Npas4 regulates medium spiny neuron physiology and gates cocaine-induced hyperlocomotion (2021)
Figure 3

Figure 3. Npas4 induction depends on NMDA receptors, voltage-dependent calcium channels, CaMKII/IV, calcineurin and nuclear calcium and is independent of PKA and MAPK signaling.

a-h, MSN cultures were stimulated with HiK (55 mM) for 1h in the presence of various pharmacologicals to dissect the synapse-to-nucleus cascades mediating transcription of Npas4, Fosb and Arc. Values were normalized to the induced condition to compare for differences in induction between treatments. Mean + SEM and one-way ANOVA with Bonferroni post-hoc correction were used in all experiments. p* < 0.05, p** < 0.01, p***<0.001, p****<0.0001. n-number indicated in each panel. a, co-incubation with the NMDA receptor blocker APV (100 mM, applied 5min before) reveals that all IEG inductions through HiK in MSNs depend on NDMA receptor activity. Mean + SEM; n = 4 independent cultures as biological replicates. b, blockade of voltage-dependent calcium channels through verapamil (30 µM, 5min before) and TTX (1 µM, 5min, to prevent verapamil-induced bursting) leads to significant reduction in gene-inductions for Npas4 and Fosb. Mean + SEM; n =4 independent cultures as biological replicates. c, inhibition of ERK1/2 through UO126 (10 µM, 20min before) leads to significant blockade of mRNA induction for Arc and Fosb, but not Npas4. Mean + SEM; n = 3 independent cultures as biological replicates. d, inhibition of p38alpha and p38beta through SB203580 or SB202190 respectively (both 10 µM and 20min before) leads to significant reductions in mRNA induction for Arc and Fosb, but not Npas4. Mean + SEM; n = 3 independent cultures as biological replicates. e, blockade of PKA activity through H-89 (10 µM, 20min before) leads to reduced induction of Arc, unaffected induction of Fosb and a superinduction of Npas4. Mean + SEM; n = 3 independent cultures as biological replicates. f, inhibition of calcineurin through co-application of Cyclosporin and FK506 (1 µM each, 20min before) leads to unaffected induction of mRNAs for Arc and Fosb, but significantly reduced levels of Npas4. Mean + SEM; n = 3 independent cultures as biological replicates. g, blockade of CaMKII/IV through application of KN-93 (1 µM, 20min before) leads to significantly reduced mRNA inductions of Arc, Fosb and Npas4. Mean + SEM; n = 4 independent cultures as biological replicates.

h, co-application of HiK and SKF reveals synergistic effects on mRNA induction of Fosb but not Arc or Npas4, Mean + SEM; n = 6 independent cultures as biological replicates.

i-n, investigating the role of nuclear calcium signaling in mRNA induction of Arc, Fosb and Npas4. i, immunohistochemical analysis of MSNs infected with rAAV-pCKIIa-GCaMP3-NLS. Infection on DIV5, fixation on DIV12. Hoe = Hoechst, D32 = anti-Darpp-32, GFP = anti-GFP, 40x magnification, scale bar = 10µm. j, nuclear calcium imaging of MSN cultures after application of different stimuli. HiK (55 mM), glutamate (Glu, 1 µM), SKF-38393 (SKF, 10 µM) or dopamine (DA, 20 µM) were applied after ~10min of baseline imaging (arrowheads). Grey traces indicate individual cells, black traces indicate mean of all imaged cells (~30) for one stimulation run. inset: y-axis: 100% ∆F/F, x-axis: 10min, n = 3 independent cultures. k, immunohistochemical analysis of MSNs infected with mCherry-NLS as control or the competitive nuclear Ca2+/CaM antagonist CaMBP4-mCherry. Infection on DIV4, fixation on DIV11. D32 = anti-Darpp-32. scale bar = 10 µm. l, MSNs expressing rAAV-mCherry-control or CaMBP4 (BP4) were treated with 55mM HiK for 1h and qPCR analysis was performed for the indicated IEGs. Mean + SEM; two-way ANOVA with Bonferroni post-hoc test, ns = not significant, p****<0.0001. n = 5 independent cultures as biological replicates. m, immunohistochemical analysis of MSNs infected with mCherry-NLS as control or the nuclear Ca2+-buffer Parvalbumin-NLS-mCherry. Infection on DIV4, fixation on DIV11. D32 = anti-Darpp-32. n, MSNs expressing rAAV-mCherry-control or PV-NLS were treated with 55mM HiK for 1h and qPCR analysis was performed for the indicated IEGs. Mean + SEM; two-way ANOVA with Bonferroni post-hoc test, p* < 0.05, p** < 0.01, p****<0.0001. n = 3 independent cultures as biological replicates.