Dynamics and mechanisms of ERK activation after different protocols that induce long-term facilitation in Aplysia

Abstract

The mitogen-activated protein kinase (MAPK) isoform extracellular signal-regulated kinase (ERK) is a key kinase involved in the induction of long-term synaptic facilitation (LTF) of the Aplysia sensorimotor synapse. Therefore, elucidating the dynamics of ERK activation after LTF-inducing protocols is critical for understanding the mechanisms underlying neuronal and synaptic plasticity. ERK activation has rich dynamic features. After a single stimulus, activation peaks 45-minute later and declines rapidly, but after two stimuli spaced 45 min apart, activation persists (Kopec et al. 2015). However, little is known about possible changes in ERK activation for periods beyond 3 h. Given its key role in long-term learning, understanding the dynamics of ERK at 3 h and beyond might provide insights into new protocols that could enhance memory retention. Three different protocols that induce LTF were used to probe the dynamics of ERK activation. The first, termed the Standard protocol, consists of five 5-min pulses of serotonin (5-HT) with regular interstimulus intervals (ISIs) of 20 min. The second, termed the Enhanced protocol, consists of five pulses of 5-HT with irregular ISIs identified with the use of computer simulations. This protocol induces greater and longer-lasting LTF than the Standard protocol. A third protocol, termed the two-pulse protocol, consists of just two 5-min pulses of 5-HT with an ISI of 45 min. Immunofluorescence revealed complex patterns of ERK activation up to 24 h after 5-HT treatment. The Standard and two-pulse protocols led to an immediate increase in active, phosphorylated ERK (pERK), which decayed within 5 h post treatment. A second wave of increased pERK was detected at 18 h post treatment. This late phase was blocked by RpcAMP (an inhibitor of protein kinase A), and by TrkB Fc and TGF-{beta} RII F antagonists. The latter two are chimeras that act via receptor sequestration. These results suggest that complex interactions among kinase pathways and growth factor cascades contribute to the late increase of ERK activity after different LTF-inducing protocols. Interestingly, ERK activity returned to basal levels 24 h after the Standard or two-pulse protocol, but remained elevated 24 h after the Enhanced protocol. This finding may help explain, in part, why the Enhanced protocol is superior to the Standard protocol in inducing long-lasting LTF.