Zebrafish cacna1da mutant allele (sa17298) and its molecular consequence. a Schematic representation of the zebrafish cacna1da transcript. Red bars represent exons and lines represent introns. Image retrieved from ensembl.org. b Schematic representation of zebrafish cacna1da and sa17298 mutation. Nucleotides in upper case are within the exon while those in lower case are within the intron. c Sample gel electrophoresis of BstEII restriction digest and resulting PCR products to determine genotype of fish/larvae. BstEII does not cut homozygous sa17298, but cuts WT into two bands (70 and 119 bp) and heterozygous sa17298 into three bands (70, 119 and 196 bp). Note the multiple bands in the BstEII digested versus the undigested corresponding samples. L: 1 kilobase DNA ladder, +/+: cacna1da^WT/WT, +/−: cacna1da^sa17298/WT, −/−: cacna1da^{sa17298/sa17298}.dcacna1da mRNA levels as measured by agarose gel electrophoresis (rt-PCR). ecacna1da mRNA levels as measured by qPCR. cacna1da mRNA expression is normalised against gapdh and rps18

Zebrafish cacna1da mutant allele (sa17298) and its molecular consequence. a Schematic representation of the zebrafish cacna1da transcript. Red bars represent exons and lines represent introns. Image retrieved from ensembl.org. b Schematic representation of zebrafish cacna1da and sa17298 mutation. Nucleotides in upper case are within the exon while those in lower case are within the intron. c Sample gel electrophoresis of BstEII restriction digest and resulting PCR products to determine genotype of fish/larvae. BstEII does not cut homozygous sa17298, but cuts WT into two bands (70 and 119 bp) and heterozygous sa17298 into three bands (70, 119 and 196 bp). Note the multiple bands in the BstEII digested versus the undigested corresponding samples. L: 1 kilobase DNA ladder, +/+: cacna1da^WT/WT, +/−: cacna1da^sa17298/WT, −/−: cacna1da^{sa17298/sa17298}.dcacna1da mRNA levels as measured by agarose gel electrophoresis (rt-PCR). ecacna1da mRNA levels as measured by qPCR. cacna1da mRNA expression is normalised against gapdh and rps18

Morphology of a WT cacna1da^WT/WTb heterozygous cacna1da^sa17298/WT and c homozygous cacna1da^{sa17298/sa17298}. Both heterozygous and homozygous mutants are morphologically indistinguishable from WT siblings. Scale bar (a–c): 1 mm

Morphology of a WT cacna1da^WT/WTb heterozygous cacna1da^sa17298/WT and c homozygous cacna1da^{sa17298/sa17298}. Both heterozygous and homozygous mutants are morphologically indistinguishable from WT siblings. Scale bar (a–c): 1 mm

Acoustic startle response and PPI. a Graph of distance travelled when presented with different auditory stimuli. Data analysed using two-way ANOVA and presented as mean ± SEM. b Preceding a startle with a prepulse stimulus decreases the startle response of larvae. Data shown as mean ± SEM. c % PPI of larvae. One-way ANOVA revealed no overall significant difference among the groups. Tukey’s multiple comparison post hoc test however showed a strong tendency of homozygous mutant larvae towards significance (i.e. enhanced PPI). cacna1da^WT/WT (N = 8), cacna1da^sa17298/WT (N = 6) and cacna1da^{sa17298/sa17298} (N = 5), p > 0.05. Error bars represent mean ± SD. ASR, acoustic startle response; Pp, prepulse; PPI, prepulse inhibition. cacna1da^WT/WT vs cacna1da^sa17298/WT: p = 0.2002, cacna1da^WT/WT vs cacna1da^{sa17298/sa17298}: p = 0.0646, cacna1da^sa17298/WT vs cacna1da^{sa17298/sa17298}: p = 0.7702

Acoustic startle response and PPI. a Graph of distance travelled when presented with different auditory stimuli. Data analysed using two-way ANOVA and presented as mean ± SEM. b Preceding a startle with a prepulse stimulus decreases the startle response of larvae. Data shown as mean ± SEM. c % PPI of larvae. One-way ANOVA revealed no overall significant difference among the groups. Tukey’s multiple comparison post hoc test however showed a strong tendency of homozygous mutant larvae towards significance (i.e. enhanced PPI). cacna1da^WT/WT (N = 8), cacna1da^sa17298/WT (N = 6) and cacna1da^{sa17298/sa17298} (N = 5), p > 0.05. Error bars represent mean ± SD. ASR, acoustic startle response; Pp, prepulse; PPI, prepulse inhibition. cacna1da^WT/WT vs cacna1da^sa17298/WT: p = 0.2002, cacna1da^WT/WT vs cacna1da^{sa17298/sa17298}: p = 0.0646, cacna1da^sa17298/WT vs cacna1da^{sa17298/sa17298}: p = 0.7702

