Purpose

Manganese-enhanced MRI¹ (MEMRI) exploits the biochemical similarity of Ca²⁺ and Mn²⁺ to map the brain’s activity in vivo. To what extend different calcium channels add to the signal increase is not yet understood. Here, we studied the role of L-type voltage-dependent Ca²⁺ channels (Ca_v1.2 and Ca_v1.3) in MEMRI contrast. We further exploit this model to study the fate of Mn²⁺ accumulation after long-term systemic application.

Methods

First, genetic deletion of the voltage-dependent channels Ca_v1.2 or Ca_v1.3 in the brain was performed as described in Langwieser et al.² and in Platzer et al.³, respectively, in order to establish a full knock-out model of the respective channel. In a second experiment, homozygous floxed CA_v1.2 mice were injected in the thalamus with adeno-associated viruses (AAV) encoding the Cre recombinase (AAV-GFP/Cre) or exclusively green fluorescent protein (AAV-GFP) as a control. AAV-mediated delivery of the Cre recombinase by AAV-GFP/Cre allows a time and region specific knockout of Ca_v1.2 after injection. Third, a local injection of Mn²⁺ into the thalamus was performed in one animal to study draining of Mn²⁺ to projection terminals. For experiments 1 and 2, animals first underwent baseline MRI (7T, 3D-T1w, TR = 50 ms, TE = 3.2 ms, 125x125x140 μm³ resolution) followed by MEMRI experiment as detailed in Grünecker et al.⁴. After baseline MRI, mice had one week of rest before they received intraperitoneal injections of 30mg/kg manganese chloride every 24 hours for eight consecutive days (8x30/24h)⁴, followed by the second MEMRI scan. For experiment 3, one animal received a single intracranial injection of Mn²⁺ in the thalamus, and was repeatedly scanned each 12h using the above mentioned MEMRI experiment.

Results

MEMRI intensities in ROIs of the hippocampus, cortex and the whole brain, pre- and post-application of an 8-days fractionated Mn²⁺ application scheme were assessed. Relative to wild-type animals, MEMRI shows a significantly reduced intensity in all three compartments only for Ca_v1.2 ko mice, but not for Ca_v1.3 ko animals (Fig. 1 for whole brain ROI), suggesting an important of Ca_v1.2 in the transportation of Mn²⁺. Region specific suppression of Ca_v1.2 expression exclusively in the thalamus does not lead to reduced accumulation in the primarily targeted brain region after repeated i.p. injection of Mn²⁺, but in the insular cortex (Fig. 2), which constitutes an axonal terminal of the thalamus. Using repeated scanning over 2 days after a single dose intracerebral delivery of Mn²⁺ in the thalamus of a wt mouse, we could show that initial accumulation inside the thalamus appears to drain towards the axonal projection terminal.

Discussion

Our results highlight a specific role of the voltage-dependent Ca_v1.2 channel in Mn²⁺ accumulation. Furthermore, after systemic fractionated application of Mn²⁺ (i.e. over an extended period of time), axonal projection terminals appear to show increased accumulation as compared with primary uptake structures. This strongly influences interpretation of MEMRI data with respect to altered activation patterns, especially when fractionated schemes or long-term Mn²⁺ delivery using osmotic mini-pumps is employed.

Conclusion:

We describe for the first time the essential role of voltage-dependent calcium transporter Ca_v1.2 in accumulation of manganese in the living mouse brain. In addition, we highlight the importance of considering intra-cerebral Mn²⁺ axonal transportation also during systemic Mn²⁺ delivery routes.

Acknowledgements

S.A-C. was supported by a Science Without Borders, CAPES, PhD stipend (BEX 9694-13-7) from Brazil