Fig. 3: Physiological properties and cholinergic neuromodulation of hippocampal CA1 neurons from WT and phosphomutant mice.

a WB validation of CRISPR-generated Cav2.3 Ser15Ala mutant mice using a pS15 phosphoantibody in cortical lysates from 5–6-week-old WT, Heterozygous S15A and Homozygous S15A mice. Example blots derive from two gels run and processed in parallel. b Quantification of total Cav2.3 (p > 0.05 One-Way ANOVA) and c Relative phospho-S15 Cav2.3 expression (One-Way ANOVA, Fisher’s LSD; n = 9, 3 mice/genotype, 3 technical replicates). d Normalised R-type Ca²⁺ current in neurons from young mice of each genotype during a step from −60 to 0 mV in presence of Na⁺, K⁺, Ca²⁺ and synaptic channel inhibitors. e R-type current inactivation tau (τ_inact) at maximal activation voltages (*p = 0.01 Two-Way Repeated Measures ANOVA, Fisher’s LSD) and f Average IV curve for all cells recorded (p > 0.05), (n = 13 neurons for both genotypes, 4–5 mice/genotype). Data for −10 mV step is shown in the inset (p = 0.06, two-tailed unpaired t test). g Membrane resting potential (V_rest, left) and input resistance (R_input, right) of adult neurons (V_rest: WT = 14, HOM S15A n = 20; R_input: WT n = 13, HOM S15A n = 17; 7 mice/genotype) in control conditions and after 10 μM carbachol (CCh) application (two-tailed paired t test). h Input/output curves for the same neurons (WT n = 9, HOM n = 13, *p < 0.028 Two-Way Repeated Measures ANOVA) and percentage of cells with CCh-induced depolarization block (inset, WT n = 13, HOM n = 21, two-tailed binomial test). i Representative control action potential trains evoked in WT (top) and HOM S15A (bottom) CA1 cells by 1s-long current injections. Membrane was held at −65 mV. j Same cells as (i) upon application of CCh and representative depolarising plateau potential (DPP), observed at some current injections in both genotypes; insets: fraction of cells with at least one DPP in each genotype group (WT n = 13, HOM n = 21, p > 0.05, two-tailed binomial test). k Same cells as (j) at higher stimulation illustrating sustained depolarization and AP block (top) or attenuation (bottom) towards the end of the stimulus and long-lasting large amplitude afterdepolarizations (ADP). Like DPPs, these ADPs were present at some current injections in a fraction of cells in both groups of mice (inset, WT n = 13, HOM n = 21, **p < 0.004, two-tailed binomial test). l Percentage of cells displaying sustained DPPs or ADPs upon stimulus termination at each current injection step. Non-linear fits (least squares regression) were compared using the extra sum-of-squares F test (p = 0.04 indicates the data cannot be adequately fit with a single Gaussian). Data is presented as mean ± S.E.M or in box plots representing minimum, maximum, median, 25/75 percentile and mean is indicated by a marker. Source data are provided as a Source Data file.