Assessment of Autism Zebrafish Mutant Models Using a High-Throughput Larval Phenotyping Platform.

In recent years, zebrafish have become commonly used as a model for studying human traits and disorders. Their small size, high fecundity, and rapid development allow for more high-throughput experiments compared to other vertebrate models. Given that zebrafish share >70% gene homologs with humans and their genomes can be readily edited using highly efficient CRISPR methods, we are now able to rapidly generate mutations impacting practically any gene of interest. Unfortunately, our ability to phenotype mutant larvae has not kept pace. To address this challenge, we have developed a protocol that obtains multiple phenotypic measurements from individual zebrafish larvae in an automated and parallel fashion, including morphological features (i.e., body length, eye area, and head size) and movement/behavior. By assaying wild-type zebrafish in a variety of conditions, we determined optimal parameters that avoid significant developmental defects or physical damage; these include morphological imaging of larvae at two time points [3 days post fertilization (dpf) and 5 dpf] coupled with motion tracking of behavior at 5 dpf. As a proof-of-principle, we tested our approach on two novel CRISPR-generated mutant zebrafish lines carrying predicted null-alleles of syngap1b and slc7a5, orthologs to two human genes implicated in autism-spectrum disorder, intellectual disability, and epilepsy. Using our optimized high-throughput phenotyping protocol, we recapitulated previously published results from mouse and zebrafish models of these candidate genes. In summary, we describe a rapid parallel pipeline to characterize morphological and behavioral features of individual larvae in a robust and consistent fashion, thereby improving our ability to better identify genes important in human traits and disorders.

AMPA receptor contribution to methylmercury-mediated alteration of intracellular Ca2+ concentration in human induced pluripotent stem cell motor neurons.

α motor neurons (MNs) are a target of the environmental neurotoxicant methylmercury (MeHg), accumulating MeHg and subsequently degenerating. In mouse spinal cord MN cultures, MeHg increased intracellular Ca2+ [Ca2+]i; the AMPA receptor (AMPAR) antagonist CNQX delayed the increase in [Ca2+]i, implicating the role of AMPARs in this response. Here we used human induced pluripotent stem cell-derived MNs (hiPSC-MNs), to characterize the role of MN AMPARs in MeHg neurotoxicity. Acute exposure to MeHg (0.1, 0.2, 0.5, 1 and 1.5 µM), fura-2 microfluorimetry, and a standard cytotoxicity assay, were used to examine MN regulation of [Ca2+]i, and cytotoxicity, respectively. Contribution of Ca2+-permeable and impermeable AMPARs was compared using either CNQX, or the Ca2+-permeable AMPAR antagonist N-acetyl spermine (NAS). MeHg-induced cytotoxicity was evaluated following a 24 h delay subsequent to 1 h exposure of hiPSC-MNs. MeHg caused a characteristic biphasic increase in [Ca2+]i, the onset of which was concentration-dependent; higher MeHg concentrations hastened onset of both phases. CNQX significantly delayed MeHg's effect on onset time of both phases. In contrast, NAS significantly delayed only the 2nd phase increase in fura-2 fluorescence. Exposure to MeHg for 1 h followed by a 24 h recovery period caused a concentration-dependent incidence of cell death. These results demonstrate for the first time that hiPSC-derived MNs are highly sensitive to effects of MeHg on [Ca2+]i, and cytotoxicity, and that both Ca2+-permeable and impermeable AMPARs contribute the elevations in [Ca2+]i.

Bridge to neuroscience workshop: An effective educational tool to introduce principles of neuroscience to Hispanics students.

