Microdialysis

This is a modification of the earlier push-pull cannula which could be used in anaesthetised animals only. The microdialysis probe which has an outside diameter of about 250 pm (Fig. 4.6) is implanted into the brain under anaesthesia and then subsequently perfused with aCSF. Solutes (including neurotransmitters) in the extracellular fluid of the brain diffuse down their concentration gradient into the probe. By taking samples of the effluent dialysate at regular intervals it is possible to monitor changes in transmitter release. This technique has been used for several years to study release of monoamines (e.g. Sharp, Umbers and Gartside 1997) but is now used to harvest acetylcholine and amino acids as well. Since the molecular cut-off of the dialysis membrane is in the region of 6-20 kDa (depending on the type of membrane used), this technique can also be used to measure release of some small neuropeptides (e.g. oxytocin and vasopressin).

One advantage of microdialysis is that it enables the study of transmitter release in specific brain areas or nuclei. To ensure its correct placement, the probe is implanted, under anaesthesia, by sterotaxic surgery. Another advantage is that the probe can be anchored in place with dental cement and experiments carried out later, in conscious freely moving animals once they have recovered from the anaesthetic. Indeed, comparison of results from studies carried out on both anaesthetised and freely moving subjects has revealed drug interactions with anaesthetics that can affect transmitter release: anaesthetic-induced changes in the regulation of noradrenaline release by ^-adrenoceptors is a case in point. It is also possible to carry out long-term

Figure 4.6 The tip of a microdialysis probe, expanded to show dialysis tubing around a steel cannula through the base of which fluid can flow out and then up and over the membrane. The length of membrane below the probe support can be altered (1-10 mm) to suit the size of the animal and the brain area being studied. Flow rates are normally below 2 pl/min

Figure 4.6 The tip of a microdialysis probe, expanded to show dialysis tubing around a steel cannula through the base of which fluid can flow out and then up and over the membrane. The length of membrane below the probe support can be altered (1-10 mm) to suit the size of the animal and the brain area being studied. Flow rates are normally below 2 pl/min or repeated studies on the same animals but this requires a slight modification of the technique. In this case, a 'guide' cannula is first implanted, under anaesthesia, through which the microdialysis probe is inserted. Unfortunately, for a variety of reasons, each microdialysis probe can be used for only a few hours and so it has to be replaced each day. However, the presence of the guide cannula makes this a relatively straightforward process that requires only light sedation of the animal.

A further advantage of microdialysis is that, unlike the push-pull cannula or the cortical cup, the perfusion medium does not come into direct contact with the tissue being studied. This reduces damage caused by turbulence as well as enzymic degradation of the transmitter. For instance, acetylcholine, but not cholinesterase, will penetrate the probe membrane. Finally, because solutes will pass out of the probe, as well as into it, the probe can also be used for infusing ions (Fig. 4.7) or drugs into specific brain regions ('reverse' or 'retro-' dialysis) so that their local effects on transmitter release can be studied. This avoids many of the problems that arise when trying to determine the synaptic actions of drugs when these are administered systemically.

The rate at which the probes are perfused with aCSF is a compromise between the time required for the solutes in the CSF to reach equilibrium with those in the probe (the slower, the better) versus the ideal time-frame for studying changes in transmitter release (the shorter, the better). In general, flow rates of around 1-2 pl/min are used and the time which elapses between taking samples is determined by how much transmitter

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