Brain slices

These can be conventional cross-sectional slices up to 0.5 mm thick, or small cubes (0.10.5 mm on each plane) or pyramids of brain tissue. The main advantage of using slices is that, either by crudely passing currents across them or, in some cases, stimulating a

Drugs And Brain Function

Figure 4.3 The distribution of neurotransmitter in different subcellular fractions, (a) Within the hypothetical brain area shown, where it is assumed that this neuronal arrangement is reproduced many times, the proportion of each neurotransmitter found in the synaptosomes, rather than the cell body (cytoplasmic) fraction, will vary considerably. On the assumption that, although concentrated in nerve terminals, the neurotransmitter will also be found in cell bodies and axons, it is likely that A will be almost entirely in synaptosomes, C mostly in the cytoplasm, while B will be more evenly divided. (b) Procedure for preparation and separation of synaptosomes. Brain tissue is homogenised and spun at 1000 x g for lOmin to remove cell debris. The supernatant (SI) is spun at 20 000 x g for lOmin and the pellet (P2), containing synaptosomes and mitochondria, is spun through two layers of sucrose for 2 h at 50 000 x g. This is 'sucrose density gradient centrifugation' and is based on the principle that an individual particle will settle in the zone of the sucrose gradient that has the same density. Thus, synaptosomes separate from other elements of the 20000xg pellet and settle at the interface between 0.8 M and 1.2 M sucrose

Figure 4.3 The distribution of neurotransmitter in different subcellular fractions, (a) Within the hypothetical brain area shown, where it is assumed that this neuronal arrangement is reproduced many times, the proportion of each neurotransmitter found in the synaptosomes, rather than the cell body (cytoplasmic) fraction, will vary considerably. On the assumption that, although concentrated in nerve terminals, the neurotransmitter will also be found in cell bodies and axons, it is likely that A will be almost entirely in synaptosomes, C mostly in the cytoplasm, while B will be more evenly divided. (b) Procedure for preparation and separation of synaptosomes. Brain tissue is homogenised and spun at 1000 x g for lOmin to remove cell debris. The supernatant (SI) is spun at 20 000 x g for lOmin and the pellet (P2), containing synaptosomes and mitochondria, is spun through two layers of sucrose for 2 h at 50 000 x g. This is 'sucrose density gradient centrifugation' and is based on the principle that an individual particle will settle in the zone of the sucrose gradient that has the same density. Thus, synaptosomes separate from other elements of the 20000xg pellet and settle at the interface between 0.8 M and 1.2 M sucrose specific neuronal tract, they can be used to study impulse-evoked release of transmitter. Because the three-dimensional integrity of the tissue is maintained, they can also be used to study modulation of transmitter release by heteroceptors (see below).

One approach, and the first to be adopted, is to study transmitter release from slices which have been preloaded with radiolabelled transmitter. In these experiments, drug-induced changes in the release of transmitter is usually monitored using the 'doublepulse' technique. This involves comparing the effects of a test drug on the amount of transmitter released in response to a reference pulse and a second identical test pulse. If all the radiolabelled transmitter that overflows in the effluent is collected, and the amount which remains in the slice at the end of the experiment is also measured, it is possible to calculate not only how much radiolabelled transmitter was originally contained in the slice but also the effects of drugs on 'fractional release', i.e. the proportion of the store of radiolabelled transmitter which is released by nerve stimulation. As with synaptosomes, however, it cannot be assumed that incubation of slices in a medium containing radiolabelled transmitter results in its even distribution throughout the slice. Also, the problem of continuous dilution of the radiolabelled store with newly synthesised (unlabelled) transmitter must be borne in mind.

Modern sensitive chromatographic and voltammetric techniques now make it possible to estimate the release of unlabelled endogenous transmitter from slices of brain tissue (commonly the hippocampus and striatum) or spinal cord (Fig. 4.4). However, whatever analytical method is used, the thickness of the slice is paramount. It is important to maintain the balance between preserving the integrity of the tissue (the thicker the slice, the better) against maintaining tissue viability by perfusion with oxygenated aCSF (the thinner the slice, the better).

TECHNIQUES in VIVO The cortical cup

The cortical cup has been used for many years to monitor changes in transmitter release induced by physiological and pharmacological stimuli (Fig. 4.5). In the past, it was used most commonly to study release of amino acids and acetylcholine. More recently, it has

Drugs And Brain Function

Figure 4.4 Release of amino acids from cortical slices exposed to 50 mM K+. Measurements by HPLC and fluorescence detection after reaction of amino acids with o-phthalaldehyde: 1, aspartate; 2, glutamate; 3, asparagine; 4, serine; 5, glutamine; 6, histidine; 7, homoserine (internal standard); 8, glycine; 9, threonine; 10, arginine; 11, taurine; 12, alanine; 13, GABA; 14, tyrosine. Glutamate concentration is almost 1 pmol/^l which represents a release rate of 30 pmol/min/mg tissue

Figure 4.4 Release of amino acids from cortical slices exposed to 50 mM K+. Measurements by HPLC and fluorescence detection after reaction of amino acids with o-phthalaldehyde: 1, aspartate; 2, glutamate; 3, asparagine; 4, serine; 5, glutamine; 6, histidine; 7, homoserine (internal standard); 8, glycine; 9, threonine; 10, arginine; 11, taurine; 12, alanine; 13, GABA; 14, tyrosine. Glutamate concentration is almost 1 pmol/^l which represents a release rate of 30 pmol/min/mg tissue

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