Region-Specific and Age-Dependent Multitarget Effects of Acetylcholinesterase Inhibitor Tacrine on Comprehensive Neurotransmitter Systems

Abstract

Regional brain distribution and metabolism of neurotransmitters and their response to drug treatment are fundamentally important for understanding the central effects of neuroactive substances. We used matrix-assisted laser desorption/ionization mass spectrometry imaging in combination with multivariate analysis to visualize in anatomical detail metabolic effects of aging and tacrine-mediated acetylcholinesterase inhibition on comprehensive neurotransmitter systems in multiple mouse brain regions of 12-week-old and 14-month-old mice. We detected age-related increases in 3,4-dihydroxyphenylacetaldehyde and histamine, indicating oxidative stress and aging deficits in astrocytes. Tacrine had a significant impact on the metabolism of neurotransmitters in both age groups; predominantly, there was an increased norepinephrine turnover throughout the brain and decreased 3-methoxy tyramine, a marker for dopamine release, in the striatum. The striatal levels of histamine were only elevated after tacrine administration in the older animals. Our results demonstrated that tacrine is a multitarget and region-specific neuroactive agent, inducing age-specific responses. Although well-studied, the complete mechanisms of the action of tacrine are not fully understood, and the current findings reveal features that may help explain its treatment-related effectiveness and central side effects.

Figure 1

Multivariate exploration of MALDI-MSI data. a, Nissl-stained coronal sections from a 14-m control animal at 0.26 mm and −1.43 mm from bregma with the analyzed brain regions annotated. b, MALDI-MS images of acetylcholine from levels 0.26 mm (upper panel) and −1.43 mm (lower panel) from bregma. Images are shown using a rainbow scale, indicating the relative abundance of acetylcholine scaled from 0 to 70%. The lateral resolution is 100 μm. c, Score plots of the first and second components of the PCA model (left) and the first and third components of the PCA model (right). Objects are colored according to age (12-w or 14-m) and treatment (tacrine or control). Gray ovals show grouping of samples according to treatment and age. d, 3D score plot of the three-principal-component PLS-DA model. Objects are colored according to age (12-w or 14-m) and treatment (tacrine or control). e, 3D loading plot of the three-component PLS-DA model. The colored spheres represent the center of each group. The gray spheres represent the significant neurotransmitters and metabolites in specific brain areas annotated by numbering. f, Diagram showing the results of significant alterations in neurotransmitters and metabolites related to age, tacrine, or both from two-way ANOVA with the FDR correction. Abbreviations: 12-w, 12-week-old; 14-m, 14-month-old; DA, dopamine; 3-MT, 3-methoxy tyramine; DOPAL, 3,4-dihydroxyphenylacetaldehyde; NE, norepinephrine; DOPEG, 3,4-dihydroxyphenylglycol; MOPEG, 3-methoxy-4-hydroxyphenylglycol; 5-HT, 5-hydroxytryptamine; 5-HIAA, 5-hydroxyindoleacetic acid; His, histamine; Amy, amygdala; BNST, bed nucleus of the stria terminalis; Cg, cingulate cortex; CPu, caudate-putamen; Hip, hippocampus; Hyp, hypothalamus; Ins, insular cortex; LS, lateral septum; MC, primary and secondary motor cortex; PALv, ventral pallidum; Pir, piriform cortex; PVT, periventricular thalamic nucleus; RSC, retrosplenial cortex; SC, primary and secondary somatosensory cortex; Th, thalamus; Tu, tubercle.

Figure 2

MALDI-MS images showing age- and tacrine-related alteration of the dopaminergic metabolic pathways in mouse brain tissue sections. a, Metabolic pathway of DA representative tissue sections (at 0.26 from bregma) from each studied group. The lateral resolution is 100 μm. The enzymes involved are annotated in boxes. b, MALDI-MS images of dopamine metabolites (at −1.43 mm from bregma) from each studied group with a lateral resolution of 50 μm. c, Heatmap based on the autoscaled log intensities generated for the selected top eight (ANOVA, P < 0.05) features. d, MALDI-MS images of 3-MT and DOPAL from each studied group in sagittal sections at 1.2 mm lateral from the midline with a lateral resolution of 100 μm. For a, b, and d, the ion intensity rainbow color scales are according to optimal visualization. Abbreviations: 12-w, 12-week-old; 14-m, 14-month-old; AD, aldehyde dehydrogenase; COMT, catechol-O-methyltransferase; MAO, monoamine oxidase, DA, dopamine; 3-MT, 3-methoxy tyramine; DOPAL, 3,4-dihydroxyphenylacetaldehyde; DOPAC, 3,4-dihydroxyphenylacetic acid; HVA, homovanillic acid; BNST, bed nucleus of the stria terminalis; Cg, cingulate cortex; CPu, caudate-putamen; Hip, hippocampus; PVT, periventricular thalamic nucleus; RSC, retrosplenial cortex.

