Tyrosine hydroxylase activation and transmitter release from central noradrenergic neurons by electrical field stimulation.

Abstract

Electrical stimulation of the noradrenergic neurons in the locus coeruleus of the rat results in a marked increase in the tyrosine hydroxylase activity of the hippocampus on the stimulated side (Roth et al., 1975). We have developed an in vitro system to further study this interesting phenomenon. Rat hippocampal slices were stimulated in an electrical field using specially built chambers which permitted a continous superfusion with Krebs Ringer Phosphate solution while stimulation was taking place. The slices were then homogenized and tyrosine hydroxylase activity and kinetic parameters were determined in the 104000 g supernate fraction. Electrical field stimulation (60 V, 4 ms, 5–20 Hz, 5 min) induced a stimulus-dependent increase in tyrosine hydroxylase activity. The increase in tyrosine hydroxylase activity was directly dependent on the number of pulses of stimulation applied. Potassium depolarization of hippocampal slices also resulted in a marked increase in the activity of the enzyme. Electrical field stimulation appears to activate tyrosine hydroxylase by causing an increase in its affinity for both substrate and pteridine cofactor and by decreasing its affinity for the endproduct inhibitor, norepinephrine. No change in Vmax was observed. The superfusion system was also used to study spontaneous and electrically evoked release of labelled norepinephrine from hippocampal slices. Electrical field stimulation for 1.0 min produced a marked increase in the release of label. Absence of calcium from the superfusion solution almost completely abolished the electrically evoked release of exogenously taken up 3H-norepinephrine. The release of 3H-norepinephrine was also found to be dependent on the number of pulses of stimulation used. The results reported support the hypothesis that depolarization of central noradrenergic neurons results in an increase in transmitter synthesis mediated in part via a kinetic activation of tyrosine hydroxylase. The concomitant use of superfusion and electrical field stimulation of hippocampal slices provides a simple system to study neurotransmitter synthesis and release in central noradrenergic neurons.