Figure 3: Primary cortical neuron cultures and hippocampal networks in BHLHE40^−/−/41^−/− mice are resistant to lithium’s stimulation dampening effect.

(A) To measure neuronal network activity in primary cortical neuron cultures, a previously published⁶⁸ Firefly luciferase reporter assay was used. This assay is based on a sensor for synapse-to-nucleus signaling named “enhanced synaptic activity response element (ESARE). (B) Ribbon plots showing the mean ESARE activity in response to bicuculline (BIC) stimulation after 24 hours. The ribbons indicate the standard error of the mean. (C) Quantification of the stimulation peak. Data are shown as box plots with whiskers extending to no more than 1.5-fold IQR. Asterisks indicate statistical significance of the interaction effect and simple effects for both genotypes tested in a two-way ANOVA. (D) The regions of interest for electrophysiology in acute slices were the anterior cingulate cortex (ACC) portion of the medial prefrontal cortex (mPFC) and the ventral Hippocampus (vHIP). (E) Shows the exemplary placement of the patch pipette in layer 2/3. Excitatory Postsynaptic Currents (EPSCs) were quantified in mPFC (F-I) and vHIP (J-M). (F, H) miniature EPSC (mEPSC) amplitude (F) and spontaneous EPSC (sEPSC) amplitude (H) in L2/3 mPFC excitatory neurons were significantly dampened in both WT and DKO, but the significant interaction of genotype and treatment (GxT) in both variables indicates a difference in lithium-induced dampening. (G, I) A significantly smaller log2-transformed foldchange (log2FC) reduction in LiCl-treated compared to Ctrl-treated neurons in both mEPSC (G) and sEPSC (I) amplitude supports this finding. (J) Average mEPSC amplitude was not significantly reduced in CA1 principal neurons. (K) However, the nominal reduction in amplitude is significantly stronger in WT compared to DKO. (L) The average sEPSC amplitude is reduced in both WT and DKO CA1 neurons in response to lithium treatment. (K) Normalized to the mean amplitude in Ctrl treated mice, we find a significantly stronger dampening effect in WT neurons compared to DKO. (N) Short-term (STP) and long-term potentiation (LTP) was assessed by high frequency stimulation (HFS) of the Schaffer collaterals and measuring fEPSP in the CA1 region of the hippocampus. Time point of stimulation is marked by an arrow. fEPSP amplitude was normalized to baseline. (O) Magnitude of STP, determined as maximal responses within first 5 min after HFS, is significantly lower in DKO mice. Treatment of lithium further reduced the STP both in WT and DKO mice, but the significant interaction indicates a difference in dampening amplitude. (P) When normalized to mean STP in Ctrl treated mice, log2FC reduction of STP amplitude is significantly larger in wildtypes compared to DKO mice. (Q) Magnitude of LTP, determined as responses between 50 and 60 min after HFS, is significantly lower in DKO mice. Treatment of lithium further reduced the LTP both in WT and DKO mice, but the significant interaction indicates a difference in dampening amplitude. (R) When normalized to the mean LTP in Ctrl treated mice, log2FC reduction of LTP amplitude is significantly larger in wildtypes compared to DKO mice. * p < 0.05; ** p < 0.01; *** p < 0.001; n.s. not significant; p-values refer to univariate two-way ANOVA with Type 2 sum-of-squares; simple effects were tested in a similar but unifactorial ANOVA procedure; WT: wildtype mice; DKO: BHLHE40^−/−/41^−/− mice; Ctrl: vehicle control; LiCl: lithium-treated; G: genotype main effect; T: treatment main effect; GxT: interaction effect; simple T: simple effects; DKO-T: DKO simple treatment effect; WT-T: WT simple treatment effect.