title: Phosphorylation-dependent low-frequency depression at phasic synapses of a crayfish motoneuron.
abstract: Extremes in presynaptic differentiation can be studied at the crayfish leg extensor muscle where, on the same muscle fiber, one motoneuron makes "phasic" depressing synapses that have a high probability of neurotransmitter release and another motoneuron makes "tonic," low-probability, facilitating synapses. The large motor axons permit intracellular access to presynaptic sites. We examined the role of phosphorylation during low-frequency depression (LFD) in the relatively little studied phasic synapses. LFD occurs with stimulation at 0.2 Hz and develops with time constants of 4 and 105 min to reach >50% depression of transmitter release in 60 min similar to long-term depression in mammals. LFD is not associated with changes in postsynaptic sensitivity to transmitter and thus is a presynaptic event, although it is not accompanied by changes in the presynaptic action potential. Blockade of protein kinases accelerated the slow phase of LFD, but stimulation of kinases reduced depression. Blockade of protein phosphatases 1A/2A reversed the slow phase. When calcineurin was inhibited, both phases of LFD were abolished, and facilitation occurred instead. Immunostaining showed calcineurin-like immunoreactivity in synaptic terminals. Recovery from LFD occurred in approximately 1 h if stimulation frequency was reduced to 0.0016 Hz. Recovery was blocked by kinase inhibition. This study shows that phosphorylation-dependent mechanisms are involved in LFD and suggests that exocytosis is controlled by conditions that shift the balance between phosphorylated and unphosphorylated substrates. The shift can occur by alteration in the relative activities of protein kinases and phosphatases.
authors.affiliation: Department of Physiology, University of Toronto, Toronto, Ontario, Canada M5S 1A8.
authors.forename: Lorelei B
authors.initials: LB
authors.lastname: Silverman-Gavrila
authors.forename: Peter M R
authors.initials: PM
authors.lastname: Orth
authors.forename: Milton P
authors.initials: MP
authors.lastname: Charlton
mesh_terms: D000818:Animals; D003400:Astacoidea; D015153:Blotting, Western / Q000379:methods; D019703:Calcineurin / Q000378:metabolism; D004307:Dose-Response Relationship, Radiation; D004330:Drosophila; D004347:Drug Interactions; D004558:Electric Stimulation / Q000379:methods; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019706:Excitatory Postsynaptic Potentials / Q000502:physiology* / Q000528:radiation effects; D007150:Immunohistochemistry / Q000379:methods; D066298:In Vitro Techniques; D009046:Motor Neurons / Q000166:cytology / Q000502:physiology*; D018482:Muscle, Skeletal / Q000166:cytology; D009433:Neural Inhibition / Q000502:physiology* / Q000528:radiation effects; D009469:Neuromuscular Junction / Q000502:physiology; D010766:Phosphorylation; D050765:Qa-SNARE Proteins / Q000378:metabolism; D013569:Synapses / Q000502:physiology*; D009435:Synaptic Transmission / Q000502:physiology*; D013997:Time Factors
chemical_list: D004791:Enzyme Inhibitors; D050765:Qa-SNARE Proteins; D019703:Calcineurin