Neurotransmitter and metabolite changes in the MA-treated mice. For all histograms, except D, n = 4 controls, 4 MA-treated. The histograms show mean ± SEM, (**) p < 0.01, (*) p < 0.05. (A) Changes in striatal dopamine. One-tailed t-test, t = 2.53, df = 6, p = 0.02. (B) Changes in striatal dihydroxyphenylacetic acid (DOPAC). One-tailed t-test, t = 3.97, df = 6, p = 0.004. (C) Changes in striatal homovanillic acid (HVA). One-tailed t-test, t = 3.84, df = 6, p = 0.004. (D) Changes in substantia nigra tyrosine hydroxylase (TH). One-tailed t-test, t = 6.18, df = 2, p = 0.013. The peak levels of substantia nigra TH from the normal and MA brains, left and right, were estimated from real-time QRT-PCR analysis of voxels G3, G4, H3, and H4 (Fig. 4B). n = 2 controls (left and right), 2 MA-treated (left and right). (E) Changes in striatal 5-hydroxytryptamine (5-HT). Two-tailed t-test, t = 0.139, df = 6, p = 0.89. (F) Changes in striatal 5-hydroxyindoleacetic acid (5-HIAA). Two-tailed t-test, t = 1.44, df = 6, p = 0.20.

Correlation matrices. (A) Spatial (voxel × voxel) correlation analyses using genes significantly differentially expressed between the anterior and posterior halves of the brain. The matrices for the normal and PD brain are shown with the data for the left and right halves separated out. The correlation between any pair of voxels as judged by gene expression levels is read by looking along the relevant row and column, and finding the intersection. The color of the corresponding element gives the correlation between that pair of voxels by reference to the scales (right). The diagonals are the autocorrelations of the gene expression patterns for each gene and are (and should be) equal to 1. All other correlations must be between 1 and −1. The numbering is such that voxels A2, A4, …, I2, I4 correspond to voxels 1, 2, …, 19, 20, respectively for the left halves of the brain, and voxels A1, A2, …, I1, I3 correspond to voxels 1, 2, …, 19, 20 for the right halves. (B) Expression (gene × gene) correlation analyses using the 6000 most strongly expressed genes when averaged across the 40 voxels. The correlation over voxels for any pair of genes is read by looking along the relevant row and column, and finding the intersection. The genes in the normal omnibus matrix (left and right halves combined) were parsimoniously ordered based on a similarity metric. The same gene order was used to construct the corresponding matrix for the MA brain, and the data were then separated into matrices for the left and right halves of the brains. (C) Monte Carlo analysis comparing similarity of gene expression matrices shown in B for left normal with right normal, and left MA with right MA. The distributions for each brain show the discrepancy between randomly selected pairs of permuted matrices quantitated using the Frobenius norm of the resulting difference matrix. The vertical lines show the differences between the actually observed left and right matrices for each brain. For both the normal and MA brains, the similarity of the gene expression matrices between the left and right halves was highly significant (p < 0.0001 for the normal brain; p = 0.005 for the MA brain). (D) Correlation analyses identify conserved networks of highly correlated gene expression clusters. The gene expression correlation matrices show the subset of genes common to both specimens that are anterior/posterior differentially expressed and display a spatial expression correlation coefficient of >0.75 with at least one other gene within the same brain. The genes in the normal omnibus matrix (left and right halves combined) were parsimoniously ordered using a similarity metric, and the same gene order was used to construct the corresponding matrix for the MA brain. The data were then separated into matrices for the left and right halves of the brains. Two mutually exclusive clusters of coregulated genes emerged: cluster 1 (genes 1–23) and cluster 2 (genes 24–55).

Putative regulatory elements shared between groups of correlated and anticorrelated genes. There were three groups of correlated (+) genes: (1) SIAT9, HIVEP2, SEPP1, SCYD1, NEFL, INA; (2) CUGBP2, MBP, CBLN1; (3) CBLN1, USP9X, PNUTL2; and one group of anticorrelated (−) genes: (1) MBP, NEFL, CBLN1, PNUTL2, USP9X. The groups are indicated by square brackets. Genes are indicated by UniGene symbol or name (http://www.ncbi.nlm.nih.gov/UniGene). Exons are indicated by short vertical lines and the direction of transcription by horizontal arrows. Homology searches were performed on human homologs of the relevant mouse genes. In all cases, the human gene has the same symbol as the mouse, except for ^ahuman NEFL = mouse Nfl, and ^bhuman PNUTL2 = mouse Sept4. The regulatory sequences responsible for correlated expression are shown as squares, those responsible for anticorrelated expression are shown as diamonds. Lines delineate the relationships between the conserved regulatory sequences. Multiple control regions frequently connected the genes. Potential binding sites are: (1) LMO2COM, (2) OCT1, (3) GATA2 and GATA3, (4) MYOD, (5) LMO2COM, (6) NFAT, (7) GFI1, (8) TCF11, (9) HFH2, (10) GFI1, (11) NFAT, (12) FREAC2, (13) OCT1, (14,15) GKLF, (16) TATA, (17) SRY, (18) TH1E47, (19) SRY, (20) HFH2, (21) BARBIE, (22) HFH2, (23) HFH3, (24) HNF3B, (25) HFH2, (26,27) HFH8 and HFH3, (28) HFH-3, (29) GATA, (30) CEBPB, (31) MYOD, (32) LMO2COM, (33) E47, (34) SRY, (35) NFAT, (36) CETS1P54. (*) No known transcription factor binding site.

