Figure 1. CaSR deletion reduces divalent-dependent excitability.

(A) Spontaneous voltage traces at RMP following the application of solutions with different divalent concentrations (T₁.₁ (upper traces), T_0.2 (middle), and T_1.1 recovery (lower)) recorded in three individual neurons with or without CaSR (conWT (black), Nes^Cre (blue) and Casr^-/- (red)). Each trace depicts 150 s of continuous acquisition. (B) Histograms of average action potential (AP) frequency (Hz) recorded using the same solutions: T_1.1, T_0.2, and T_1.1 recovery. Individual recordings represented by open circles linked with lines and average is represented with a bar. From left to right graphs depict conWT (n = 18), Nes^Cre (n = 21), and Casr^-/- (n = 18). ANOVA: Post-hoc tests (Sidak compensated for multiple comparisons here and in all later figures) showed that action potential frequency increased in conWT (p=0.0009) and Nes^Cre (P, 0.0001), but not Casr^-/- (p=0.6697) neurons when changing from T_1.1 to T_0.2 (Figure 1—source data 1). (C) Baseline average action potential frequency in T_1.1. was unaffected by genotype (p>0.999). (D) Average action potential frequency with T_0.2 application was the same in conWT and Nes^Cre (p=0.9831) and higher than in Casr^-/- neurons (p=0.013 and 0.0033, respectively). (E) Plot of effect of external divalent concentration and CaSR on RMP. Two-way RM ANOVA indicates that increasing [Ca²⁺]_o (F (1, 37)=31.65, p<0.0001) and CaSR deletion (F (1, 37)=19.1, p<0.0001) hyperpolarized the RMP without an interaction (F (1, 37)=1.035, p=0.3155). Post-hoc testing indicated RMP was depolarized with the switch to T_0.2 in both Nes^Cre and Casr^-/-neurons (p<0.0001 and p=0.0066 for 21 and 18 recordings respectively; Figure 1—source data 1). (F) Plot of average action potential threshold in T_1.1 and T_0.2 in Nes^Cre and Casr^-/- neurons elicited as per panel G. Two-way RM ANOVA indicates that reducing [Ca²⁺]_o hyperpolarized the action potential threshold (F (1, 27)=56.48, p<0.0001) but that genotype had no effect (F (1, 27)=2.284, p=0.1424). [Ca²⁺]_o was highly effective in both Nes^Cre and Casr^-/-neurons (p<0.0001 and p=0.0003 for 19 and 10 recordings, respectively). Individual neuron values are represented by open circles linked by lines and averages by filled circles. (G) Exemplar action potentials elicited by current injection in a Nes^Cre (blue) and a Casr^-/- neuon (red) in T_1.1 (unbroken) and T_0.2 (broken). Action potential threshold is indicated by +for the first action potential elicited by current injection (50–200 pA) under the same conditions as panel E. (H) Histogram summarizing effects of divalents on action potential frequency in Nes^Cre and Casr^-/- neurons after a current injection to counter divalent-dependent depolarization following T_0.2 application. Two-way RM ANOVA indicates that reducing [Ca²⁺]_o increases the action potential frequency (F (1, 35)=11.54, p=0.0017) and that this is significant in the Nes^Cre but not Casr^-/-neurons (p=0.0075 and 0.1555 for 21 and 16 recordings, respectively). Inset shows average membrane potential after the current injection. (I) Histogram summarizing effects of divalents on action potential frequency in Nes^Cre and Casr^-/- neurons after current injection in T_1.1 to depolarize membrane potential to value recorded in T_0.2. Two-way RM ANOVA indicates that reducing [Ca²⁺]_o increases the action potential frequency (F (1, 35)=45.09, p=0.0004) and that this is significant in the Nes^Cre but not Casr^-/-neurons (p=0.0044 and 0.056 for 21 and 16 recordings, respectively). Inset shows average membrane potential after the current injection.

Figure 1—figure supplement 1. Casr expression levels reduced in Casr^-/- neurons.

Casr expression levels shown as delta delta with actin used as normalizing gene. Average values of 1.0, 0.02, and 0.65 for Nes^Cre, Casr^-/-, and conWT respectively with each genotype reflecting the average data from six cultures each represented by triplicate samples. The Kruskal-Wallis test indicates differences between genotypes (p=0.0002) with Dunn’s multiple comparison showing Casr expression levels are lower in Casr^-/- (p=0.0016), but not conWT (p=0.7739), than in Nes^Cre neocortical cultures.

Figure 1—figure supplement 2. CaSR deletion reduces divalent-dependent excitability following the generation of action potentials elicited by current injections.

(A) The neurons were held at RMP in T_1.1 (zero basal current injection) or at the same potential in T_0.2 (hyperpolarizing current injection as described in Figure 1H). The current injection is shown in the upper row. The broken horizontal line denotes 0 mV. Reduced divalent concentrations T_0.2 increased action potentials in Nes^Cre and Casr^-/- neurons. (B) Action potentials were plotted versus the current injection for Nes^Cre neurons (n = 19). (C) Action potentials were plotted versus the current injection for Casr^-/- neurons (n = 11).