Multiphoton Imaging to assess Chloride homeostasis

Effect of GABA on intracellular chloride viewed with fluorescence lifetime imaging (FLIM)

During neurodevelopment, the effect of the neurotransmitter GABA on neurons changes from excitatory to inhibitory. This shift is due to a change in the intracellular concentration of chloride (Cl-) ions that alter the current polarity of the GABA-gated ion channel known as the GABAA receptor. Thus, Cl- ions enter into neurons in the mature central nervous system (CNS), while the opposite occurs in the immature CNS. Disruptions in this transformation are linked to various neurodevelopmental disorders.

MQAE is a membrane dye used to monitor Cl- levels, and the intracellular concentration of Cl- ions directly influences the dye’s fluorescence intensity and lifetime. In this study, Justin Hamel of Dr. Antoine Godin's laboratory at Université Laval's Centre de recherche CERVO Brain research Centre used primary neurons from rat hippocampus, incubated with an extracellular solution containing MQAE to enable it to pass through the cell membrane. 60 seconds after the start of imaging, GABA was then added to the extracellular solution. Exposure to GABA opens the GABAA channels, making them permeable to Cl- ions that then follow the electrochemical gradient of the cell. As these neurons are relatively immature, their high basal intracellular Cl- concentration causes Cl- ions to flow out of them. This decrease in intracellular Cl- ion concentration leads to an increase in MQAE fluorescence intensity and lifetime.

The video showing the change in Cl- level was acquired at 31 Hz, using Bliq’s Video-rate Multiphoton System (VMS) with a 25X water immersion objective. The wavelength used for excitation was 750 nm, and the acquisition was for 600 seconds.

The video of Justin Hamel won the second prize in the image contest recently launched at the CERVO research center, University Laval.