SVD delineates anatomical regions of the brain. The SVD was performed using the anterior/posterior differentially expressed subset of genes. The spatial patterns resulting from the first, second, third, and fourth PCs are shown. Alongside are the first 400 members of the corresponding gene vectors. The ordinate represents the contribution by the relevant gene to the variation of the vector spatial pattern, and the abscissa represents the genes in decreasing order of differential expression. The pseudocolor scales give the level of expression of the relevant gene vector in the spatial patterns.

Voxelation scheme. The slices are labeled A through J, from anterior to posterior. Slice A corresponds to section 8 (6.94 mm interaural, 3.14 mm bregma) of the Mouse Brain Library (Rosen et al. 2000; Williams 2000; http://www.nervenet.org/MBL/mbl.html), slice B to section 11 (5.78 mm interaural, 1.98 mm bregma), slice C to section 12 (5.50 mm interaural, 1.70 mm bregma), slice D to section 14 (4.39 mm interaural, 0.74 mm bregma), slice E to section 16 (3.80 mm interaural, 0.00 mm bregma), slice F to section 17 (2.98 mm interaural, −0.82 mm bregma), slice G to section 20 (1.68 mm interaural, −2.12 mm bregma), slice H to section 24 (0.00 mm interaural, −3.80 mm bregma), slice I to section 28 (−1.31 mm interaural, −5.09 mm bregma), slice J to section 32 (−2.44 mm interaural, −6.24 mm bregma). The anterior–posterior coordinates of the Mouse Brain Library (interaural and bregma relative distances) are as described (Franklin and Paxinos 1997). Odd-numbered voxels (e.g., A1, A3, B1) are from the right side of the brain, and even-numbered voxels (e.g., A2, A4, B2) are from the left.

Spatial gene expression patterns for the subset of correlated genes. (A) Spatial expression patterns of the genes shown in Figure 3D for the normal and MA brains. The relative level of expression of any gene in any voxel is read by looking along the relevant row and column, finding the intersection, and referring to the scales. The voxel numbering in the columns of the matrix is such that voxels A1, A2, A3, …, I2, I3, I4 correspond to voxels 1, 2, 3, …, 37, 38, 39, 40, respectively. The genes are in the same order as for Figure 3D. The two clusters of genes are apparent, and although these have highly conserved patterns of expression within the normal and PD brains, these patterns are somewhat divergent between the two brains. (B) Level of TH expression as judged using real-time QRT-PCR. TH expression levels are found to peak in voxels corresponding to the olfactory bulb (A1–A4, voxels 1–4) and the substantia nigra (G3, G4, corresponding to voxels 27, 28; and H3, H4 corresponding to voxels 31, 32). However, the level of TH in the substantia nigra of the PD brain is substantially decreased compared with the normal brain (Fig. 1D). (C) The Nfl gene is present as two separate spots on the microarrays, corresponding to genes 39 and 50 in A. This provided an opportunity to assess within-array replicability, which was excellent (for the normal brain, r = 0.96, F_[1,38] = 443.65, p < 0.0001; for MA brain, r = 0.90, F_[1,38] = 153.80, p < 0.0001). (D) Expression pattern of the precerebellin-1 gene in the normal and PD brain. The line drawing shows the anatomy of the midlevel transverse section employed, which corresponds to section 8 of the Mouse Brain Library (interaural 5.40 mm, bregma −4.60 mm). (Olf) Olfactory lobes; (Str) striatum; (Hi) hippocampus; (Ce) cerebellum. Imaging software smoothed the expression patterns over the voxels. Gene expression levels can be deduced by reference to the pseudocolor scales.

Differentially expressed genes. (A) Scatterplots show mean expression levels across the 40 voxels in the normal and MA brains on a logarithmic scale (log₂) for genes where p < 10⁻⁹. The comparisons within brains (left and right normal; left and right MA) provide another assessment of replicability (for normal brain, r = 0.90, F_[1,34] = 143.98, p < 0.0001; for MA brain, r = 0.98, F_[1,34] = 817.76, p < 0.0001). The comparison between brains (left normal and left MA, right normal and right MA) shows the differences in expression between genes repressed and induced in the MA brain. Red line: twofold above equivalent expression level; green line: equivalent expression level; blue line: twofold below equivalent expression level. (B) Mean expression levels across the 40 voxels of the normal and MA brains on a logarithmic scale (log₂ ±SEM). (Red) Normal; (blue) MA. The genes are ranked from most (gene 1) to least significant (gene 36; p < 10⁻⁹). To allow replicability to be assessed, the data for the left and right halves of each brain were separated out as the left and right data points, respectively, for each gene. No significant differences were found comparing left normal with right normal (two-tailed t test on log₂-transformed data, t = 0.012, df = 70, p = 0.99) and left MA with right MA (two-tailed t test on log₂-transformed data, t = −0.33, df = 70, p = 0.75). (C) Confirmation of gene expression differences for selected genes by real-time QRT-PCR analysis of voxel RNA. Expression levels are shown relative to controls and normalized using 18S RNA. The bars indicate mean ± SEM. (***) p < 0.001, (**) p < 0.01, two-tailed t-test. (D) Gene categories in the entire 9000-gene data set (9K) and in those genes significantly different (diff) between the control and MA brains (p < 0.001) when averaged across all 40 voxels. Genes involved in cell/cell interactions (cytoskeleton, extracellular matrix, and cell adhesion) were significantly overrepresented in the regulated subset (p = 0.02), but apoptosis-related genes were not (p = 0.39). The bars show mean ± SD, (*) p < 0.05, as judged using Monte Carlo statistics. The control category represents 15 randomly chosen genes to show the validity of the Monte Carlo analysis. As expected, this category showed no significant difference in frequency between the 9K and regulated data sets (p = 0.48).