Enhanced long-term potentiation and impaired learning in mice lacking alternative exon 33 of CaV1.2 calcium channel

Abstract

The CACNA1C (calcium voltage-gated channel subunit alpha 1 C) gene that encodes the Ca_V1.2 channel is a prominent risk gene for neuropsychiatric and neurodegenerative disorders with cognitive and social impairments like schizophrenia, bipolar disorders, depression and autistic spectrum disorders (ASD). We have shown previously that mice with exon 33 deleted from Ca_V1.2 channel (Ca_V1.2-exon 33^-/-) displayed increased Ca_V1.2 current density and single channel open probability in cardiomyocytes, and were prone to develop arrhythmia. As Ca²⁺ entry through Ca_V1.2 channels activates gene transcription in response to synaptic activity, we were intrigued to explore the possible role of Cav1.2_Δ33 channels in synaptic plasticity and behaviour. Homozygous deletion of alternative exon 33 resulted in enhanced long-term potentiation (LTP), and lack of long- term depression (LTD), which did not correlate with enhanced learning. Exon 33 deletion also led to a decrease in social dominance, sociability and social novelty. Our findings shed light on the effect of gain-of-function of Ca_V1.2_Δ33 signalling on synaptic plasticity and behaviour and provides evidence for a link between Ca_V1.2 and distinct cognitive and social behaviours associated with phenotypic features of psychiatric disorders like schizophrenia, bipolar disorder and ASD.

Fig. 1. Exon 33^-/- mice displayed enhanced LTP and reinforced E-LTP to L-LTP.

A Schematic representation of a transverse hippocampal slice showing the positioning of electrodes in the CA1 region of hippocampus. One stimulating electrode S1 was placed in the stratum radiatum to stimulate Schaffer collateral fibres and a recording electrode ‘rec’ was placed in the CA1 apical dendritic region. B STET in synaptic input S1 resulted in a statistically significant potentiation that maintained for 180 min in WT mice (P = > 0.05, n = 9). C STET in Exon 33^−/− mice also resulted in a long-lasting LTP with a higher percentage of potentiation than WT (Fig. 1B) from 30 min and remained stable for 180 min (U-test, 30 min, P = 0.002; 60 min, P = 0.002; 120 min, P = 0.002;180 min, P = 0.009, n = 9). Dotted line represents Fig B for comparison. D WTET in WT resulted only in a short-lasting LTP that decayed to the baseline (Wilcox, 15 min, P = 0.03, n = 7). E WTET in Exon 33^−/− mice resulted in a long-lasting LTP that remained significant until 180 min (P = > 0.05, n = 7). Dotted graph represents D for comparison. The dotted line at 100% represents a line for reference. Error bars in all graphs indicate ±SEM. Analog traces represent typical fEPSPs of input S1 recorded 15 min before (dotted line), 30 min after (dashed line), and 180 min (solid line) after tetanisation. Bar graph represents the comparison of potentiation between WT and Exon 33^−/− mice at 60 min post tetanisation. Three solid arrows represent the time of induction of L-LTP by STET for the induction of late-LTP. Single arrow represents the time point of induction of E-LTP by WTET. Scale bars: vertical, 2 mV; horizontal, 3 ms. STET-strong tetanisation, WTET-weak tetanisation.

Fig. 2. Exon 33^-/- mice displayed lack of LTD.

A Induction of LTD by using SLFS in WT mice, resulted in a stable depression that was statistically significant throughout the recorded time period of 180 min (P = > 0.05, n = 7). B SLFS was delivered to Exon 33^−/− mice which resulted in a potentiation after 30 min and was stable for 180 min (n = 7). Dotted graph represents A for comparison. A significant difference was found between WT and Exon 33^−/− mice from 25 min (Utest, 25 min, P = 0.004; 60 min, P = 0.001; 120 min, P = 0.001; 180 min, P = 0.001). C Induction of E-LTD using WLFS in WT mice showed a depression that was short lasting (85 min, P = 0.03, n = 8) which then returned to baseline levels. D Application of WLFS did not show any significant depression throughout the recording time period of 180 min in Exon 33^-/- mice (P = < 0.05, n = 7). Dotted graph represents C for comparison. The bar graphs represents the comparison of potentiation/depression at 60 min time points between WT and to Exon 33^−/− mice. Single arrow represents the time point of application of SLFS and WLFS. SLFS- strong low frequency stimulation, WLFS- weak low frequency stimulation. Analog traces and scale bars as in Fig. 1. Error bars in all graphs indicate ±SEM.

