Effects of electrical stimulation of rat midbrain on 5-hydroxytryptamine synthesis as determined by a sensitive radioisotope method.

5-Hydroxytryptamine (5-HT) synthesis has been determined in the rat brain by measuring the 5-[3H]HT formed from [3H]tryptophan in the presence of monoamine oxidase inhibitor. Electrical stimulation in the region of the midbrain raphe nucleus increased formation of 5-[3H]HT by over 100 per cent, although the level of endogenous 5-HT and the concentration and specific activity of tryptophan were unchanged; the results are interpreted in terms of a two-compartment model. The optimum stimulation parameters were determined. Three days after a single dose of the tryptophan hydroxylase inhibitor p-chlorophenylalanine, stimulation increased 5-HT synthesis by the same percentage as in untreated animals. It was also found that after the end of an hour's stimulation, synthesis returned to control values in under an hour. These results suggest that the rise in synthesis of the amine on stimulation is not due to induction of tryptophan hydroxylase, but more likely to an increase in the activity of existing enzyme. MIDBRAIN ON 5-HYDROXYTRYPTAMINE SYNTHESIS SEVERAL methods have been used to measure 5-HT turnover in the rat brain: these include the rate of accumulation of the amine after monoamine oxidase (MAO) inhibition (NEFF and TOZER, 1968), catabolism of intracisternally-injected 5-HT (SIMMONDS, 1970), administration of loading doses of L-tryptophan to saturate tryptophan hydroxylase, the rate-limiting enzyme (ASHCROFT, ECCLESTON and CRAWFORD, 1965) and the application of steady-state kinetics to the accumulation of 5-[14C]HT from [14C]tryptophan infused intravenously (LIN, COSTA, NEFF, WANG and NGAI, 1969). All of these methods have disadvantages. Measurement of synthesis by the rate of accumulation of the amine after MA0 inhibition assumes that the drug acts immediately; the use of intracisternally-injected 5-HT is unsatisfactory as it may enter cells where it is normally absent (SIMMONDS, 1970) and in the case of tryptophan loading, a true indication of turnover is not obtained because the 5-hydroxyindole acetic acid (5-HIAA) which accumulates is constantly being transported out of the brain. The method of LIN et al. (1969) comes the closest to normal physiological conditions, but has the practical disadvantage that whenever 5-HT turnover has to be measured under different conditions, such as when a drug is given, the curve of tryptophan specific activity in plasma has to be determined as its shape may alter. We have developed a simple method of measuring relative rates of 5-HT synthesis in rat brain which overcomes most of these problems. It involves the administration of an MA0 inhibitor (pargyline) followed by [3H]tryptophan and, after a fixed length of time, the amount of 5-[3H]HT synthesized is measured. We have used this method to investigate the effects of electrical stimulation in the rat midbrain on synthesis of P.J.S. is an M.R.C. Scholar. Abbreviations used: PCP, p-chlorophenylalanine; MAO, monoamine oxidase; 5-HIAA, 5hydroxyindole acetic acid; PPO, 2,5-diphenyloxazole ; POPOP, p-bis-[2-(5 phenyloxazolyl)]-benzene ; r, coefficient of linear correlation.