Locomotor activity of WT and heterozygous sa17298 zebrafish larvae at 6 dpf, tracked independently under different illumination conditions. a Tracking in the dark reveals no difference in locomotor activity between cacna1da^WT/WT (N = 16) and cacna1da^sa17298/WT (N = 15). b Tracking in 100% light reveals a statistically significant difference in locomotor activity between cacna1da^WT/WT (N = 21) and cacna1da^sa17298/WT (N = 22). ***p < 0.001. Data analysed using Mann Whitney U test and represented as mean ± SD

Locomotor activity of WT and heterozygous sa17298 zebrafish larvae at 6 dpf, tracked independently under different illumination conditions. a Tracking in the dark reveals no difference in locomotor activity between cacna1da^WT/WT (N = 16) and cacna1da^sa17298/WT (N = 15). b Tracking in 100% light reveals a statistically significant difference in locomotor activity between cacna1da^WT/WT (N = 21) and cacna1da^sa17298/WT (N = 22). ***p < 0.001. Data analysed using Mann Whitney U test and represented as mean ± SD

Behaviour of WT and heterozygous sa17298 zebrafish larvae at 6 dpf in the light-dark transition test. Each dot represents an individual larval measurement. Data analysed using non-RM two-way ANOVA followed by Tukey’s post hoc test. Data represented as mean ± SD. a Light-dark transition elicits an increase in locomotor activity in both genotypes with locomotor difference between cacna1da^WT/WT and cacna1da^sa17298/WT reaching statistical significance in both illumination states. * (p < 0.05), ** (p < 0.01): [cacna1da^WT/WT vs cacna1da^sa17298/WT] in the dark, #### (p < 0.0001): [cacna1da^WT/WT vs cacna1da^WT/WT] and [cacna1da^sa17298/WT vs cacna1da^sa17298/WT]. b Zone preference of WT and heterozygous sa17298 larvae (thigmotaxis) represented as % total distance moved in the outer zone in the light-dark transition test

Behaviour of WT and heterozygous sa17298 zebrafish larvae at 6 dpf in the light-dark transition test. Each dot represents an individual larval measurement. Data analysed using non-RM two-way ANOVA followed by Tukey’s post hoc test. Data represented as mean ± SD. a Light-dark transition elicits an increase in locomotor activity in both genotypes with locomotor difference between cacna1da^WT/WT and cacna1da^sa17298/WT reaching statistical significance in both illumination states. * (p < 0.05), ** (p < 0.01): [cacna1da^WT/WT vs cacna1da^sa17298/WT] in the dark, #### (p < 0.0001): [cacna1da^WT/WT vs cacna1da^WT/WT] and [cacna1da^sa17298/WT vs cacna1da^sa17298/WT]. b Zone preference of WT and heterozygous sa17298 larvae (thigmotaxis) represented as % total distance moved in the outer zone in the light-dark transition test

Startle response to dark flashes. a Baseline locomotor activity 21 s prior to onset of startle stimuli, ***p < 0.001. Data represented as mean ± SD. b Mean distance moved in response to startle stimuli. Unpaired Student’s t test showed cacna1da^WT/WT moved less than cacna1da^sa17298/WT during the 90-s startle stimulation, *p < 0.05. Data represented as mean ± SD. c Distance moved in response to startle stimuli represented as mean ± SEM. Paired Student’s t test analysis between cacna1da^WT/WT and cacna1da^sa17298/WT with statistical significance represented as *p < 0.05, ^#p < 0.01, ^p < 0.001. Red arrow: onset of startle stimulus (dark flashes)

Startle response to dark flashes. a Baseline locomotor activity 21 s prior to onset of startle stimuli, ***p < 0.001. Data represented as mean ± SD. b Mean distance moved in response to startle stimuli. Unpaired Student’s t test showed cacna1da^WT/WT moved less than cacna1da^sa17298/WT during the 90-s startle stimulation, *p < 0.05. Data represented as mean ± SD. c Distance moved in response to startle stimuli represented as mean ± SEM. Paired Student’s t test analysis between cacna1da^WT/WT and cacna1da^sa17298/WT with statistical significance represented as *p < 0.05, ^#p < 0.01, ^p < 0.001. Red arrow: onset of startle stimulus (dark flashes)

