Journal: EMBO Reports

Article Title: Npas4 regulates medium spiny neuron physiology and gates cocaine‐induced hyperlocomotion

doi: 10.15252/embr.202051882

Figure Lengend Snippet: DARPP‐32 (D32) immunostaining shows that striatal neuron cultures consist mainly of D32‐positive medium spiny neurons (MSNs). 40× magnification, scale bar = 50 µm. MAP2 marks neurons, Hoechst (Hoe) stains DNA. n = 3 cultures from independent mouse litters. 100× magnification images show intracellular distribution of D32 in D32‐positive neurons (D32 +). D32‐negative neuron (D32 ‐) are shown for comparison. Scale bar = 10 µm. MSN cultures at DIV11 were treated for 2 h with either 10 µM of the D1‐agonist SKF‐38393 (SKF) alone or with SKF and 10 µM of the selective D1‐antagonist SCH‐23390 (SCH). qPCR was performed to determine the relative mRNA levels of the immediate early genes cFos and Arc. Mean + SEM; one‐way ANOVA with Holm‐Sidak post‐hoc correction; ** P < 0.01, **** P < 0.0001; n = 3 independent cultures as biological replicates. MSNs were depolarized with potassium chloride (HiK, 55 mM) or treated with the D1‐agonist SKF‐38393 (10 µM) for 1, 2 or 4 h. Subsequently, qPCR was performed to assess immediate early gene mRNA levels. Mean + SEM; two‐way ANOVA with Bonferroni post‐hoc test; indicated P ‐values for induction over control for each treatment; * P < 0.05, ** P < 0.01, *** P < 0.001; n = 4 independent cultures as biological replicates. To assess differences in mRNA load induced by HiK vs SKF, the values from the experiments in panel (D) were summed up over time for each treatment and the sums for each treatment were divided by each other to yield the ratio I HiK / I SKF . Values for this ratio were compared through one‐way ANOVA with Tukey's post‐hoc test; mean + SEM; * P < 0.05, *** P < 0.001; n = 4 independent cultures as biological replicates. Immunohistochemistry analysis shows that cells in human iPSC‐derived forebrain organoids express the forebrain neuron marker FOXG1 (green) and the neuron differentiation marker MAP2 (red). Hoe = Hoechst. Scale bar = 30 µm. Differential induction of Npas4 through HiK and not forskolin in hiPSC‐derived forebrain organoids. Organoids were treated with HiK (55 mM) or the PKA activator forskolin plus TTX (Fsk, 10 µM; TTX, 1 µM) for 1 h before qPCR analysis was performed to assess mRNA levels of Npas4 and Arc. Mean + SEM; n = 7 organoids in control condition, n = 5 organoids for HiK, n = 5 organoids for Fsk/TTX. One‐way ANOVA with Tukey's post‐hoc test, ns = not significant, * P < 0.05, *** P < 0.001.

Article Snippet: Hyperlocomotion upon cocaine treatment in naive animals depends on activity changes in D1‐dopamine receptor (D1R)‐expressing MSNs (Pierce & Kalivas, ; Hikida et al , ) and striatal D1 and NMDA receptors were shown to be necessary for full expression of the acute locomotion increase after cocaine administration (Pulvirenti et al , ; McGregor & Roberts, ) as well as for cocaine‐dependent locomotor sensitization (Brown et al , ) (Kita et al , ).

Techniques: Immunostaining, Comparison, Control, Immunohistochemistry, Derivative Assay, Marker

Journal: EMBO Reports

Article Title: Npas4 regulates medium spiny neuron physiology and gates cocaine‐induced hyperlocomotion

doi: 10.15252/embr.202051882

Figure Lengend Snippet: A–G MSN cultures were stimulated with HiK (55 mM) for 1 h 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 µM, applied 5 min 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, 5 min before) and TTX (1 µM, 5 min, 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, 20 min 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 20 min 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, 20 min 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, 20 min 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, 20 min 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 signalling 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, 40× magnification, scale bar = 10 µm. (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 ˜10 min 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: 10 min, n = 3 independent cultures. (K) immunohistochemical analysis of MSNs infected with mCherry‐NLS as control or the competitive nuclear Ca 2+/ 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 1 h 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 Ca 2+ ‐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 1 h 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.

Article Snippet: Hyperlocomotion upon cocaine treatment in naive animals depends on activity changes in D1‐dopamine receptor (D1R)‐expressing MSNs (Pierce & Kalivas, ; Hikida et al , ) and striatal D1 and NMDA receptors were shown to be necessary for full expression of the acute locomotion increase after cocaine administration (Pulvirenti et al , ; McGregor & Roberts, ) as well as for cocaine‐dependent locomotor sensitization (Brown et al , ) (Kita et al , ).

Techniques: Incubation, Activity Assay, Inhibition, Immunohistochemical staining, Infection, Imaging, Control, Expressing

