Enjoyable Gut Neuroscience

The idea behind the electrochemical technique is that when a compound is oxidized, the transfer of electrons can be detected, and quantified. This provides a direct and accurate measure of the number of molecules of compound (i.e., the concentration) at a specific place and at a specific time. When a compound is oxidized it donates (i.e., loses) electrons. This can occur when the compound is near to a surface that is held at a high potential voltage. A surface commonly used in biological electrochemistry is that of a carbon fiber electrode. These electrodes are of a low resistance and the potential at the surface can be clamped or varied by a voltage clamp amplifier similar to those used to examine channel activity in oocytes (e.g., Axon GeneClamp). The current generated by the amplifier negates any change in voltage at the electrode tip and is proportional to the amount of current generated by oxidation of the compound.

Advantages: There are several major advantages to using electrochemical techniques. First, the technique can positively identify the compound being detected. The major families of catechol and amine neurotransmitters have oxidation potentials that differentiate them from other biological compounds that also oxidize (e.g., ascorbic acid). When combined with other chemical or pharmacological techniques, the identity of the compound detected can usually be narrowed down to a single compound. Second, the technique is robust. A properly treated electrode can be used for several hours. In the CNS, electrodes are routinely implanted and recordings made for 5 or 6 hours (Daws et al., 1997). Third, the technique has a very high temporal and spatial resolution. The concentrations of compounds such as 5-HT can be detected with sub-millisecond time resolution. This allows the kinetics of transmitter release to be assessed and to be followed in time or with the addition of pharmacological agents. In addition, the size of the recording electrode surface directly controls the area from which recordings take place - the smaller the electrode, the smaller the area recorded from. Thus, an area of only a few tens of microns can be examined (i.e., single cell localization).

Challenges: There are several major technical challenges inherent to using electrochemical techniques in a biological system. First, the identity of the compound being detected can not always be determined exactly. Second, because the surface of the electrode is usually small, proteins and other biological material can quickly foul the surface. These processes are worse when the electrode is held at a working potential where oxidized byproducts can stick to the surface. Third, gathering data at a high speed (e.g., at 1 microsecond intervals) often precludes the identification of a compound. Conversely, the identification of a compound may take several seconds such that subsequent recordings yield data points at 10 second intervals. Finally, a limitation is that only compounds that oxidize within a fairly narrow range of voltages can be examined. The oxidation of water and of molecular oxygen form the upper and lower boundaries, respectively. Beyond these boundaries, the large signals produced by water and oxygen make detection of biological compounds difficult if not impossible. All of the catechol and amine neurotransmitters have oxidations potentials that lie between these limits.

Solutions: The approaches used in my lab overcome these challenges by using chemical and pharmacological techniques to supplement the electrochemical techniques. Further, two major variants of the electrochemical technique are used. First, cyclic voltammetry, which measures current during a voltage ramp, is used to identify the major species of compound being oxidized. Second, chronoamperometry, which measures current at a fixed holding potential, will be used to measure release events and other very fast events. Finally, as noted, 5-HT oxidizes well within the range of voltages delineated by water and oxygen.