Objective: To manipulate the activity of dopaminergic (DA) neurons in culture using pharmacological and molecular tools to promote their survival.

Background: Parkinson’s disease is a neurodegenerative condition caused by progressive degeneration of DA neurons in the Substantia Nigra pars compacta. Although the causes that lead to DA neurons death remain unknown, their degeneration may involve oxidative stress, disturbed calcium homeostasis and mitochondrial dysfunction. Recent findings suggest that electrical activity could play an important role in determining their survival. Pharmacological blockade of the electrical activity in vitro can induce cell death while increasing electrical activity induces neuron survival, probably involving the increase of intracellular Ca2+ levels. However, some evidences suggest that an excessive increase in intracellular Ca2+ can generate toxic conditions for DA neurons.

Methods: Primary DA cultures were obtained from ventral mesencephalon of mouse embryos dissected at gestational day 13 (E13.5). Pharmacological treatments were carried out using Nicotine alone or combined with Tetraethylammonium (TEA), an agent that blocks voltage-dependent K+ channels, thus increasing electrical activity. These treatments were combined with transduction with either inhibitory or excitatory heterologous Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) that were delivered into postmitotic neurons by lentiviral (LV) particles. Cultures were treated with clozapine-n-oxide (CNO). After treatments neurons were fixed at days in vitro (DIV) 2, 5, 9 or 14th and then immunostained for tyrosine hydroxylase. The number of DA neurons was determined by direct quantification in an epifluorescent microscope.

Results: We reproduced the in vitro model of DA degeneration in mouse cell culture and were able to artificially modulate their electrical activity using molecular tools to control neuronal survival. Nicotine and TEA promoted DA survival at DIV 14, and enhancement of electrical activity with excitatory M3 DREADD prevented DA cell death when treated with CNO.

Conclusions: DREADDs delivered with LV vectors are efficient to transduce DA neurons in vitro and represent a good approach to manipulate their activity in order to study the mechanism of activity-mediated survival. Further work is under way to demonstrate the role of artificial electrical manipulation on the survival of DA neurons in vitro and vivo.

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MDS Abstracts - https://www.mdsabstracts.org/abstract/modulating-neuronal-activity-in-dopaminergic-neurons-to-promote-their-survival/