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Sci Rep. 2017 Jul 26;7(1):6521. doi: 10.1038/s41598-017-06919-0.

Generation and validation of novel adeno-associated viral vectors for the analysis of Ca2+ homeostasis in motor neurons.

Author information

1
Department of Biomedical Science, University of Padova, Padova, Italy.
2
CNR Neuroscience Institute, Padova, Italy.
3
Department of Integrative Medical Biology (IMB), Umeå Universitet, 901 87, Umeå, SE, Sweden.
4
Department of Medicine, and Venetian Institute of Molecular Medicine, Padova, Italy.
5
Department of Biomedical Science, University of Padova, Padova, Italy. alessandro.bertoli@unipd.it.

Abstract

A finely tuned Ca2+ homeostasis in restricted cell domains is of fundamental importance for neurons, where transient Ca2+ oscillations direct the proper coordination of electro-chemical signals and overall neuronal metabolism. Once such a precise regulation is unbalanced, however, neuronal functions and viability are severely compromised. Accordingly, disturbed Ca2+ metabolism has often been claimed as a major contributor to different neurodegenerative disorders, such as amyotrophic lateral sclerosis that is characterised by selective motor neuron (MN) damage. This notion highlights the need for probes for the specific and precise analysis of local Ca2+ dynamics in MNs. Here, we generated and functionally validated adeno-associated viral vectors for the expression of gene-encoded fluorescent Ca2+ indicators targeted to different cell domains, under the transcriptional control of a MN-specific promoter. We demonstrated that the probes are specifically expressed, and allow reliable local Ca2+ measurements, in MNs from murine primary spinal cord cultures, and can also be expressed in spinal cord MNs in vivo, upon systemic administration to newborn mice. Preliminary analyses using these novel vectors have shown larger cytosolic Ca2+ responses following stimulation of AMPA receptors in the cytosol of primary cultured MNs from a murine genetic model of ALS compared to the healthy counterpart.

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