles

References

    1. Ansorge O., Daniel S. E. and Pearce R. K. B. (1997). Neuronal loss and plasticity in the supraoptic nucleus in Parkinson's disease. Neurology 49, 610-613. 10.1212/WNL.49.2.610 - DOI - PubMed
    1. Araki T., Mikami T., Tanji H., Matsubara M., Imai Y., Mizugaki M. and Itoyama Y. (2001). Biochemical and immunohistological changes in the brain of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mouse. Eur. J. Pharm. Sci. 12, 231-238. 10.1016/S0928-0987(00)00170-6 - DOI - PubMed
    1. Arkadir D., Bergman H. and Fahn S. (2014). Redundant dopaminergic activity may enable compensatory axonal sprouting in Parkinson disease. Neurology 82, 1093-1098. 10.1212/WNL.0000000000000243 - DOI - PubMed
    1. Baker H., Kobayashi K., Okano H. and Saino-Saito S. (2003). Cortical and striatal expression of tyrosine hydroxylase mRNA in neonatal and adult mice. Cell. Mol. Neurobiol. 23, 507-518. 10.1023/A:1025015928129 - DOI - PubMed
    1. Baquet Z. C., Bickford P. C. and Jones K. R. (2005). Brain-derived neurotrophic factor is required for the establishment of the proper number of dopaminergic neurons in the substantia nigra pars compacta. J. Neurosci. 25, 6251-6259. 10.1523/JNEUROSCI.4601-04.2005 - DOI - PMC - PubMed

Publication types

LinkOut - more resources

Feedback