Nano-Amperometric Electrochemical Biosensors for Quantitative Measurements of Transient Release By Living Neurons Application to Glutamate Release
a PASTEUR, Chemistry Department, École Normale Supérieure, PSL Sorbonne University, CNRS, 24 rue Lhomond, 75005 Paris, France,
b State Key Laboratory of Physical Chemistry of Solid Surfaces, Xiamen University, Xiamen, 361005, China.
Glutamate (Glu) is a crucial fundamental excitatory neurotransmitter released through vesicular exocytosis in the central nervous system. Hence, quantitative measurements and interpretation of intravesicular Glu and of transient exocytotic release contents directly from individual living neurons are highly desired for understanding the mechanisms (full or sub-quantal release?) of synaptic transmission and plasticity. However, this could not be achieved so far due to the lack of adequate experimental strategies relying on selective and sensitive Glu nanosensors.
We will show that a novel electrochemical Glu nanobiosensor based on a single SiC nanowire (I,II) is prone to selectively measure in real-time Glu fluxes released via exocytosis by large Glu vesicles (ca. 125 nm diameter) present in single hippocampal axonal varicosities as well as their intravesicular content before exocytosis by IVIEC (III).1
Combination of these two series of measurements revealed a sub-quantal release mode in living hippocampal neurons, viz., only ca. one third to one half of intravesicular Glu molecules are released by individual vesicles during exocytotic events.
Importantly, this fraction remained practically the same when hippocampal neurons were pretreated with L-Glu-precursor L-glutamine, while it significantly increased after zinc treatment, although in both cases the intravesicular contents before release were drastically affected.
This work will also serve to discuss the meaning and adequacy of pre-calibrations performed in bulk solutions to assess the analytical properties of enzyme-based electrochemical nanosensors aimed to detect fast transient release events.
Still, we will take advantage of this example to demonstrate the general difficulties and analytical dangers associated to the use calibration curves performed in bulk solutions to analyze data in vivo with micro- or nanometric enzymatic electrochemical sensors.2,3