Lentiviral-based expression system of ghrelin receptor in neurons.

MUSTAFÁ ER; LOPEZ SOTO JE; MARTÍNEZ DAMONTE V; RAINGO J; RODRÍGUEZ SS

Huerta Grande

Congreso; XXVIII Congreso Anual de la Sociedad Argentina de Investigación en Neurociencia; 2013

Sociedad Argentina de Neurociencia

Growth hormone secretagogue receptor type 1a (GHSR1a) activity modulates neuronal circuits that control appetite and energy expenditure, but the molecular mechanisms involved are currently unknown. GHSR1a is a G protein-coupled receptor that shows constitutive activity and ligand-evoked activation. Our research is focused in GHSR1a actions on the communication between neurons, specifically at the presynaptic level. Recently, we found that neuronal calcium channels, structures that control neurotransmitter release, are inhibited by GHSR1a constitutive and ligand evoked activity.  Here we aim to develop a system suitable to evaluate the effect of GHSR1a on synaptic activity in mouse embryonic neuronal cultures. In order to measure electrical synaptic activity in vitro we need to express GHSR1a at native levels in a large amount of neurons and to identify the neurons expressing the receptor. Here we present a lentivirus-based expression system of GHSR1a tagged with yellow fluorescent protein (YFP) and an analog system for the GHSR1aA204E mutant that lacks constitutive activity. High neuronal transduction rates plus a weak promoter that ensure expression levels close to the native condition are some of the advantages of lentiviral systems. Furthermore, we used a third generation system to guarantee a high level of biosecurity. Here we present our clone strategy, protocols development and some preliminary data demonstrating the utility of our system.