Fig 2: Effect of gramicidin A on ERK phosphorylation and localization in BCE cells. (a) Double-stained images of pERK and nuclei (H: Hoechst) for different conditions (C: control; KGln; Gra; Gra-Cho; Cho). (b) Plot of percentage of pERK positive nuclei. In agreement with (a), the percentage of pERK-positive nuclei is significantly larger for Gra and Gra-Cho compared to control and the other treatments (∗p < 0.05; ∗∗∗p < 0.0001). Although Cho determines a higher global immunofluorescent signal of pERK with respect to control (see (a)), its distribution is diffuse throughout the cytoplasm with a low nuclear component. (c) Western blots of pERK in cell lysates for all the conditions. Similarly to (a), the blots reveal an important increase in pERK in response to the gramicidin or gramicidin-choline treatments and a somewhat augmented signal for choline alone. KGln did not determine any modification. Asterisks indicate significant differences with respect to control (∗∗∗p < 0.0001). The signal for Cho is also significantly different with respect to Gra (p < 0.01) and Gra-Cho (p < 0.05) (not indicated in the figure). Load and transfer controls and quantifications were done as described in Section 2.5. Bar: 30 μm.

Fig 3: Effect of S1P on p38 and AKT activation in CMSCs. (a) The effect of S1P stimulation for 5 min was assessed after Gi inhibition by 200 ng/ml PTX analyzing p38 activation state by western blot, *P<0.05, n=3. The panel above the graph shows a representative experiment. (b) The activation state of PKC isoforms was analyzed by western blot after treatment with 5 μM S1P, n=3. (c) AKT activation state was analyzed after treatment with S1P at different concentrations by western blot. n=7. A representative experiment is shown

Fig 4: Distinct P-ERK antibodies reveal differential presynaptic responses to 5HT. (A) Overview of the two-trial training paradigm in Aplysia. (A, Top) A tactile stimulus delivered to the animal’s tail causes tail withdrawal mediated by a predominantly monosynaptic reflex arc (SN–MN). Training stimuli (electric shocks) produce a global release of 5HT throughout the Aplysia CNS, causing sensitization of the reflex. These responses can be modeled using direct 5HT applications to SN–MN cocultures. Schematic diagram of an MN paired with three SNs represents the image from C. (A, Bottom) A single training trial (or application of 5HT) does not induce LTM or LTF and causes only transient ERK phosphorylation in ganglia. Two trials (or 5HT applications) spaced 45 min apart induce a sustained elevation of P-ERK, as well as LTM and LTF lasting >24 h. (B) Activating TxY motif of MAPKs (Left) and the principal P-ERK antibodies used in this study (Right). panP-ERK (CST, 9101) recognizes all forms of ERK phosphorylated at the TxY motif, whereas dualP-ERK (Sigma, M8159) is selective for dually phosphorylated, and thus active, ERK. (C) Immunofluorescence in fixed SN–MN cocultures reveals distinct localization of panP-ERK and dualP-ERK signals. Note that the z plane is below the nuclei to highlight neurites. (Scale bars, 50 µm.) (D) Changes in panP-ERK and dualP-ERK in presynaptic SNs in response to 5HT treatment. (D, Top) Overview of the treatment protocols. (D, Bottom) Immunofluorescence targeting panP-ERK and dualP-ERK and in MN-paired SNs. The overlay includes DAPI signal to indicate the location of the nucleus. (Scale bars, 10 µm.) (E) Quantification of changes in panP-ERK and dualP-ERK densities, and the dual:panP-ERK density ratio in the SN nucleus (n = 16 to 20). (F) Quantification of changes in panP-ERK and dualP-ERK densities, and the dual:panP-ERK density ratio in the SN cell-body cytosol (n = 16 to 20). Asterisks indicate significance in Dunnett’s post hoc tests following one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. All data are shown as means ± SEM.

Fig 5: Mixed LTF protocols are sensitive to trial order. (A) LTF was induced in SN–MN cocultures using two spaced applications of 5HT (intertrial interval [ITI] 45 min) at 2 or 50 µM in various combinations. (A, Top Left) Overview of the paradigms used. (A, Top Right) EPSPs were recorded before (pre) and 16 to 24 h after the treatment (post). (A, Bottom) Quantification of changes in EPSP amplitudes (n = 11 to 16). Asterisks above bars indicate significant deviation from baseline in a single-sample t test. Asterisks between bars indicate significance in Dunnett’s post hoc tests following one-way ANOVA. (B) Same as A, except LTF was induced using five spaced applications of 5HT (ITI 15 min) at various concentrations (n = 14 to 18). Asterisks above bars indicate significant deviation from baseline in a single-sample t test. Asterisks between bars indicate significance in a two-tailed unpaired Student’s t test. (C, Top Left) Overview of treatment protocols. LTF was induced in SN–MN cocultures using the weak–strong paradigm. PKA was activated using a pulse containing forskolin and the phosphodiesterase inhibitor IBMX, applied 45 min before or 45 min after the first weak pulse of 5HT. (C, Right) EPSPs were recorded before (pre) and 16 to 24 h after the treatment (post). (C, Bottom) Quantification of changes (n = 15 to 17). (D, Top Left) Overview of treatment protocols. ERK phosphorylation was activated in psMEK-expressing presynaptic SNs by 1-h irradiation with 502-nm light either alone or in combination with a single 5HT pulse (50 µM) delivered immediately before or immediately after irradiation. (D, Top Right) EPSPs were recorded before (pre) and 16 to 24 h after the treatment (post). (D, Bottom) Quantification of changes (n = 15 to 36). (Scale bars, 50 ms/5 mV.) Asterisks in C and D indicate significance in two-tailed unpaired Student’s t tests. *P < 0.05, **P < 0.01, ***P < 0.001. All data are shown as means ± SEM.