Effects of neuroactive drugs on the locomotor activity of 6-dpf WT and heterozygous sa17298 larvae. Larvae were exposed to different neuroactive drugs. Each dot represents individual larval measurement. Data analysed using non-RM two-way ANOVA followed by Tukey’s post hoc test. Data represented as mean ± SD. a 2-h RISP, b 24-h RISP c 2-h HALO and d 2-h VPA. HALO, haloperidol; RISP, risperidone; VPA, valproic acid. *p < 0.05, **p < 0.01, ***p < 0.001 [cacna1da^WT/WT vs cacna1da^sa17298/WT] in respective groups. ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, ^####p < 0.0001 [cacna1da^WT/WT vs cacna1da^WT/WT] and [cacna1da^sa17298/WT vs cacna1da^sa17298/WT]

Effects of neuroactive drugs on the locomotor activity of 6-dpf WT and heterozygous sa17298 larvae. Larvae were exposed to different neuroactive drugs. Each dot represents individual larval measurement. Data analysed using non-RM two-way ANOVA followed by Tukey’s post hoc test. Data represented as mean ± SD. a 2-h RISP, b 24-h RISP c 2-h HALO and d 2-h VPA. HALO, haloperidol; RISP, risperidone; VPA, valproic acid. *p < 0.05, **p < 0.01, ***p < 0.001 [cacna1da^WT/WT vs cacna1da^sa17298/WT] in respective groups. ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, ^####p < 0.0001 [cacna1da^WT/WT vs cacna1da^WT/WT] and [cacna1da^sa17298/WT vs cacna1da^sa17298/WT]

Effects of neuroactive drugs on the behaviour of 6-dpf WT and heterozygous sa17298 larvae in the light-dark test. Larvae were exposed to different neuroactive drugs. Each dot represents individual larval measurement. Data analysed using two-way ANOVA followed by multiple comparison t test. Data represented as mean ± SD. a 2-h RISP, b 24-h RISP, c 2 h HALO and d 2-h VPA. ctl, control; HALO, haloperidol; RISP, risperidone; VPA, valproic acid. *p < 0.05, **p < 0.01, ***p < 0.001 [cacna1da^WT/WT vs cacna1da^sa17298/WT] in respective groups. ^###p < 0.001, ^####p < 0.0001 [cacna1da^WT/WT vs cacna1da^WT/WT] and [cacna1da^sa17298/WT vs cacna1da^sa17298/WT]

Effects of neuroactive drugs on the behaviour of 6-dpf WT and heterozygous sa17298 larvae in the light-dark test. Larvae were exposed to different neuroactive drugs. Each dot represents individual larval measurement. Data analysed using two-way ANOVA followed by multiple comparison t test. Data represented as mean ± SD. a 2-h RISP, b 24-h RISP, c 2 h HALO and d 2-h VPA. ctl, control; HALO, haloperidol; RISP, risperidone; VPA, valproic acid. *p < 0.05, **p < 0.01, ***p < 0.001 [cacna1da^WT/WT vs cacna1da^sa17298/WT] in respective groups. ^###p < 0.001, ^####p < 0.0001 [cacna1da^WT/WT vs cacna1da^WT/WT] and [cacna1da^sa17298/WT vs cacna1da^sa17298/WT]

Time series graph of the effects of neuroactive drugs on the behaviour of 6-dpf WT and heterozygous sa17298 larvae in the startle response to dark flashes test. Larvae were exposed to different neuroactive drugs. Each dot represents individual larval measurement. Data represented as mean ± SEM. a 2-h RISP, b 24-h RISP, c 2-h HALO, d 2-h VPA. HALO, haloperidol; RISP, risperidone; VPA, valproic acid

Time series graph of the effects of neuroactive drugs on the behaviour of 6-dpf WT and heterozygous sa17298 larvae in the startle response to dark flashes test. Larvae were exposed to different neuroactive drugs. Each dot represents individual larval measurement. Data represented as mean ± SEM. a 2-h RISP, b 24-h RISP, c 2-h HALO, d 2-h VPA. HALO, haloperidol; RISP, risperidone; VPA, valproic acid