Brain Research, 85 (1975) 135-139 135 ,f~ Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands Differential effects of caffeine, D-amphetamine and methylphenidate on individual raphe cell fluorescence: a microspectrofluorimetric demonstration MARK A. GEYER, W. JOHN DAWSEY AND ARNOLD J. MANDELL Department of Paychiatry, University of California, San Diego, La Jolla, Calif'. 92037 (U.S.A.) (Accepted November 5th, 1974) Caffeine, amphetamine and methylphenidate may alter the activity of seroton- ergic pathways in the brain in addition to activating central catecholaminergic sys- tems 3,6. Caffeine increases the concentrations of brain 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA)I, z. It has been speculated that this effect might be due to inhibition ofphosphodiesterase (PDE), stimulation ofcatecholamine release, or to a more direct effect on 5-HT neurons 1,2. In studies of another stimulant, several reports have indicated that the behavioral effects of amphetamine are altered by manipulations of serotonergic systems 13. Amphetamine also increases the firing rates of some 5-HT-containing neurons in the raphe~. We recently reported inhibition of tryptophan hydroxylase activity in some brain regions shortly after injection of D- amphetamine 8. Using the resolution and anatomical specificity of semiquantitative micro- spectrofluorimetry we assessed localized changes in the transmitter content of central serotonergic neurons, and now report confirmation of Berkowitz and Spector'sl, e observations that caffeine increases intraneuronal 5-HT. To test the possibility that this phenomenon results from a release of presynaptic catecholamines, we compared the effect of caffeine with those of o-amphetamine and methylphenidate. Our results indicate that caffeine's effect is unrelated to catecholamine release. Eighteen male Sprague-Dawley rats (Carworth, 120-140 g) were housed with a 12:12 h light-dark cycle and free access to food and water. Before experimentation, they were deprived of food for 24 h. One hour before decapitation in a counter- balanced order, each rat received an intraperitoneal injection of isotonic saline (N - 5), or 50 mg/kg caffeine (Sigma; N ~ 5), or 25 mg/kg methylphenidate hydrochloride (CIBA; N = 4), or 7.5 mg/kg D-amphetamine sulfate (Sigma; N =- 4). This dose of caffeine was one of the standard doses used by Berkowitz and Spectort, 2, while the doses olD-amphetamine and methylphenidate were selected to produce roughly com- parable patterns of stereotypic behavior (Segal, personal communication, 1974) and substantial catecholamine release 10. Immediately after sacrifice the brains were removed