Voltammetry

This can be carried out in vitro (in brain slices, cultured cell preparations) or in vivo and involves penetrating the experimental tissue with a carbon-fibre electrode of 5-30 p,m in diameter (Fig. 4.9). This serves as an oxidising electrode and the Faradaic current generated by the oxidation of solutes on the surface of the electrode is proportional to their concentration. Obviously, only neurotransmitters which can be oxidised can be measured in this way so the technique is mainly limited to the study of monoamines and their metabolites. The amplitude of each peak on the ensuing voltammogram is a measure of solute concentration and individual peaks can be identified because different

z 0 H—'—'—I—-—-—I—-—-—I—'—'—I-

0 60 120 180 240

Time (min)

Figure 4.8 Noradrenaline concentration in dialysis samples from probes implanted in the rat frontal cortex. Spontaneous efflux of noradrenaline is stable throughout a 4h sampling period ('extended basals') but is increased markedly when either the noradrenaline reuptake inhibitor, desipramine (5 pM), or the ^-adrenoceptor antagonist, atipamezole (0.5 pM), is infused into the extracellular fluid via the microdialysis probe ('retrodialysis')

solutes oxidise at different potentials. Changes in the concentration of transmitters are monitored by rapid cycles of voltage scans (e.g. Palij and Stamford 1994). Since a complete scan takes only about 20 ms, the time resolution with voltammetry is much better than with microdialysis and is suitable for studying rapid, transient changes in transmitter release.

One difficulty with this method is that all oxidisable solutes in the external medium will be incorporated into the voltammogram and interfering peaks can be a problem. In fact, the concentration of monoamine metabolites and oxidisable solutes can be considerably greater than those of the parent amines which can be difficult to distinguish as a result. Ascorbic acid and uric acid are particularly problematic in this respect, although recent work suggests that an increase in the concentration of extracellular ascorbic acid could be a marker for the early phase of cerebral ischaemia. In general, voltammetry is most useful for measuring rapid (subsecond) changes in monoamine release. Under these circumstances, slower changes in the metabolites and other compounds do not interfere. Another problem is that the life of the electrode is limited by progressive 'poisoning' which diminishes its sensitivity. As a rule, voltammetric electrodes are best suited to 'acute' rather than 'chronic' measurements.

Biosensors

As the term suggests, the use of biosensors to measure transmitter release rests on exploiting a biological response which is proportional to the amount of transmitter in

Figure 4.9 Basics of voltammetry. (a) The cut tip of the microelectrode surrounded by the glass insulation, (b) Input voltage waveform to the potentiostat (—1.0 to +1.4V versus Ag/AgCl, 480 V/s). (c) Background current-corrected cyclic voltammogram of catecholamine obtained by plotting the Faradaic current against the input voltage waveform. The areas marked O and R are the oxidation and reduction currents. (d) Typical dopamine release and reuptake event following local electrical stimulation of a striatal slice. The trace is a plot of the oxidation peak height against time, calibrated for dopamine. (Figure and legend kindly supplied by J. A. Stamford)

Figure 4.9 Basics of voltammetry. (a) The cut tip of the microelectrode surrounded by the glass insulation, (b) Input voltage waveform to the potentiostat (—1.0 to +1.4V versus Ag/AgCl, 480 V/s). (c) Background current-corrected cyclic voltammogram of catecholamine obtained by plotting the Faradaic current against the input voltage waveform. The areas marked O and R are the oxidation and reduction currents. (d) Typical dopamine release and reuptake event following local electrical stimulation of a striatal slice. The trace is a plot of the oxidation peak height against time, calibrated for dopamine. (Figure and legend kindly supplied by J. A. Stamford)

the sample and which can be quantified. One of the earliest biosensors was the dorsal wall muscle of the leech which contracts in the presence of nM concentrations of acetylcholine. Others are the bioluminescent proteins, such as aequorin, which fluoresce in the presence of Ca2+. Within a reasonable range, the fluorescence intensity is proportional to the cation concentration and so it can be used to monitor the increase in the intracellular concentration of Ca2+ during excitation of nerve terminals. More recently, biosensors have been developed which comprise electrodes coated with glucose oxidase or lactate oxidase. The activity of these enzymes generates a current that can be used to quantify the concentration of glucose and lactate on the surface of the electrode. This work is playing an important part in research on brain metabolism during neuronal activity. So far, these electrodes are used in 'on-line' assays of samples collected by microdialysis but might be adapted for measurements in vivo in the future.

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