Modeling SCN1A Epilepsy with Dual Isogenic Pairs of Human iPSC-derived Neurons

Over 1250 mutations in SCN1A, the Nav1.1 voltage-gated sodium channel gene, are associated with a variety of seizure disorders including Dravet syndrome (DS) and genetic epilepsy with febrile seizure plus (GEFS+). Individuals with SCN1A genetic epilepsy exhibit a broad range of seizure phenotypes and many of them are not well controlled by conventional anti-convulsant therapy. How specific mutations alter sodium channel function in a way that contributes to seizure generation is still largely unexplored. An understanding of cellular mechanisms is important for future studies targeted at developing more effective patient-specific therapies. Previous studies in models including Xenopus oocytes, human embryonic kidney cells, mouse, Drosophila and zebrafish have provided some important insights into functional changes in sodium currents and firing properties associated with a number of SCN1A mutations. However, they also reveal that the same mutation can have distinct effects in the different models. By combining recent advances in stem cell reprogramming technology and gene editing tools it is now possible explore how specific gene mutations alter activity in human neurons. To evaluate changes in neuronal activity associated with a specific mutation, independent of genetic background, we generated two pairs of isogenic human iPSC lines by CRISPR/Cas9 editing. One pair is a control line from an unaffected sibling, and the mutated control homozygous for the GEFS+ K1270T SCN1A mutation. The second pair is a GEFS+ patient line heterozygous for the K1270T mutation, and the corrected patient line. To detect mutation-associated changes by comparing electrophysiological properties between cell lines, it is necessary to differentiate iPSC into neurons that could both fire action potentials and form synaptic connections. While there were a number of protocols for differentiation of iPSCs into neurons in the literature, there was a lot of variability in the time course and degree of differentiation even from plating to plating within one cell line. Starting with the published protocols we identified conditions that support robust reproducible differentiation from plating to plating and between iPSCs lines into cultures with GABAergic and glutamatergic neurons. The iPSC-derived neuronal cultures from both isogenic pairs of iPSCs that we generated contained a similar proportion of GABAergic and glutamatergic neurons. By comparing the electrophysiological properties in inhibitory and excitatory iPSC-derived neurons from these pairs, we found the K1270T mutation causes gene dosage-dependent and cell type-specific alterations in evoked firing and sodium currents that result in hyperactive neural networks. We also identified differences associated with genetic background and interaction between the mutation and genetic background. Dual isogenic iPSC-derived neuronal cultures provide an efficient strategy to evaluate the causality of a single gene mutation independent of genetic background and to develop patient-specific anti-seizure therapies

A reinforcement learning algorithm shapes maternal care in mice

The neural substrates for processing classical rewards such as food or drugs of abuse are well-understood. In contrast, the mechanisms by which organisms perceive social contact as rewarding and subsequently modify their interactions are unclear. Here we tracked the gradual emergence of a repetitive and highly-stereotyped parental behavior and show that trial-by-trial performance correlates with the history of midbrain dopamine (DA) neuron activity. We used a novel behavior paradigm to manipulate the subject’s expectation of imminent pup contact and show that DA signals conform to reward prediction error, a fundamental component of reinforcement learning (RL). Finally, closed-loop optogenetic inactivation of DA neurons at the onset of pup contact dramatically slowed emergence of parental care. We conclude that this prosocial behavior is shaped by an RL mechanism in which social contact itself is the primary reward

A dopaminergic reward prediction error signal shapes maternal behavior in mice

How social contact is perceived as rewarding and subsequently modifies interactions is unclear. Dopamine (DA) from the ventral tegmental area (VTA) regulates sociality, but the ongoing, unstructured nature of free behavior makes it difficult to ascertain how. Here, we tracked the emergence of a repetitive stereotyped parental retrieval behavior and conclude that VTA DA neurons incrementally refine it by reinforcement learning (RL). Trial-by-trial performance was correlated with the history of DA neuron activity, but DA signals were inconsistent with VTA directly influencing the current trial. We manipulated the subject's expectation of imminent pup contact and show that DA signals convey reward prediction error, a fundamental component of RL. Finally, closed-loop optogenetic inactivation of DA neurons at the onset of pup contact dramatically slowed emergence of parental care. We conclude that this component of maternal behavior is shaped by an RL mechanism in which social contact itself is the primary reward

Astrocyte-enriched feeder layers from cryopreserved cells support differentiation of spontaneously active networks of human iPSC-derived neurons

BackgroundHuman induced pluripotent stem cell (hiPSC)-derived neuronal cultures are a useful tool for studying the mechanisms of neurological disorders and developing novel therapeutics. While plating hiPSC-derived neuronal progenitors onto glial feeder layers prepared from rodent cortex has been reported to promote functional differentiation of neuronal networks, this has not been examined in detail.New methodHere we describe a method of using cryopreserved cells from primary cultures for generation of mouse astrocyte-enriched, neuron-free feeder layers that grow from 10% to 100% confluence in 1 week.ResultsElectrophysiological analysis demonstrated that compared to biochemical substrates alone, astrocyte-enriched feeder layers support more rapid differentiation of hiPSC-derived progenitors into excitable neurons that form spontaneously active networks in culture. There was a positive correlation between the degree of astroglial confluence at the time of progenitor plating and the average frequency of postsynaptic currents 3 weeks after plating. One disadvantage to plating on 100% confluent feeder layers was a high incidence of the astroglial layer with the overlying neurons detaching from the coverslips during transfer to the recording chamber.Comparison with existing method(s)Prevailing methods using primary glial feeder layers can result in possible contamination with rodent neurons and an unpredictable rate of growth. We provide a reliable method of generating mouse astroglial feeder layers from cryopreserved primary cultures to support differentiation of hiPSC-derived neurons.ConclusionsThe ability to make astrocyte-enriched feeder layers of defined confluence from cryopreserved primary cultures will facilitate the use of human stem cell derived neuronal cultures for disease modeling

Reproducible and efficient generation of functionally active neurons from human hiPSCs for preclinical disease modeling.