Fig. 3. Behavioural tagging is impaired in Exon 33^-/- mice.

A Schematic diagram of experimental protocol used for control experiments in BT. Mice were given weak IA training by giving a weak foot shock consisting of 0.3 mA for 2 s. Step-down latency was tested at 1 h, 24 h, and 7 d post-IA training. The cut-off time for step-down latency was 4 min. B Schematic diagram of the experimental protocol used for BT paradigm. Mice were given weak IA training, 1 h after NE (10 min), by providing a weak foot shock. Step-down latency was tested at 1 h, 24 h, and 7 d post-IA. Associative memory was observed only in WT mice (blue bars) exposed to NE. Memory measured 1 h after IA training showed memory retention in WT mice, but not in Exon 33^−/− mice (WT no-NE: P = < 0.0001; WT NE: P = < 0.0001; Exon 33^−/− no-NE: P = 0.99; Exon 33^−/− NE: P = > 0.9999 (n = 7 for all groups) (C 1 h). Memory measured at 24 h shows LTM only in WT mice with NE, but not in WT mice without NE and Exon 33^−/− mice (WT no-NE: P = 0.97; WT NE: P = < 0.0001; Exon 33^−/− no-NE: P = 0.99; Exon 33^−/− NE: P = 0.99) (C 24 h). Similarly at 7 day, remote memory was seen only in WT mice with NE showing that Exon 33^−/− mice was unable to acquire and retain memory (WT no-NE: P = > 0.9999; WT NE: P = < 0.0001; Exon 33^−/− no-NE: P = > 0.9999; Exon 33^−/− NE: P = > 0.9999) (C 7 day). Bar graphs representing WT is shown as open bars and Exon 33^−/− mice as patterned bars (n = 7 from all groups). Error bars indicate ±SEM. Asterisks indicate significant differences between groups (ns = not significant, **p < 0.01, ***p < 0.001, ****p < 0.0001). Error bars indicate ±SEM. Asterisks indicate significant differences between groups. ns represents non-significant (ns) groups.

Fig. 4. Exon33^-/- mice displayed deficits in social dominance, sociability and social preference.

A Schematic diagram of Tube Dominance Test. Two mice were placed on either side of a clear Plexiglass tube separated by removable separators. As they enter the tube from the release sites at opposite ends, they will interact at the middle of the tube. One of the mice must retreat for the other mice to continue. The dominant mice (grey) shows greater aggression and force the subordinate mice (white) to retreat. The test ends when one mouse pushed the other out of the tube. Winning was scored as binary win lose. B Exon 33^−/− mice had significantly less wins than WT when tested against WT mice. Student t-test was used for statistical analysis (WT n = 8, Exon 33^-/- n = 8, p = 0.026). C Schematic diagram of social interaction test. The subject mice (white) will be allowed to freely interact with two grid cups in the open field box. On day 1, one empty grid cup and one with Mouse 1 (dark grey) was placed in the box. On day 2, one grid cup will be with the familiar Mouse 1 (dark grey), which has been introduced on day 1 and the other grid cup with a new Mouse 2 (light grey). D on day 1, WT mice spent more time sniffing at the grid cup with mouse, while Exon 33^−/− mice spent a similar amount of time at both grid cups. Two-way ANOVA with multiple comparisons was used for statistical analysis (WT n = 13, Exon 33^-/- n = 13, F (1, 48) = 8.064, WT p = 0.0065). E On day 2, when a new Mouse 2 was introduced in the grid cup, WT mice spent more time sniffing at the grid cup with Mouse 2 (new) than Mouse 1 (familiar), while Exon 33 − /− mice still spent an equal amount of time at the two grid cups. Two-way ANOVA with multiple comparisons was used for statistical analysis (WT n = 9, Exon 33^-/- n = 13, F (1, 30) = 2.757, WT p = 0.035).