Distinguishing core from penumbra by lipid profiles using Mass Spectrometry Imaging in a transgenic mouse model of ischemic stroke.

Detecting different lipid profiles in early infarct development may give an insight on the fate of compromised tissue. Here we used Mass Spectrometry Imaging to identify lipids at 4, 8 and 24 hours after ischemic stroke in mice, induced by transient middle cerebral artery occlusion (tMCAO). Combining linear transparency overlay, a clustering pipeline and spatial segmentation, we identified three regions: infarct core, penumbra (i.e. comprised tissue that is not yet converted to core), and surrounding healthy tissue. Phosphatidylinositol 4-phosphate (m/z = 965.5) became visible in the penumbra 24 hours after tMCAO. Infarct evolution was shown by 2D-renderings of multiple phosphatidylcholine (PC) and Lyso-PC isoforms. High-resolution Secondary Ion Mass Spectrometry, to evaluate sodium/potassium ratios, revealed a significant increase in sodium and a decrease in potassium species in the ischemic area (core and penumbra) compared to healthy tissue at 24 hours after tMCAO. In a transgenic mouse model with an enhanced susceptibility to ischemic stroke, we found a more pronounced discrimination in sodium/potassium ratios between penumbra and healthy regions. Insight in changes in lipid profiles in the first hours of stroke may guide the development of new prognostic biomarkers and novel therapeutic targets to minimize infarct progression.

Characterization of acetylcholine release and the compensatory contribution of non-Ca(v)2.1 channels at motor nerve terminals of leaner Ca(v)2.1-mutant mice.

The severely ataxic and epileptic mouse leaner (Ln) carries a natural splice site mutation in Cacna1a, leading to a C-terminal truncation of the encoded Ca(v)2.1 alpha(1) protein. Ca(v)2.1 is a neuronal Ca(2+) channel, mediating neurotransmitter release at many central synapses and the peripheral neuromuscular junction (NMJ). With electrophysiological analyses we demonstrate severely reduced ( approximately 50%) neurotransmitter release at Ln NMJs. This equals the reduction at NMJs of Cacna1a null-mutant (Ca(v)2.1-KO) mice, which display a neurological phenotype remarkably similar to that of Ln mice. However, using selective Ca(v) channel blocking compounds we revealed a compensatory contribution profile of non-Ca(v)2.1 type channels at Ln NMJs that differs completely from that at Ca(v)2.1-KO NMJs. Our data indicate that the residual function and presence of Ln-mutated Ca(v)2.1 channels precludes presynaptic compensatory recruitment of Ca(v)1 and Ca(v)2.2 channels, and hampers that of Ca(v)2.3 channels. This is the first report directly showing at single synapses the deficits and plasticity in transmitter release resulting from the Ln mutation of Cacna1a.

Gene dosage-dependent transmitter release changes at neuromuscular synapses of CACNA1A R192Q knockin mice are non-progressive and do not lead to morphological changes or muscle weakness.

Ca(v)2.1 channels mediate neurotransmitter release at the neuromuscular junction (NMJ) and at many central synapses. Mutations in the encoding gene, CACNA1A, are thus likely to affect neurotransmitter release. Previously, we generated mice carrying the R192Q mutation, associated with human familial hemiplegic migraine type-1, and showed first evidence of enhanced presynaptic Ca(2+) influx [Neuron 41 (2004) 701]. Here, we characterize transmitter release in detail at mouse R192Q NMJs, including possible gene-dosage dependency, progression of changes with age, and associated morphological damage and muscle weakness. We found, at low Ca(2+), decreased paired-pulse facilitation of evoked acetylcholine release, elevated release probability, and increased size of the readily releasable transmitter vesicle pool. Spontaneous release was increased over a broad range of Ca(2+) concentrations (0.2-5mM). Upon high-rate nerve stimulation we observed some extra rundown of transmitter release. However, no clinical evidence of transmission block or muscle weakness was found, assessed with electromyography, grip-strength testing and muscle contraction experiments. We studied both adult ( approximately 3-6 months-old) and aged ( approximately 21-26 months-old) R192Q knockin mice to assess effects of chronic elevation of presynaptic Ca(2+) influx, but found no additional or progressive alterations. No changes in NMJ size or relevant ultrastructural parameters were found, at either age. Our characterizations strengthen the hypothesis of increased Ca(2+) flux through R192Q-mutated presynaptic Ca(v)2.1 channels and show that the resulting altered neurotransmitter release is not associated with morphological changes at the NMJ or muscle weakness, not even in the longer term.