Studies of Excitability in a Model Peptidergic System: The Roles of Cyclic AMP, Protein Phosphorylation and Serotonin During Afterdischarge in the Bag Cell Neurons of Aplysia californica

Abstract

The polypeptide hormone-secreting bag cell neurons from the abdominal ganglion of Aplysia can be induced to fire repetitively when triggered by a brief electrical stimulus to the afferent pathway. This thesis investigates the mechanism of this afterdischarge by employing biochemical, pharmacological and electrophysiological approaches. The description of bag cell afterdischarge, its modulation by the transmitters serotonin and dopamine and evidence for the role of cyclic AMP in the genesis of afterdischarge is presented in Chapter 1. Bag cell afterdischarge is shown to be inhibited by the application of serotonin and lengthened by the application of dopamine or the methylxanthine phosphodiesterase inhibitors. Cyclic AMP undergoes a 2-3 fold increase in the bag cell clusters during an electrically-stimulated afterdischarge but not in matched controls where equivalent electrical stimulation did not elicit afterdischarge. As further evidence for a role for cyclic AMP in the genesis of afterdischarge, afterdischarges were obtained in unstimulated preparations by the extracellular application of the cyclic AMP analogues, 8-benzylthio-cyclic AMP and 8-methylthio-cyclic AMP. Chapter 2 describes protein phosphorylation in bag cell tissues under a number of different conditions. The presence of an endogenous, cyclic AMP-dependent protein kinase activity is demonstrated in crude membranes prepared from bag cells and the substrate specificity for this activity is shown to be similar to that of protein kinase catalytic subunit prepared from bovine heart. Increases in phosphorylation of a 33,000 dalton and 21,000 dalton phosphoprotein are shown to occur during electrically-stimulated afterdischarge in bag cells. The 21,000 dalton substrate is shown to be apparently specific to bag cell tissues and an amino acid composition and partial amino acid sequence of this protein is presented. Chapter 3 presents evidence that serotonin, within the physiological range reported by other workers for Aplysia (0.1-1.0 μM) brings about a rapid inhibition of an ongoing afterdischarge. This inhibition is antagonized by the stereospecific blocker of serotonin action, D-butaclamol but not its inactive isomer, L-butaclamol. Serotonergic inhibition is shown to be associated with decreased bag cell action potential duration and height and an increased threshold to spike generation. Evidence is presented that the second, calcium-dependent phase of bag cell afterdischarge is most sensitive to the action of the transmitter and that the potassium channel blocker, tetraethylammonium can overcome serotonin's inhibitory effect. This raises the possibility that serotonin may cause inhibition of bag cell afterdischarge by increasing potassium conductance. The possible functional role of serotonin inhibition of egg-laying is discussed.</p