Increased locomotor activity via regulation of GABAergic signalling in foxp2 mutant zebrafish—implications for neurodevelopmental disorders

Abstract

Recent advances in the genetics of neurodevelopmental disorders (NDDs) have identified the transcription factor FOXP2 as one of numerous risk genes, e.g. in autism spectrum disorders (ASD) and attention-deficit/hyperactivity disorder (ADHD). FOXP2 function is suggested to be involved in GABAergic signalling and numerous studies demonstrate that GABAergic function is altered in NDDs, thus disrupting the excitation/inhibition balance. Interestingly, GABAergic signalling components, including glutamate-decarboxylase 1 (Gad1) and GABA receptors, are putative transcriptional targets of FOXP2. However, the specific role of FOXP2 in the pathomechanism of NDDs remains elusive. Here we test the hypothesis that Foxp2 affects behavioural dimensions via GABAergic signalling using zebrafish as model organism. We demonstrate that foxp2 is expressed by a subset of GABAergic neurons located in brain regions involved in motor functions, including the subpallium, posterior tuberculum, thalamus and medulla oblongata. Using CRISPR/Cas9 gene-editing we generated a novel foxp2 zebrafish loss-of-function mutant that exhibits increased locomotor activity. Further, genetic and/or pharmacological disruption of Gad1 or GABA-A receptors causes increased locomotor activity, resembling the phenotype of foxp2 mutants. Application of muscimol, a GABA-A receptor agonist, rescues the hyperactive phenotype induced by the foxp2 loss-of-function. By reverse translation of the therapeutic effect on hyperactive behaviour exerted by methylphenidate, we note that application of methylphenidate evokes different responses in wildtype compared to foxp2 or gad1b loss-of-function animals. Together, our findings support the hypothesis that foxp2 regulates locomotor activity via GABAergic signalling. This provides one targetable mechanism, which may contribute to behavioural phenotypes commonly observed in NDDs.

Loss-of-Function Models of the Metabotropic Glutamate Receptor Genes Grm8a and Grm8b Display Distinct Behavioral Phenotypes in Zebrafish Larvae (Danio rerio)

“…Comparing to our previous findings we noted that the developmental distribution of grm8a resembles the one of gad1a and gad1b at 36 hpf ( Lüffe et al, 2021 ), especially in the medulla oblongata (MO, Figures 4A,B ). Hence, we hypothesized that grm8a expression might correlate with the distribution of GABAergic markers as seen for other species and brain regions before ( Ferraguti et al, 2005 ).…”

“…To confirm colocalization in single cells we used double RNA ISH for gad1a (red) and grm8a (blue) at 36 hpf on whole-mounts and cryosections. Due to the largely similar pattern of gad1a and gad1b expression ( Lüffe et al, 2021 ), the technical benefits and the broader expression of grm8a compared to grm8b ( Figures 1A,B , 2 , 3) , we decided to assess the colocalization of grm8a and gad1a expression.…”

“…In order to investigate the behavioral consequences of grm8a and grm8b loss-of-function we performed locomotor tracking at 5 dpf as described previously ( Lüffe et al, 2021 ). Briefly, we placed larvae in a 12-well dish and recorded locomotor activity in a commercial setup for 5 min after 5 min of habituation.…”

“…Whole-mount RNA ISH was performed according to the methods published previously ( Thisse and Thisse, 2008 ; Lechermeier et al, 2019 , 2020 ; Lüffe et al, 2021 ). Briefly, specimens were fixed in 4% PFA, washed in PBS containing 0.1% Tween-20 (PBST) and dehydrated in methanol (MeOH) and finally stored in 100% MeOH at –20°C.…”

Exploring the Contribution to ADHD of Genes Involved in Mendelian Disorders Presenting with Hyperactivity and/or Inattention

“…One of the loci associated with ADHD in the largest GWAS meta-analysis pointed to FOXP2 , another member of the same family [ 4 ]. FOXP2 forms homo- and/or heterodimers with FOXP1 and FOXP4 [ 42 , 43 ], and regulates neuron maturation and locomotor activity [ 44 ]. Thus, these three genes seem to have a pleiotropic role in ADHD and its related comorbidities.…”