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Fig. 1. Glutamate signaling through ionotropic receptors is necessary and sufficient to induce glutamate sensitivity of cultured myocytes. (A) Glutamate-induced Fluo-4 AM fluorescence intensity in acetylcholine-sensitive myocytes (arrowheads) in response to neurotransmitter superfusion of a neuron-myocyte coculture. (B) Digitized fluorescence of six myocytes in response to 5-s superfusion with 5 mM glutamate + 3 µM pancuronium or 1 µM acetylcholine following 18–24 h exposure to 1 µM glutamate in 2 mM Ca2+ culture medium. Traces are offset for clarity. (C) The incidence of glutamate sensitivity of myocytes in neuron-myocyte culture depends on the culture medium. Basal sensitivity in neuron-myocyte cultures in 2 mM Ca2+ increased in the absence of Ca2+ that increases the incidence of glutamatergic neurons but did not decrease significantly in the presence of 1 µM veratridine that decreases the incidence of glutamatergic neurons, ANOVA with Tukey’s range test [F(2, 25) = 7.947]. Myocytes grown in the absence of neurons are unaffected by culture condition. (D) The combination of 50 µM AP5 and 15 µM NBQX, but not either inhibitor alone, decreased incidence of glutamate responses in myocyte-only cultures grown in the presence of 1 µM glutamate. ANOVA [F(4, 100) = 13.37, P < 0.0001] with Dunnett’s post hoc test. (C and D) n ≥ 8 cultures per group with 8–10 myocytes per culture. Values are mean ± SEM, **P < 0.01, ***P < 0.001, ****P < 0.0001. See also SI Appendix, Figs. S1 and S2.
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Fig. 2. Metabotropic glutamate receptors do not stimulate glutamate-induced sensitivity of myocytes. Antagonists (Ant) to type I mGluRs (100 µM AIDA) but not type II mGluRs (100 µM LY341495) or type III mGluRs (100 µM MSOP) enhanced the incidence of glutamate sensitivity in the absence of exogenous glutamate (ANOVA with Dunnett’s post hoc test compared to control F(6, 80) = 2.207; a, P < 0.05), with no additional gain in sensitivity in the presence of glutamate (ANOVA F(6, 103) = 5.091; P < 0.05). The increased incidence of glutamate sensitivity in response to glutamate was blocked in myocytes cultured in the presence of agonists (Ago) for each class of mGluR (I, 100 µM CHPG; II, 100 µM LY354740; III, 20 µM L-AP4) (two-tailed unpaired t tests between glu+ and glu- pairs). n ≥ 8 cultures per group with 8–10 myocytes per culture. Values are mean ± SEM. **P < 0.01, ****P < 0.0001.
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Fig. 3. Glutamate signaling to ionotropic receptors is necessary and sufficient to induce NMDA receptor up-regulation in myocytes in vivo. (A) Agarose beads loaded with drugs were implanted at stage 21 (22.5 h) and animals raised to stage 40 (2.8 d). (B) Glutamate delivered by agarose beads stimulated an increase in GluN1 immunoreactivity (% of labeled area within indicated borders) in trunk myocytes. (C) Bead delivery of 10 μM AMPA and 10 μM NMDA also induced expression of GluN1 in myocytes, ANOVA [F(2, 51) = 26.8] with Dunnett’s post hoc test. (D) Bead delivery of 0.5 mM AP5 plus 0.15 mM NBQX abrogated the glutamate-induced increase in GluN1 expression, ANOVA [F(3, 86) = 31.47]. n = 4 independent experiments for each graph. The lower right number on each bar is the number of embryos examined. Values are mean ± SEM. ****P < 0.0001. See also SI Appendix, Fig. S3.
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Fig. 4. Inhibitors of adenylate cyclase and MAP kinases suppress glutamate-induced glutamate sensitivity of myocytes. Coincubation of glutamate with pharmacological inhibitors identified adenylate cyclase (AC; 5 μM SQ22536), MEK1/2 (10 μM U0126), JNK (5 μM SP600125), and p38 (10 μM SB203580) as mediators of the increase in glutamate sensitivity, ANOVA [F(9, 131) = 5.265] with Tukey’s post hoc test. n ≥ 8 cultures per group and 8–10 myocytes per culture. Values are mean ± SEM. ****P < 0.0001.
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Fig. 5. p38 is required for glutamate-induced glutamate sensitivity of myocytes and up-regulation of NMDA receptors. (A) MO-mediated knockdown of p38β or p38γ in vitro abolished the glutamate-mediated increased sensitivity of myocytes to glutamate without affecting baseline sensitivity, unpaired t tests between −glu and +glu pairs. n ≥ 5 cultures per group with 8–10 myocytes per culture. (B) Western blots of cultured myocytes exposed to glutamate were probed for p-p38, p38, and actin. (C) The ratio of phosphorylated versus total p38 increased briefly after glutamate exposure. Values are from three experimental replicates, one-sample t tests to expected mean of 1 (control). (D) MO-mediated p38β knockdown in vivo eliminated the glutamate-induced increase in GluN1 expression, ANOVA [F(3, 77) = 18.98] with Tukey’s post hoc test. (E) Quantification of D. n = 4 independent experiments for each bar. Values are mean ± SEM. *P < 0.05, ****P < 0.0001.
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Fig. 6. JNK is required for glutamate-induced myocyte glutamate sensitivity and NMDA receptor up-regulation. (A) MO-mediated knockdown of JNK1 in vitro abolished the glutamate-mediated increased sensitivity of myocytes to glutamate (ANOVA F(3, 35) = 6.87 with Tukey’s post hoc test). n ≥ 8 cultures per group with 8–10 myocytes per culture. (B) Western blots of cultured myocytes exposed to glutamate were probed for p-JNK, JNK, and actin. (C) The ratio of phosphorylated to total JNK decreased temporarily after glutamate exposure. Values are from three experimental replicates; one-sample t tests to expected mean of 1 (control). (D) MO-mediated JNK knockdown in vivo eliminated the effect of glutamate on increased GluN1 expression, ANOVA [F(3, 81) = 43.74] with Tukey’s post hoc test. (E) Quantification of D. n = 4 independent experiments. Values are mean ± SEM. *P < 0.05, ***P < 0.001, ****P < 0.0001.
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Fig. 7. MEF2 is required for NMDA receptor subunit up-regulation in vivo. (A) GluN1 up-regulation is abolished by MEF2C MO-mediated knockdown. (B) Quantification of A, n = 4 independent experiments. Values are mean ± SEM, ANOVA [F(3, 75) = 21.67] with Tukey’s post hoc test. ****P < 0.0001.
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Fig. 8. Model of neurotransmitter-receptor matching. Glutamate activation of ionotropic receptors (iGluR) deactivates JNK through an unknown intermediary to regulate jun/ATF transcription factors that bind to the AP-1 site in the GluN1 promoter. In parallel, iGluR stimulation leads to activation of adenylate cyclase (AC) to generate cAMP that activates p38 in combination with metabotropic actions of NMDARs. Ca2+ binding Ca2+-mediated activation of PI3K, and p38-mediated phosphorylation of MEF2 dimers facilitate DNA binding and transactivation. MEF2 requires SP1/3 for efficient DNA binding to the GluN1 promoter. The combined actions of these pathways create a feedforward cycle that allows sustained exposure to glutamate to up-regulate and maintain NMDA receptor subunit GluN1 expression.
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