Sensitization of the gill withdrawal reflex results from presynaptic facilitation at the excitatory synapses made by sensory neurons on gill motor neurons. Facilitation is accompanied by an increase in the duration of the action potential in sensory cells because of the depression of a K+ current. This results in an increasd influx of CA2+ and a greater release of transmitter from sensory neurons. There is evidence that serotonin is the facilitating transmitter and that the depression of the K+ current by serotonin mediated by cAMP-dependent protein phosphorylation. To test further the role of the cAMP-dependent protein kinase and of protein phosphorylation in sensitization, we have attempted to prevent or reverse the development of the electrophysiological correlates that accompany sensitization. We have pressure-injected sensory neurons with a specific and a stable protein inhibitor of the cAMP-dependent protein kinase both before and after the application of serotonin or the activation of the facilitator neurons. The increase in spike broadening that accompanies facilitation was prevented or diminished by injection of the inhibitor. Moreover, injection of the inhibitor could reverse fully the developed spike broadening produced by prior application of serotonin. These observations strenthen the evidence for the involvement of protein phosphorylation in presynaptic facilitation. Phosphorylation of the substrate protein evidently is quite labile and does not persist after the kinase is inhibited. Thus, the time course of short term sensitization appears to be determined by an active kinase. We think that it is likely that the mechanism for maintaining the kinase in an active form resides in the slow decay of the cAMP produced by the action of serotonin or the facilitator neurons on the sensory cells.

Ca(2+)-activated and Ca(2+)-independent protein kinase Cs (PKCs) are present in the nervous system of the marine mollusk Aplysia californica (Kruger et al., 1991). Sensitizing stimuli or application of the facilitatory transmitter 5-HT to intact isolated ganglia produces the presynaptic facilitation of sensory-to-motor neuron synapses that underlies behavioral sensitization, which is a simple form of learning. Activation of PKC can also produce this presynaptic facilitation (Braha et al., 1990). To determine which type of PKC is activated, we developed a sensitive and selective assay to measure both Ca(2+)-activated and Ca(2+)-independent PKC activities in crude supernatant and membrane fractions of nervous tissue. This assay is based on the specific binding of the Ca(2+)-activated PKCs to phosphatidylserine vesicles in the presence of Ca2+ and makes use of a novel synthetic peptide with sequences conforming to phylogenetically conserved pseudosubstrate regions of the Ca(2+)-independent kinases. We provide evidence that the presynaptic facilitation is produced by a Ca(2+)-activated isoform: application of 5-HT increases the amount of the Ca(2+)-activated PKC activity associated with the membrane. Under these conditions, no increase in Ca(2+)-independent kinase activity is seen.

We have isolated the cDNA for a tyrosine kinase receptor that is expressed in the nervous system of Aplysia californica and that is similar to the vertebrate insulin receptor. Binding studies and immunocytochemical staining show that the receptor is abundant in the bag cell neurons. Application of vertebrate insulin to clusters of bag cell neurons stimulates the phosphorylation of the receptor on tyrosine residues, and exposure of isolated bag cell neurons to insulin produces an increase in height and a decrease in duration of the action potentials that can be detected within 15-30 min. These effects were not seen with insulin-like growth factor-1. In voltage-clamped neurons, insulin produces an increase in the amplitude of the voltage-dependent Ca2+ current that can be blocked by preincubation with herbimycin A, an inhibitor of tyrosine kinases. Insulin also enhances a delayed K+ current. We suggest that insulin-like peptides regulate the excitability of the bag cell neurons.

We isolated cDNA clones from an Aplysia sensory-cell library encoding two isoforms of protein kinase C (PKC). Several isozyme-specific regions are conserved in the Aplysia kinases, notably the variable regions V5 in the Ca(2+)-dependent PKC (Apl I) and V1 in the Ca(2+)- independent PKC (Apl II). Neuronal proteins with the properties expected of these two isoforms can be identified with antibodies raised against peptides synthesized from the amino acid sequences deduced from the clones. Sacktor and Schwartz (1990) measured the proportion of kinase activity that can be translocated to membrane in Aplysia sensory neurons and ganglia by stimuli that produce the presynaptic facilitation underlying behavioral sensitization. Much less Apl I and Apl II are translocated, suggesting that still other isoforms of PKC exist in these cells.