Multiplexing FSCV and iGluSnFR3 Sensors Reveals Adenosine Transiently Inhibits Stimulated Dopamine and Glutamate Release

Simultaneous measurements of different neurotransmitters are challenging, but necessary, to understand neurotransmitter interactions in the brain. Genetically encoded sensors have a high spatial resolution, but there are still limited colors, while electrochemistry provides high time resolution but only for a limited number of electroactive analytes. Here, we multiplexed fast-scan cyclic voltammetry (FSCV) and genetically encoded fluorescence sensors to simultaneously measure adenosine, dopamine, and glutamate to investigate the spatial and temporal profiles of adenosine neuromodulation. A genetically encoded glutamate sensor (iGluSnFR3.v857) was expressed in the caudate-putamen region and then in a brain slice, a carbon fiber microelectrode (CFME) was implanted to monitor electrically stimulated dopamine release near cells expressing the glutamate sensor. Glutamate and dopamine release were inversely correlated, with areas with high stimulated glutamate release displaying low stimulated dopamine release and vice versa. Exogenous adenosine was applied locally to the brain slice, lasting 30 s, resulting in a transient inhibitory effect on both electrically stimulated dopamine and glutamate release. Inhibition by adenosine was observed only within a 250 μm distance, showing regional inhibition effects. Dopamine and glutamate release recovered 10 min after adenosine injection. The A₁ receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) blocked the adenosine inhibition of both glutamate and dopamine release, indicating adenosine has a global transient inhibition effect on dopamine and glutamate release via A₁ receptor modulation. This study shows that multiplexing FSCV and fluorescence sensors (iGluSnFR3.v857) allows simultaneous monitoring of multiple neurotransmitters and reveals an overall transient inhibition by adenosine in the caudate.