Serotonin Regulates the Secretion and Autocrine Action of a Neuropeptide to Activate MAPK Required for Long-Term Facilitation in Aplysia

Abstract

In Aplysia, long-term facilitation (LTF) of sensory neuron synapses requires activation of both protein kinase A (PKA) and mitogen-activated protein kinase (MAPK). We find that 5-HT through activation of PKA regulates secretion of the sensory neuron-specific neuropeptide sensorin, which binds autoreceptors to activate MAPK. Anti-sensorin antibody blocked LTF and MAPK activation produced by 5-HT and LTF produced by medium containing sensorin that was secreted from sensory neurons after 5-HT treatment. A single application of 5-HT followed by a 2 hr incubation with sensorin produced protein synthesis-dependent LTF, growth of new presynaptic varicosities, and activation of MAPK and its translocation into sensory neuron nuclei. Inhibiting PKA during 5-HT applications and inhibiting receptor tyrosine kinase or MAPK during sensorin application blocked both LTF and MAPK activation and translocation. Thus, long-term synaptic plasticity is produced when stimuli activate kinases in a specific sequence by regulating the secretion and autocrine action of a neuropeptide.

Ribosomal Protein S6 Kinase Is a Critical Downstream Effector of the Target of Rapamycin Complex 1 for Long-term Facilitation in Aplysia

“…In particular, whereas short-term facilitation (STF) is independent of protein synthesis, protein synthesis inhibitors block both long-term facilitation (LTF) of the SN-MN synapse (3) and long-term memory for sensitization (8), making this a relevant system for examining the role of translational control in memory formation. Moreover, in Aplysia SNs, exogenous treatment with the neurotransmitter responsible for inducing facilitation, 5-hydroxytryptamine (5-HT), leads to the activation of TORC1 (9 -11), and bath application of the TORC1 inhibitor, rapamycin, blocks LTF measured at 24 h (24-h LTF) (12)(13). When specifically applied to the synapse, however, rapamycin spares 24-h LTF but blocks the stabilization of newly grown varicosities as well as a stabilized phase of LTF, measured at 72 h (72-h LTF), at that particular synapse (14).…”

“…In particular, whereas short-term facilitation (STF) is independent of protein synthesis, protein synthesis inhibitors block both long-term facilitation (LTF) of the SN-MN synapse (3) and long-term memory for sensitization (8), making this a relevant system for examining the role of translational control in memory formation. Moreover, in Aplysia SNs, exogenous treatment with the neurotransmitter responsible for inducing facilitation, 5-hydroxytryptamine (5-HT), leads to the activation of TORC1 (9 -11), and bath application of the TORC1 inhibitor, rapamycin, blocks LTF measured at 24 h (24-h LTF) (12)(13) 3 The abbreviations used are: TOR, target of rapamycin; TORC1, target of rapamycin complex 1; SN, sensory neuron; MN, motor neuron; STF, short-term facilitation; LTF, long-term facilitation; 5-HT, 5-hydroxytryptamine; 24-h LTF, LTF measured at 24 h; 72-h LTF, LTF measured at 72 h; 4E-BP, 4E-binding protein; S6K, S6 kinase; eIF4E, eukaryotic initiation factor 4E; eIF4G, eukaryotic initiation factor 4G; L-LTP, late long-term potentiation; mGluR-LTD, metabotropic glutamate receptor-dependent long-term depression; Rheb, Ras homologue enriched in brain; ApRheb, Aplysia orthologue of Rheb; eGFP, enhanced green fluorescent protein; mRFP, monomeric red fluorescent protein; eCFP, enhanced cyan fluorescent protein; PKC, protein kinase C; eEF2, eukaryotic elongation factor 2; ASW, artificial sea water; EPSP, excitatory post-synaptic potential; UTR, untranslated region; eYFP, enhanced yellow fluorescent protein; IRES, internal ribosomal entry site; ELH, egg-laying hormone; GST, glutathione S-transferase; raptor, regulatory-associated protein of TOR; TOS, TOR signaling; TOP, terminal oligopyrimidine tract; eEF2K, eukaryotic elongation factor 2 kinase; cap, 5Ј-m 7 -GTP cap; cap:IRES ratio, the ratio of cap-dependent translation to IRES-dependent translation; NA, numerical aperture; PBS, phosphate-buffered saline; WT, wild type. …”

Protein Kinase C Regulates Local Synthesis and Secretion of a Neuropeptide Required for Activity-Dependent Long-Term Synaptic Plasticity

“…Immunocytochemistry was used to monitor the expression and distribution of sensorin throughout the sensory neurons and total p42/44 MAPK or phosphorylated p42/44 MAPK in the cell bodies of sensory neurons ( Hu et al, 2004aHu et al, , 2006. Cultures at various times after stimuli or after the application of control solutions were rinsed briefly in artificial seawater, fixed in 4% paraformaldehyde, and processed as described previously (Liu et al, 2003;Hu et al, 2004a).…”

“…Immunocytochemistry was used to monitor the expression and distribution of sensorin throughout the sensory neurons and total p42/44 MAPK or phosphorylated p42/44 MAPK in the cell bodies of sensory neurons ( Hu et al, 2004aHu et al, , 2006. Cultures at various times after stimuli or after the application of control solutions were rinsed briefly in artificial seawater, fixed in 4% paraformaldehyde, and processed as described previously (Liu et al, 2003;Hu et al, 2004a). Cultures were exposed to rabbit polyclonal antibody specific for sensorin (1:1000) or total p42/44 MAPK and phospho-p42/44 MAPK (1:200; Cell Signaling Technology, Beverly, MA) diluted in 2% normal goat serum in 0.01 M PBS with 0.3% Triton X-100 at 4°C for 24 h. The incubated cultures were washed in 0.01 M PBS and incubated in FITC-conjugated goat anti-rabbit IgG (1:200; Sigma) at 4°C for 4 h. After washing in 0.01 M PBS, cultures were imaged directly with the appropriate filter set for detecting the fluorescent signal.…”

Identification of a cis-acting element that localizes mRNA to synapses

“…To identify such cis-acting LEs, we generated and expressed chimeric reporters to study the localization of the mRNA encoding the Aplysia sensory neuron (SN)-specific peptide transmitter, sensorin (19). Release of sensorin from the SN is required for both synapse formation and long-term facilitation (19,20). The localization of sensorin mRNA is regulated by synapse formation, such that it is diffusely localized in neurites of isolated SNs (which do not form synapses) but concentrates at synapses in SNs paired with target motor neurons (MNs) (19).…”