Figure 1. Activity-dependent repression of nrxn1α in primary cortical neuron cultures.

(A) RT-PCR analysis of nrxns and other gene transcripts in primary cortical neuron cultures 24 hours after stimulation by a high K⁺ concentration (51 mM, 10 min) (left panel). Real-time qPCR analysis revealed a decrease in nrxn1α and nrxn3β mRNA after high K⁺ stimulation (full-length gels in Fig. S1; n = 4 biological samples; Student’s t-test, **P < 0.01, ns, no significance). (B,C) Primary cortical neuron cultures were subjected to transient stimulation by KCl or NaCl on DIV9 (B) (full-length gels in Fig. S1). The nrxn1α expression was analyzed using RT-PCR after stimulation (C) (n = 13 biological samples for KCl stimulation; n = 3 biological samples for NaCl stimulation; One-way ANOVA followed by Dunnett’s post hoc test, *P < 0.05, **P < 0.01, ns, no significance). (D,E) Primary cortical neuron cultures were infected by lenti-virus LV: CamKIIα-ChR2-EGFP at DIV 1 and stimulated with LED (50 Hz, 5 ms/pulse, 10 min). Expression of nrxn1α was quantified using qPCR (n = 9 for normal group, n = 3 for TTX treatment group; two -way ANOVA followed by a Bonferroni post hoc test, ***P < 0.001, ns, no significance). (F) Levels of egr1 expression induced by KCl stimuli (grey) or optical stimuli (black) (n = 8 for control group, n = 5 for KCl stimuli group, n = 5 for light stimuli group; *P < 0.05, one -way ANOVA followed by Tukey’s post hoc test, *P < 0.05, ***P < 0.001). (G) Luciferase assay revealed activity-dependent repression of nrxn1α promoter activity. The nrxn1α promoter is cloned to drive the expression of firefly luciferase in pGL3-enhancer. pTK-renilla luciferase was co-transfected as the control. Luciferase activity was measured 36 hours after optogenetic stimulation. The ratio of Firefly/Renilla luciferase activity was reported (n = 6 for normal group, n = 3 for TTX treatment group; two-way ANOVA with Bonferroni post hoc test, **P < 0.01).