¹Boehringer Ingelheim, Neuroscience & Mental Health, Biberach, Germany, ²Boehringer Ingelheim, Global DMPK, Biberach, Germany, ³University Clinic Ulm, Department of Neurology, Ulm, Germany, ⁴University Clinic Ulm, German Center for Neurodegenerative Diseases (DZNE), Ulm, Germany

Background: Tetrabenazine (TBZ) is a known inhibitor of the vesicular monoamine transporter 2 (VMAT2), resulting in reduced monoaminergic signaling, including dopamine. Previous internal behavioral studies have demonstrated that TBZ exerts a noticeably stronger impact on motivational behavior in rats compared to mice. However, the underlying neurochemical mechanisms responsible for these species-specific differences remain unclear. This study examines how TBZ affects dopamine transmission in both rats and mice using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). This method measures neurotransmitter (and metabolite) levels in brain section with high spatial resolution (~50 µm).

Aims & Objectives: The primary objective was to assess the effects of TBZ on dopamine levels in the brains of rats and mice following treatment with varying doses, utilizing MALDI-MSI. The study aimed to focus on the reduction of monoamines and explore the therapeutic potential of TBZ. In-house behavioral data indicated that TBZ dose-dependently reduced effort-based responses in rats, whereas mice exhibited no such effect at equivalent doses. To investigate these species-specific differences, higher TBZ doses were tested in mice, and basal dopamine levels were compared between the two species. Ultimately, the goal was to evaluate interspecies variations and refine preclinical models for better translational relevance.

Method: A MALDI-MSI workflow was used to detect neurotransmitters in rodent brain tissue, using FMP-10 derivatization to label, among other molecules, neurotransmitters. The study included Wistar rats (n=4 per group) and C57BL/6 mice (n=6 per group). Animals were euthanized 90 minutes after TBZ (mice: 2.5, 5, 10 mg/kg i.p.; rats: 0.25, 0.75, 1.5 mg/kg i.p.) or vehicle treatment, and brains were extracted and stored at -80°C until further processing. Brain sections were collected from striatal and nucleus accumbens regions (the latter in mice only). Spray coating with FMP-10 was performed using an HTX sprayer. Samples were then analyzed in a Q-ToF Mass Spectrometer (timsTOF fleX MALDI-2), followed by spectral analysis and visualization of neurotransmitter distribution. Co-registration with hematoxylin and eosin (H&E) stained images enabled virtual microdissection, allowing for precise localization and quantification of neurotransmitter presence.

Results: In rats, TBZ produced a dose-dependent, linear decrease in dopamine levels (r² = 0.97), confirming its potent effect on dopaminergic transmission. In contrast, mice displayed a non-linear reduction in dopamine, with striatal r² = 0.82 and nucleus accumbens r² = 0.59, indicating a less predictable response. Furthermore, baseline dopamine levels in the mouse dorsal striatum were found to be 1.74 times higher than in rats. These findings highlight significant species-specific differences in both dopamine baseline levels and response to TBZ.

Discussion & Conclusions: The combination of MSI and FMP-10 derivatization enables the quantification of neurotransmitters in brain sections with high spatial resolution. The observed behavioral differences between rats and mice were mirrored by species-differences at the neurochemical level. These findings underscore the importance of considering species-specific neurochemical profiles when interpreting results from preclinical models and provide a robust basis for future investigations focused on the dopaminergic system.