ATP is an important neurotransmitter and modulator that can act at two classes of receptors within the CNS. As many of the functions of ATP are incompletely understood, the ability to measure ATP release from the CNS in real time is a valuable tool to further understand its functions. Here we present a micro-biosensor capable of rapid in situ detection of ATP in neuronal tissue.

Building on our previous work (Llaudet et al. 2003) we have entrapped 2 enzymes, glycerol kinase and glycerol-3-phosphate oxidase, in a thin robust layer around a Pt microelectrode. The system is based on the amperometric detection of peroxide produced by the oxidation of glycerol-3-phosphate; in the first step of the enzyme cascade glycerol kinase catalyses the transfer of a phosphate from ATP to glycerol, which therefore needs to be present at concentrations of at least 500 ÁM. The modified electrode shows impressive selectivity, sensitivity (150 mA/M/cm₂) and speed of response (10 s 10-90 % signal) towards ATP. The sensor is independent of glycerol concentrations above 500 ÁM and of oxygen, but while the magnitude of the response to ATP is independent of pH, over the range pH 6-8, the base line current shows a slight pH dependence. The electrode size can range from 25 to 100 Ám in diameter and 0.5 to 2 mm in length which allows them to be inserted into most tissues without causing significant damage.

We have tested this new biosensor by measuring the release of ATP from the spinal cord of Xenopus embryos during swimming. Xenopus embryos were prepared under MS222 anaesthesia for extracellular ventral root recordings using previously described methods (Dale, 1995). The sensor was aligned with the ventral part of the spinal cord and simultaneous ventral root recordings made. We detected transient ATP release throughout the swimming episode that was phase-locked to ventral root discharge on each motor cycle. With the aid of this new biosensor we have for the first time provided direct evidence for the rhythmic release of ATP during spinal cord activity.