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Fluorescence-activated cell sorting of dopaminergic neurons from the substantia nigra of mice as a new approach to study their functioning in norm and pathologyстатья
- Авторы: Troshev D., Blokhin V., Ukrainskaya V., Kolacheva A., Ugrumov M.
- Журнал: FEBS open bio
- Год издания: 2022
- Издательство: John Wiley & Sons Inc.
- Местоположение издательства: United States
- Номер статьи: P-01.2-044
- DOI: 10.1002/2211-5463.13440
- Аннотация: The study of the molecular mechanisms of the functioning of dopaminergic neurons (DNs) of the nigrostriatal system of the brain is among highest priorities in neuroscience, since they play a key role in the regulation of motor behavior, and their death leads to the development of Parkinson's disease (PD). The aim of this work was to develop a method for selection a pure population of DNs from the substantia nigra (SN) and to assess the possibility of studying these neurons in norm and pathology. To this end, three objectives were solved. First, we found the optimal conditions for selecting DNs from SN of intact mice using two fluorescent markers, DRAQ5 (nuclear marker) and GBR-BP (original non-commercial stain). The latter is internalized into DAs via the dopamine transporter. Optimal conditions included using GBR-BP at a concentration of 50 nM and staining of DN in vibratome sections of SN prior to cell dissociation. When solving the second issue, it was proved that the sorted neurons are dopaminergic. Indeed, these cells were shown to express the genes for the synthesis of dopamine and dopamine transporter. The most convincing evidence is the demonstration that tyrosine hydroxylase was detected immunohistochemically in 97% of sorted neurons. When solving the third issue, we obtained evidence that in DNs sorted from SN cell suspension, it is possible to quantify changes in at least the expression of genes for proteins involved in dopamine metabolism and neurotransmission in animals in pathology, in this work, when modeling PD. Thus, when modeling the early clinical stage of PD in MPTP-treated mice, unidirectional and similar changes in the number of DNs and the expression of genes for tyrosine hydroxylase and dopamine transporter were found in sorted DNs and in DNs in the whole SN.
- Добавил в систему: Трошев Дмитрий Вадимович