Neuroscience as a discipline is rarely covered in educational institutions in Puerto Rico. In an effort to overcome this deficit we developed the Bridge to Neuroscience Workshop (BNW), a full-day hands-on workshop in neuroscience education. BNW was conceived as an auxiliary component of a parent recruitment program called Bridge to the PhD in Neuroscience Program (BPNP). The objectives of BNW are to identify promising students for BPNP, and to increase awareness of neuroscience as a discipline and a career option. BNW introduces basic concepts in neuroscience using a variety of educational techniques, including mini-lectures, interactive discussions, case studies, experimentation, and a sheep brain dissection. Since its inception in 2011 BNW has undergone a series of transformations that continue to improve upon an already successful and influential educational program for underrepresented minorities. As of Fall 2018, we have presented 21 workshops, impacting 200 high school and 424 undergraduate students. BNW has been offered at University of Puerto Rico (UPR)-Arecibo, UPR-Cayey, UPR-Humacao, Pontificia Universidad Católica de Ponce, and Universidad Interamericana de Puerto Rico-Arecibo. A pre-and post evaluation was given to evaluate material comprehension and thus measure effectiveness of our one-day interactive workshop. Our results suggest that both high school and undergraduate students have little prior knowledge of neuroscience, and that participation in BNW improves not only understanding, but also enthusiasm for the discipline. Currently, our assessment has only been able to evaluate short-term effects (e.g. comprehension and learning). Therefore, our current focus is developing methods capable of determining how participation in BNW impacts future academic and career decisions.

Evaluating a Gene-Environment Interaction in Amyotrophic Lateral Sclerosis: Methylmercury Exposure and Mutated SOD1.

PURPOSE OF REVIEW: Gene-environment (GxE) interactions likely contribute to numerous diseases, but are often difficult to model in the laboratory. Such interactions have been widely hypothesized for amyotrophic lateral sclerosis (ALS); recent controlled laboratory studies are discussed here and hypotheses related to possible mechanisms of action are offered. Using methylmercury exposure and mutated SOD1 to model the impacts of such an interaction, we interpret evidence about their respective mechanisms of toxicity to interrogate the possibility of additive (or synergistic) effects when combined. RECENT FINDINGS: Recent work has converged on mechanisms of calcium-mediated glutamate excitotoxicity as a likely contributor in one model of a gene-environment interaction affecting the onset and progression of ALS-like phenotype. The current experimental literature on mechanisms of metal-induced neuronal injury and their relevant interactions with genetic contributions in ALS is sparse, but we describe those studies here and offer several integrative hypotheses about the likely mechanisms involved.

Effects of methylmercury on spinal cord afferents and efferents-A review.

Methylmercury (MeHg) is an environmental neurotoxicant of public health concern. It readily accumulates in exposed humans, primarily in neuronal tissue. Exposure to MeHg, either acutely or chronically, causes severe neuronal dysfunction in the central nervous system and spinal neurons; dysfunction of susceptible neuronal populations results in neurodegeneration, at least in part through Ca2+-mediated pathways. Biochemical and morphologic changes in peripheral neurons precede those in central brain regions, despite the fact that MeHg readily crosses the blood-brain barrier. Consequently, it is suggested that unique characteristics of spinal cord afferents and efferents could heighten their susceptibility to MeHg toxicity. Transient receptor potential (TRP) ion channels are a class of Ca2+-permeable cation channels that are highly expressed in spinal afferents, among other sensory and visceral organs. These channels can be activated in numerous ways, including directly via chemical irritants or indirectly via Ca2+ release from intracellular storage organelles. Early studies demonstrated that MeHg interacts with heterologous TRP channels, though definitive mechanisms of MeHg toxicity on sensory neurons may involve more complex interaction with, and among, differentially-expressed TRP populations. In spinal efferents, glutamate receptors of the N-methyl-D-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and possibly kainic acid (KA) classes are thought to play a major role in MeHg-induced neurotoxicity. Specifically, the Ca2+-permeable AMPA receptors, which are abundant in motor neurons, have been identified as being involved in MeHg-induced neurotoxicity. In this review, we will describe the mechanisms that could contribute to MeHg-induced spinal cord afferent and efferent neuronal degeneration, including the possible mediators, such as uniquely expressed Ca2+-permeable ion channels.