N-methyl-D-aspartate receptors (NMDARs) are glutamate-gated ion channels that play critical roles in neuronal development and nervous system function. Here, we developed a model to study NMDARs in early development in zebrafish, by generating CRISPR-mediated lesions in the NMDAR genes, grin1a and grin1b, which encode the obligatory GluN1 subunits. While receptors containing grin1a or grin1b show high Ca 21 permeability, like their mammalian counterpart, grin1a is expressed earlier and more broadly in development than grin1b. Both grin1a 2/2 and grin1b 2/2 zebrafish are viable. Unlike in rodents, where the grin1 knockout is embryonic lethal, grin1 double-mutant fish (grin1a 2/2 ; grin1b 2/2), which lack all NMDAR-mediated synaptic transmission, survive until ;10 d dpf (days post fertilization), providing a unique opportunity to explore NMDAR function during development and in generating behaviors. Many behavioral defects in the grin1 double-mutant larvae, including abnormal evoked responses to light and acoustic stimuli, prey-capture deficits, and a failure to habituate to acoustic stimuli, are replicated by short-term treatment with the NMDAR antagonist MK-801, suggesting that they arise from acute effects of compromised NMDAR-mediated transmission. Other defects, however, such as periods of hyperactivity and alterations in place preference, are not phenocopied by MK-801, suggesting a developmental origin. Together, we have developed a unique model to study NMDARs in the developing vertebrate nervous system.

Background Autism spectrum disorder (ASD), like many neurodevelopmental disorders, has complex and varied etiologies. Advances in genome sequencing have identified multiple candidate genes associated with ASD, including dozens of missense and nonsense mutations in the NMDAR subunit GluN2B, encoded by GRIN2B. NMDARs are glutamate-gated ion channels with key synaptic functions in excitatory neurotransmission. How alterations in these proteins impact neurodevelopment is poorly understood, in part because knockouts of GluN2B in rodents are lethal. Methods Here, we use CRISPR-Cas9 to generate zebrafish lacking GluN2B (grin2B−/−). Using these fish, we run an array of behavioral tests and perform whole-brain larval imaging to assay developmental roles and functions of GluN2B. Results We demonstrate that zebrafish GluN2B displays similar structural and functional properties to human GluN2B. Zebrafish lacking GluN2B (grin2B−/−) surprisingly survive into adulthood. Given the prevalence of social deficits in ASD, we assayed social preference in the grin2B−/− fish. Wild-type fish develop a strong social preference by 3 weeks post fertilization. In contrast, grin2B−/− fish at this age exhibit significantly reduced social preference. Notably, the lack of GluN2B does not result in a broad disruption of neurodevelopment, as grin2B−/− larvae do not show alterations in spontaneous or photic-evoked movements, are capable of prey capture, and exhibit learning. Whole-brain imaging of grin2B−/− larvae revealed reduction of an inhibitory neuron marker in the subpallium, a region linked to ASD in humans, but showed that overall brain size and E/I balance in grin2B−/− is comparable to wild type. Limitations Zebrafish lacking GluN2B, while useful in studying developmental roles of GluN2B, are unlikely to model nuanced functional alterations of human missense mutations that are not complete loss of function. Additionally, detailed mammalian homologies for larval zebrafish brain subdivisions at the age of whole-brain imaging are not fully resolved. Conclusions We demonstrate that zebrafish completely lacking the GluN2B subunit of the NMDAR, unlike rodent models, are viable into adulthood. Notably, they exhibit a highly specific deficit in social behavior. As such, this zebrafish model affords a unique opportunity to study the roles of GluN2B in ASD etiologies and establish a disease-relevant in vivo model for future studies.

Autism Spectrum Disorder (ASD), like many neurodevelopmental disorders, has complex and varied etiologies. Advances in genome sequencing have identified multiple candidate genes associated with ASD, including dozens of missense and nonsense mutations in the NMDAR subunit GluN2B, encoded by GRIN2B. NMDARs are glutamate-gated ion channels with key synaptic functions in excitatory neurotransmission. How alterations in these proteins impact neurodevelopment is poorly understood, in part because knockouts of GluN2B in rodents are lethal. Here, we demonstrate that zebrafish GluN2B displays similar structural and functional properties to human GluN2B. Using CRISPR-Cas9, we generated fish lacking all functional GluN2B (grin2B-/-) and surprisingly found that they survive into adulthood. Given the prevalence of social deficits in ASD, we assayed social preference in the grin2B-/- fish. Wild-type fish develop a strong social preference by 3 weeks post fertilization (wpf). In contrast, grin2B-/- fish at this age exhibit significantly reduced social preference. This phenotype is specific for GluN2B and not due to general NMDAR dysfunction, as frameshift mutations in other NMDAR subunits do not generate social deficits. Notably, the lack of GluN2B does not result in a broad disruption of neurodevelopment, as grin2B-/- larvae do not show alterations in spontaneous or photic-evoked movements, are capable of prey capture, and exhibit learning capabilities. Whole-brain imaging of grin2B-/- larvae revealed a reduction in inhibitory neurons in the subpallium, a region linked to ASD in humans, but showed that overall brain size and E/I balance in grin2B-/- is comparable to wild-type. Together, these findings highlight the specific role of GluN2B in ASD etiologies and establishing a disease-relevant in vivo model for future studies.

ABSTRACTNMDA receptors (NMDARs) are glutamate-gated ion channels that play critical roles in neuronal development and nervous system function. Pharmacological antagonism is an invaluable tool to study NMDARs, but is experimentally limited. Here, we developed a model to study NMDARs in early development in zebrafish, by generating CRISPR-mediated lesions in the NMDAR genes, grin1a and grin1b, which encode the obligatory GluN1 subunits. While receptors containing grin1a or grin1b show high Ca2+ permeability, like their mammalian counterpart, grin1a is expressed earlier and more broadly in development than grin1b. Both grin1a−/− and grin1b−/− zebrafish are viable as adults. Unlike in rodents, where the grin1 knockout is embryonic lethal, grin1 double mutant fish (grin1a−/−; grin1b−/−), which lack all NMDAR-mediated synaptic transmission, survive until about 10 days post fertilization, providing a unique opportunity to explore NMDAR function during development and in generating behaviors. Many behavioral defects in the grin1 double mutant larvae, including abnormal evoked responses to light and acoustic stimuli, prey capture deficits and a failure to habituate to acoustic stimuli, are replicated by short-term treatment with the NMDAR antagonist MK-801, suggesting they arise from acute effects of compromised NMDAR-mediated transmission. Other defects, however, such as periods of hyperactivity and alterations in place preference, are not phenocopied by MK-801, suggesting a developmental origin. Taken together, we have developed a unique model to study NMDARs in the developing vertebrate nervous system.