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.
Evolution shapes the brain, as it does the body, to allow an organism to adapt to its ecological niche by building upon inherited, conserved traits and developing new, divergent ones. Recently, some investigators have proposed that brains may not have evolved from a single common ancestor, but rather may have evolved more than once. Therefore, molecules, cells and genes that evolved earlier for other purposes may have been co‐opted for use in building nervous systems independently. Prior to the emergence of bilaterians, invertebrate nervous systems displayed radial symmetric nerve net formations, but the beginnings of nervous system condensation were there. In bilaterian nervous systems we see great variety; invertebrates of this group display simple ganglion/nerve cord formations as well as highly elaborated brains. Vertebrates have the most elaborated brains of all metazoans with complex subdivisions, some of which are highly conserved and others highly variable. Key Concepts Brains have evolved to enable organisms to compete successfully in different environmental niches, and the adaptations they show reflect the demands of those environments. Recently, some investigators have proposed that brains may not have evolved from a single common ancestor, but rather may have evolved more than once. In this view, which is controversial, the common ancestor of all invertebrates did not have a nervous system, and the nervous system developed separately in Ctenophora, on the one hand, and Cnidaria and Bilateria on the other. Some evidence suggests that, even within Bilateria, nervous systems may have evolved separately in different groups. Attempts to understand the origins of the nervous system must take into account the possibility that similar characteristics in present‐day nervous systems are not homologous as derivatives of a structure in the common ancestor, but rather, they have arisen separately in different lineages because of exaptation of pre‐existing traits in a common ancestor. Invertebrate brains show a wide variety of structures from nerve nets to condensed ganglia to fully elaborated brains. All vertebrates have the same subdivisions of the brain: the hindbrain, midbrain and forebrain. The hindbrain is relatively conservative, retaining a recognisable structure in spite of many variations, whereas the forebrain shows more variability among vertebrate groups. Elaborate nervous systems and the capacity for complex learning have developed independently in some molluscs, arthropods and vertebrates. The presence of elaborated brains in disparate groups illustrates that evolution of the brain has not shown a trend from simple to complex across metazoan taxa, but rather has occurred as a result of diverse environmental pressures that have led to the same elaborate structures more than once.
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.
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