Journal: EMBO Reports

Article Title: Npas4 regulates medium spiny neuron physiology and gates cocaine‐induced hyperlocomotion

doi: 10.15252/embr.202051882

Figure Lengend Snippet: Time course of AAV injections, habituation and treatments to assess the impact of Npas4 knockdown on MSN neuronal morphology under basal conditions and after repeated cocaine injections. Representative images of AAV‐shscr‐GFP‐ and AAV‐shNpas4‐GFP‐infected cells together with the sparse labelling of D1R‐MSNs achieved through a co‐injection of AAV‐Flex‐tdTomato and AAV‐PPTA‐Cre (to label D1 MSNs) or AAV‐ENK‐Cre (to label D2 MSNs) in a 1/10,000 ratio. Scale bar = 100 µm. Representative confocal images of staining for Npas4 expression (red) in the NAc of mice infected with AAV shscr‐GFP or AAV‐shNpas4‐GFP 1 h after cocaine treatment. Scale bar = 20 µm. Note the presence of Npas4‐positive neurons infected with the AAV shscr‐GFP (arrowheads) and not in cells infected with the AAV‐shNpas4. Quantifications of GFP‐positive and Npas4‐positive cells 1 h post cocaine treatment. Data were normalized so that the number of GFP‐ and Npas4‐positive cells was set to 100%. 20 fields of view were analysed from n = 3 mice in the case of AAV‐shscr‐GFP and 30 fields of view from n = 3 mice for AAV‐shNpas4‐GFP. Mean + SEM; unpaired Student's t ‐test; * P < 0.05, n = 3 mice per group. Confocal images of tdTomato‐labelled D1‐MSNs expressing shscr‐GFP or shNpas4‐GFP (left panels; scale bar: 20 µm). 3D volume‐renderings of MSN dendrites from the NAc shell (right panels; scale bar: 2 µm). Quantification of spine density in D1‐MSNs, defined as the number of spines normalized to 10µm of dendrite for saline control and after cocaine injection. Mean + SEM; two‐way ANOVA, Bonferroni post‐hoc test, * P < 0.05; ** P < 0.01; *** P < 0.001. n = 40 neurons from 4 mice in each experimental group. Confocal images of tdTomato‐labelled D2‐MSNs expressing shscr‐GFP or shNpas4‐GFP (left panels; scale bar: 20 µm). 3D volume‐renderings of MSN dendrites from the NAc shell (right panels; scale bar: 2 µm). Quantification of spine density in D2‐MSNs, defined as the number of spines normalized to 10µm of dendrite for saline control and after cocaine injection. Mean + SEM; two‐way ANOVA, Bonferroni post‐hoc test, all non‐significant. n = 40 neurons from 4 mice in each experimental group.

Article Snippet: Hyperlocomotion upon cocaine treatment in naive animals depends on activity changes in D1‐dopamine receptor (D1R)‐expressing MSNs (Pierce & Kalivas, ; Hikida et al , ) and striatal D1 and NMDA receptors were shown to be necessary for full expression of the acute locomotion increase after cocaine administration (Pulvirenti et al , ; McGregor & Roberts, ) as well as for cocaine‐dependent locomotor sensitization (Brown et al , ) (Kita et al , ).

Techniques: Knockdown, Infection, Injection, Staining, Expressing, Saline, Control

Journal: EMBO Reports

Article Title: Npas4 regulates medium spiny neuron physiology and gates cocaine‐induced hyperlocomotion

doi: 10.15252/embr.202051882

Figure Lengend Snippet: A, B The datasets from Fig were analysed for spine head diameter and plotted in a cumulative distribution. Around 2,000 spines from 10 neurons from four mice per group were analysed. (A) D1‐MSNs. (B) D2‐MSNs. Two‐way ANOVA with Bonferroni post‐hoc tests. Scrambled saline vs scrambled cocaine: P < 0.004 with a mean difference of 0.016 microns. This mean difference constitutes a negligible effect, in spite of statistical significance. For ANOVA statistics, please see Table .

Article Snippet: Hyperlocomotion upon cocaine treatment in naive animals depends on activity changes in D1‐dopamine receptor (D1R)‐expressing MSNs (Pierce & Kalivas, ; Hikida et al , ) and striatal D1 and NMDA receptors were shown to be necessary for full expression of the acute locomotion increase after cocaine administration (Pulvirenti et al , ; McGregor & Roberts, ) as well as for cocaine‐dependent locomotor sensitization (Brown et al , ) (Kita et al , ).

Techniques: Saline

Journal: EMBO Reports

Article Title: Npas4 regulates medium spiny neuron physiology and gates cocaine‐induced hyperlocomotion

doi: 10.15252/embr.202051882

Figure Lengend Snippet: A–D Scatter plot of passive and active membrane properties of MSNs in ex vivo slice preparations from shRNA‐AAV‐infected mice, plotted for MSNs as D1‐positive (D1+), D1‐negative (D1‐) and those two populations pooled (all). (A) rheobase; mean ± SEM. (B) Firing rate estimated at twice the value of the rheobase; mean ± SEM. (C) input/output gain values recorded in shscr‐ or shNpas4‐injected mice, mean ± SEM. The increased rheobase and lower input‐output gain indicate a lower excitability for MSNs in the Npas4‐knockdown group. (D) facilitation at corticostriatal synapses examined in MSNs with paired‐pulse stimulation at 20 and 4 Hz. Representative current traces and scatter plots of paired‐pulse experiments illustrate a potent facilitation in MSNs in mice injected with scr‐shRNA and a significant decrease of the facilitation in mice injected with shNpas4, with a stronger effect in D1‐negative MSNs. Mean ± SEM. Data information: For n ‐numbers, please see Table . Mann–Whitney‐Test, * P < 0.05, ** P < 0.01.

Article Snippet: Hyperlocomotion upon cocaine treatment in naive animals depends on activity changes in D1‐dopamine receptor (D1R)‐expressing MSNs (Pierce & Kalivas, ; Hikida et al , ) and striatal D1 and NMDA receptors were shown to be necessary for full expression of the acute locomotion increase after cocaine administration (Pulvirenti et al , ; McGregor & Roberts, ) as well as for cocaine‐dependent locomotor sensitization (Brown et al , ) (Kita et al , ).

Techniques: Membrane, Ex Vivo, shRNA, Infection, Injection, Knockdown, MANN-WHITNEY