Aim: 

Local translation of mRNAs has been proposed as an important mechanism used by neurons to obtain synapse-specific modifications during long-lasting forms of plasticity. However little is known about the temporal requirement of local protein synthesis for the stable maintenance of learning-related synaptic growth on the one hand and the role of this new growth for the persistence of the changes in synaptic effectiveness that accompany memory storage on the other.

Methods: 

Toward this aim, we have used Aplysia culture where a single bifurcated sensory neuron makes synaptic contacts with two spatially separated motor neurons. By applying inhibitors of protein synthesis to the synapse at later time points e.g. 24 hr and 72 hr after 5-HT-training, we have been able to examine directly the role of local protein synthesis in the stabilization, as opposed to the induction, of the long-term synaptic changes that accompany long-term facilitation (LTF).

Results: 

We find that local application of emetine at 24 hr blocks LTF and the 5-HT-induced increase in sensory neuron varicosity number but when given at 72 hr inhibiting local protein synthesis has no effect on either. To explore the labile nature of the 5-HT-induced synaptic growth, we examined the selective effects of emetine on two different populations of sensory neuron varicosities: preexisting varicosities and 5-HT-induced newly formed varicosities. We found that at the branch that received emetine 24 hr after 5-HT treatment, only 38.1% of the 5-HT-induced newly formed varicosities were maintained at 72 hr versus 81.6% of the preexisting varicosities. One reason the 5-HT-induced newly formed varicosities are labile may reflect, in part, the undifferentiated nature of their presynaptic compartment. We tested this hypothesis by utilizing the activity-sensitive fluorescent protein, ecliptic synapto-PHluorin (synPH) to quantitatively analyze the number of synPH-active (competent for evoked transmitter release) and synPH-inactive (not competent for evoked transmitter release) 5-HT-induced newly formed varicosities at 24 hr. After local application of emetine, we did image the same synaptic area at 72 hr and determined that 63.9% of the 5-HT-induced newly formed varicosities that were synPH-active at 24 hr remained stable and, therefore, were present at 72 hr versus only 29.1% of the synPH-inactive varicosities.

Conclusion: 

Our finding that the 5-HT-induced newly formed sensory neuron varicosities are sensitive to disruption for a critical period lasting several days indicates that the newly formed varicosities require a sustained increase in the local synthesis of proteins to acquire the more stable properties of mature varicosities and this stabilization of learning-induced synaptic growth leads to the persistence of LTF.