The use of human induced pluripotent stem cell (hiPSC)-derived neuronal cultures to study the mechanisms of neurological disorders is often limited by low efficiency and high variability in differentiation of functional neurons. Here we compare the functional properties of neurons in cultures prepared with two hiPSC differentiation protocols, both plated on astroglial feeder layers. Using a protocol with an expandable intermediate stage, only a small percentage of cells with neuronal morphology were excitable by 21-23days in culture. In contrast, a direct differentiation strategy of the same hiPSC line produced cultures in which the majority of neurons fired action potentials as early as 4-5days. By 35-38days over 80% of the neurons fired repetitively and many fired spontaneously. Spontaneous post-synaptic currents were observed in ~40% of the neurons at 4-5days and in ~80% by 21-23days. The majority (75%) received both glutamatergic and GABAergic spontaneous postsynaptic currents. The rate and degree of maturation of excitability and synaptic activity was similar between multiple independent platings from a single hiPSC line, and between two different control hiPSC lines. Cultures of rapidly functional neurons will facilitate identification of cellular mechanisms underlying genetically defined neurological disorders and development of novel therapeutics

Reproducible and efficient generation of functionally active neurons from human hiPSCs for preclinical disease modeling

The use of human induced pluripotent stem cell (hiPSC)-derived neuronal cultures to study the mechanisms of neurological disorders is often limited by low efficiency and high variability in differentiation of functional neurons. Here we compare the functional properties of neurons in cultures prepared with two hiPSC differentiation protocols, both plated on astroglial feeder layers. Using a protocol with an expandable intermediate stage, only a small percentage of cells with neuronal morphology were excitable by 21–23 days in culture. In contrast, a direct differentiation strategy of the same hiPSC line produced cultures in which the majority of neurons fired action potentials as early as 4–5 days. By 35–38 days over 80% of the neurons fired repetitively and many fired spontaneously. Spontaneous post-synaptic currents were observed in ~40% of the neurons at 4–5 days and in ~80% by 21–23 days. The majority (75%) received both glutamatergic and GABAergic spontaneous postsynaptic currents. The rate and degree of maturation of excitability and synaptic activity was similar between multiple independent platings from a single hiPSC line, and between two different control hiPSC lines. Cultures of rapidly functional neurons will facilitate identification of cellular mechanisms underlying genetically defined neurological disorders and development of novel therapeutics. Keywords: Induced pluripotent stem cells, hiPSC-derived neurons, Functional differentiation, Reproducibility, Disease modelin

Near-infrared fluorophore IR-61 improves the quality of oocytes in aged mice via mitochondrial protection

Maternal aging is associated with a decline in oocyte quality, which leads to the decreased fertility. Therefore, developing approaches to reduce aging-induced deterioration of oocyte quality in older women is important. Near-infrared cell protector-61 (IR-61), a novel heptamethine cyanine dye, has the potential for antioxidant effects. In this study, we found that IR-61 can accumulate in the ovaries and improved ovarian function of naturally aged mice; it also increased the oocyte maturation rate and quality by maintaining the integrity of the spindle/chromosomal structure and reducing the aneuploidy rate. In addition, the embryonic developmental competence of aged oocytes was improved. Finally, RNA-sequencing analysis indicated that IR-61 might perform the beneficial effects on aged oocytes by regulating mitochondrial function, this was confirmed by immunofluorescence analysis of mitochondrial distribution and reactive oxygen species. Taken together, our findings demonstrate that IR-61 supplementation in vivo can increase oocyte quality and protect oocytes from aging-induced mitochondrial dysfunction, and thus could improve the fertility of older women and efficiency of assisted reproductive technology

Interneuron Dysfunction in a New Mouse Model of SCN1A GEFS.

Advances in genome sequencing have identified over 1300 mutations in the SCN1A sodium channel gene that result in genetic epilepsies. However, it still remains unclear how most individual mutations within SCN1A result in seizures. A previous study has shown that the K1270T (KT) mutation, linked to genetic epilepsy with febrile seizure plus (GEFS+) in humans, causes heat-induced seizure activity associated with a temperature-dependent decrease in GABAergic neuron excitability in a Drosophila knock-in model. To examine the behavioral and cellular effects of this mutation in mammals, we introduced the equivalent KT mutation into the mouse (Mus musculus) Scn1a (Scn1aKT) gene using CRISPR/Cas9 and generated mutant lines in two widely used genetic backgrounds: C57BL/6NJ and 129X1/SvJ. In both backgrounds, mice homozygous for the KT mutation had spontaneous seizures and died by postnatal day (P)23. There was no difference in mortality of heterozygous KT mice compared with wild-type littermates up to six months old. Heterozygous mutants exhibited heat-induced seizures at ∼42°C, a temperature that did not induce seizures in wild-type littermates. In acute hippocampal slices at permissive temperatures, current-clamp recordings revealed a significantly depolarized shift in action potential threshold and reduced action potential amplitude in parvalbumin (PV)-expressing inhibitory CA1 interneurons in Scn1aKT/+ mice. There was no change in the firing properties of excitatory CA1 pyramidal neurons. These results suggest that a constitutive decrease in inhibitory interneuron excitability contributes to the seizure phenotype in the mouse model