Figure 3

Histaminergic metabolic changes associated with aging and tacrine. a, MALDI-MS images of His and N-MH at coronal brain levels 0.20 mm from bregma (upper panels) and −1.43 mm from bregma (lower panels). The lateral resolution is 100 μm for images at level 0.20 mm and 50 for images at level −1.43. The ion intensity rainbow color scales are scaled according to the optimal visualization of each metabolite. b, Heatmap based on the auto-scaled log intensities, generated for the selected top nine (ANOVA, P < 0.05) features. c, Comparison of His levels (log ion intensity) in the PALv between each experimental group using one-way ANOVA with Tukey’s post hoc test. d, Comparison of the N-MH/His ratio in the CPu between each experimental group using one-way ANOVA with Tukey’s post hoc test. Means and standard deviation are shown. *P < 0.05, **P < 0.01. Abbreviations: 12-w, 12-week-old; 14-m, 14-month-old; Cg, cingulate cortex; CPu, caudate-putamen; N-MH, N-methylhistamine; His, histamine; Hyp, hypothalamus; LS, lateral septum; PALv, ventral pallidum.

Figure 4

Changes in NE metabolism in response to age and tacrine administration. a, MALDI-MS images of NE metabolic pathway from coronal mouse brain tissue sections (bregma −1.43 mm) at a lateral resolution of 50 μm. b, MALDI-MS images of NE and related metabolites (bregma 1.26) at lateral resolution of 100 μm. c, Heatmap based on the auto-scaled log intensities generated for the top 30 (ANOVA, P < 0.05) features. d, MALDI-MS images of DOPEG and MOPEG from each studied group in sagittal sections at 1.2 mm lateral from the midline at a lateral resolution of 100 μm. e, Correlation (Pearson coefficient r) of RSC levels of DOPEG and MOPEG with the corresponding levels in Cg and PALv, respectively. Abbreviations: 12-w, 12-week-old; 14-m, 14-month-old; AR, aldehyde reductase; COMT, catechol-O-methyltransferase; MAO-A, monoamine oxidase A; DOPEG, 3,4-dihydroxyphenylglycol; NE, norepinephrine; MOPEG, 3-methoxy-4-hydroxyphenylglycol; Amy, amygdala; BNST, bed nucleus of the stria terminalis; Cg, cingulate cortex; CPu, caudate-putamen; Hyp, hypothalamus; Ins, insular cortex; MC, primary and secondary motor cortex; PALv, ventral pallidum; PVT, paraventricular thalamic nucleus; RSC, retrosplenial cortex; SC, primary and secondary somatosensory cortex; Th, thalamus.

Figure 5

Effect of age and tacrine administration on regional monoaminergic metabolism. a, Dot plot of the log-transformed ratio between the RMS-normalized ion intensity of HVA and DA in multiple brain regions of the investigated groups. b, Overlaid MALDI-MS images of DA (magenta) and HVA (green) in coronal mouse brain tissue sections (−1.1 to −1.6 mm from bregma) of the four investigated groups. c, Dot plot of the log-transformed ratio between the RMS-normalized ion intensity of MOPEG and NE in multiple brain regions of the investigated groups. d, Overlaid MALDI-MS images of NE (magenta) and MOPEG (green) in coronal mouse brain tissue sections (−1.1 to −1.6 mm from bregma) of the four investigated groups. e, Dot plot of the log-transformed ratio between the RMS-normalized ion intensity of 5-HIAA and 5-HT in multiple brain regions of the investigated groups. f, Overlaid MALDI-MS images of 5-HT (magenta) and 5-HIAA (green) in coronal mouse brain tissue sections (−1.1 to −1.6 mm from bregma) of the four investigated groups. Abbreviations: 12-w, 12-week-old; 14-m, 14-month-old; DA, dopamine; HVA,; homovanillic acid; NE, norepinephrine; MOPEG, 3-methoxy-4-hydroxyphenylglycol; 5-HT, 5-hydroxytryptamine; 5-HIAA, 5-hydroxyindoleacetic acid; Amy, amygdala; BNST, bed nucleus of the stria terminalis; Cg, cingulate cortex; CPu, caudate-putamen; Hip, hippocampus; Hyp, hypothalamus; Ins, insular cortex; LS, lateral septum; MC, primary and secondary motor cortex; Tu, olfactory tubercle; PALv, ventral pallidum; Pir, piriform cortex; PVT, paraventricular thalamic nucleus; RSC, retrosplenial cortex; SC, primary and secondary somatosensory cortex; Th, thalamus.