Synaptosomes

Synaptosomes are 'pinched-off' nerve terminals which become severed from the parent axon during gentle homogenisation of brain tissue and then subsequently reseal. They

Figure 4.1 Turnover of classical neurotransmitters. At normal rates of neuronal activity, endogenous stores of neurotransmitter are maintained at constant (steady-state) levels, indicating that the supply of new neurotransmitter (through synthesis) meets the demand (determined by release and metabolism). Consequently, the rate of the depletion (A) of the endogenous store of transmitter after inhibition of its synthesis indicates turnover rate and is described by the equation:

Figure 4.1 Turnover of classical neurotransmitters. At normal rates of neuronal activity, endogenous stores of neurotransmitter are maintained at constant (steady-state) levels, indicating that the supply of new neurotransmitter (through synthesis) meets the demand (determined by release and metabolism). Consequently, the rate of the depletion (A) of the endogenous store of transmitter after inhibition of its synthesis indicates turnover rate and is described by the equation:

where [T] is the tissue concentration at time t; [T]0 is the transmitter concentration at time 0; and k is the rate constant for the efflux of transmitter. When plotted semi-logarithmically (B), the exponential decline in tissue stores of transmitter gives a straight line described by the equation:

At steady-state there is no net loss of transmitter from the system and so the rate of synthesis of transmitter equals the rate of its efflux. Thus:

turnover rate (TOR) = k x tissue transmitter concentration at time 0

For monoamines, turnover rate is only an approximate measure of release rate because of recycling of released transmitter and spontaneous metabolism of the endogenous store are separated as a crude synaptosomal pellet from undisrupted tissue by differential centrifugation of the brain homogenates (Fig. 4.3). After resuspension of the synaptosomal pellet, they are perfused with artificial cerebrospinal fluid (aCSF) and the concentration of transmitter in the effluent used as an index of its release.

The main advantage of using synaptosomes is that they are free from any influence of the parent axon. Another is that, since the volume of extracellular space (the incubation medium) is functionally infinite, transmitter will not accumulate near the synapto-somes. This means that reuptake of released transmitter is unlikely to occur and that, under drug-free conditions, transmitter release will not be modified by activation of auto- or heteroceptors (see below).

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Figure 4.2 The intraneuronal stores of monoamines are maintained by synthesis from precursors taken in with the diet. The pool is depleted by release of transmitter and some spontaneous metabolism of intraneuronal transmitter. Released monoamines are inactivated by reuptake on membrane-bound transporters. Following reuptake, some transmitter might be recycled while the remainder is metabolised. Some transmitter escapes the reuptake process and overflows from the synapse in the extracellular fluid

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Figure 4.2 The intraneuronal stores of monoamines are maintained by synthesis from precursors taken in with the diet. The pool is depleted by release of transmitter and some spontaneous metabolism of intraneuronal transmitter. Released monoamines are inactivated by reuptake on membrane-bound transporters. Following reuptake, some transmitter might be recycled while the remainder is metabolised. Some transmitter escapes the reuptake process and overflows from the synapse in the extracellular fluid

A disadvantage of using synaptosomes is that they cannot be used to study transmitter release evoked by propagated nerve impulses, but the release, like that from intact neurons, is Ca2+-dependent and K+-sensitive. Pharmacological studies using synaptosomes have also provided evidence that the amount of transmitter that is released following their depolarisation is regulated by the activation of presynaptic receptors.

If the amount of transmitter released into the effluent is too small to be measured by a conventional neurochemical assay, the endogenous store can be 'tagged' by incubating the synaptosomes with radiolabelled transmitter (usually 3H). After washing the synaptosomes, the release of 3H into the perfusate is then measured using (extremely sensitive) liquid scintillation counting. This approach rests on the assumption that the radiolabelled transmitter is taken up only by the 'right' synaptosomes (e.g. that only those derived from noradrenergic neurons will take up [3H]noradrenaline) which is not always the case. Another unjustified assumption is that the radiolabelled transmitter mixes freely with the endogenous (unlabelled) store. Finally, as it is the radiolabel, rather than the actual transmitter itself, which is being measured it must be established that the label remains attached to the neurotransmitter. For instance, [3H]glutamate can be turned into [3H]GABA or [3H]glutamine and so care must be taken to ensure that any findings with 3H refer to release of [3H]glutamate rather than that of its metabolites.

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