CRISPR-Cas9 mutagenesis and behavioral parameters of adgrl3.1^−/− larvae.

A Cartoon showing position of forward and reverse sgRNA constructs used to create the novel adgrl3.1^−/− line. B The base pair sequence of adgrl3.1 that is deleted by sgRNA injection. The nucleotides shown in orange were not removed and so are retained in the mutant version of adgrl3.1. C The CRISPR guide RNAs were designed to target the different splice variants of adgrl3.1 found in the Ensembl database. (NMD: non-sense mediated decay). D Swim velocity of homozygous wild type (WT, n = 184) and homozygous adgrl3.1^−/− (HOM, n = 179) larvae throughout the whole recording period. The data are pooled from four experiments. The time between 13:30 and 18:00 shows alternating intervals of 30-min lights-off (grey shaded bar) and lights-on phases, followed by constant light-on from 18:00 to 22:00. For the night time, from 22:00 to 08:00 the next morning, the lights were turned off (grey shaded bar). E The average distance moved for the five 30-min lights-off periods demonstrates that homozygous adgrl3.1^−/− larvae moved significantly more than wild-type larvae. F Sleep parameters, expressed as fold change of wild-type larvae, were examined throughout the night, during 10-h lights-off phase. No significant differences between genotypes were observed in any of the five sleep parameters. * denote significant differences.

Effects of ADHD therapeutics on adgrl3.1^−/− mutants.

adgrl3.1^−/− larvae were treated with three different concentrations (1 µM, 10 µM, and 30 µM) of A clonidine, B atomoxetine, and C guanfacine and compared to larvae treated with 0.3% DMSO. Average distance moved during the five 30-min periods of lights-on and sleep parameters during the night was analyzed. Clonidine, atomoxetine and guanfacine all reduced the distance moved during lights-on significantly for all three concentrations. Clonidine, atomoxetine, and guanfacine all significantly altered the sleep dynamics at multiple parameters during the night. Wild-type (WT) larvae treated with 0.3% DMSO are included for visual comparison. * denote significant differences.

Effects of putative ADHD therapeutics on adgrl3.1^−/− mutants.

adgrl3.1^−/− larvae were treated with five different concentrations (0.1 µM, 1 µM, 10 µM, 30 µM, and 100 µM) of A aceclofenac, B amlodipine, C doxazosin and D moxonidine and compared to larvae treated with 0.3% DMSO. Average distance moved during the five 30-min periods of lights-on and sleep parameters during the night were analyzed. Aceclofenac, amlodipine, doxazosin and moxonidine all reduced the distance moved during lights-on significantly for various concentrations. Aceclofenac, amlodipine, doxazosin and moxonidine all significantly altered the sleep dynamics at multiple parameters during the night. Wild-type (WT) larvae treated with 0.3% DMSO are included for visual comparison. * denote significant differences.

Effects of moxonidine in spontaneous alternation rodent assay.

A significant difference was found between mice exposed to scopolamine and naïve mice (saline) in which scopolamine-exposed mice showed decrease in spontaneous alterations. Treatment with donepezil and atomoxetine rescued this effect. Moxonidine also rescued this effect in mice in a dose-dependent manner, showing significantly higher spontaneous alterations in mice treated with 0.3 mg/kg and 1 mg/kg treatment but no significant differences were observed between mice treated with 0.1 mg/kg moxonidine compared to scopolamine treated mice. * denote significant differences.

Motility and sleep effects of I1 receptor agonist LNP599.

adgrl3.1^−/− larvae were treated with five different concentrations (0.1 µM, 1 µM, 10 µM, 30 µM, and 100 µM) of LNP599 and compared to larvae treated with 0.3% DMSO. Average distance moved during the five 30-min periods of lights-on and sleep parameters during the night were analyzed. LNP599 reduced the distance moved during lights-on significantly in a dose dependent manner for 10 µM, 30 µM, and 100 µM. LNP599 significantly altered all sleep parameters at various concentrations. Wild type (WT) larvae treated with DMSO are included for visual comparison. * denote significant differences.