The nervous system is vulnerable to perturbations during specific developmental periods. Insults during such susceptible time windows can have long-term consequences, including the development of neurological diseases such as epilepsy. Here we report that a pharmacological intervention timed during a vulnerable neonatal period of cortical development prevents pathology in a genetic epilepsy model. By using mice with dysfunctional Kv7 voltage-gated K(+) channels, which are mutated in human neonatal epilepsy syndromes, we demonstrate the safety and efficacy of the sodium-potassium-chloride cotransporter NKCC1 antagonist bumetanide, which was administered during the first two postnatal weeks. In Kv7 current-deficient mice, which normally display epilepsy, hyperactivity and stereotypies as adults, transient bumetanide treatment normalized neonatal in vivo cortical network and hippocampal neuronal activity, prevented structural damage in the hippocampus and restored wild-type adult behavioral phenotypes. Furthermore, bumetanide treatment did not adversely affect control mice. These results suggest that in individuals with disease susceptibility, timing prophylactically safe interventions to specific windows during development may prevent or arrest disease progression.

De novo mutations in voltage- and ligand-gated channels have been associated with an increasing number of cases of developmental and epileptic encephalopathies, which often fail to respond to classic antiseizure medications. Here, we examine two knock-in mouse models replicating de novo sequence variations in the HCN1 voltage-gated channel gene, p.G391D and p.M153I (Hcn1G380D/+ and Hcn1M142I/+ in mouse), associated with severe drug-resistant neonatal- and childhood-onset epilepsy, respectively. Heterozygous mice from both lines displayed spontaneous generalized tonic-clonic seizures. Animals replicating the p.G391D variant had an overall more severe phenotype, with pronounced alterations in the levels and distribution of HCN1 protein, including disrupted targeting to the axon terminals of basket cell interneurons. In line with clinical reports from patients with pathogenic HCN1 sequence variations, administration of the antiepileptic Na+ channel antagonists lamotrigine and phenytoin resulted in the paradoxical induction of seizures in both mouse lines, consistent with an effect to further impair inhibitory neuron function. We also show that these variants can render HCN1 channels unresponsive to classic antagonists, indicating the need to screen mutated channels to identify novel compounds with diverse mechanism of action. Our results underscore the necessity of tailoring effective therapies for specific channel gene variants, and how strongly validated animal models may provide an invaluable tool towards reaching this objective.

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De novo mutations in voltage- and ligand-gated channels have been associated with an increasing number of cases of developmental and epileptic encephalopathies, which often fail to respond to classic antiseizure medications. Here, we examine two knock-in mouse models replicating de novo mutations in the HCN1 voltage-gated channel gene, p.G391D and p.M153I (Hcn1G380D/+ and Hcn1M142I/+ in mouse), associated with severe drugresistant neonatal- and childhood-onset epilepsy, respectively. Heterozygous mice from both lines displayed spontaneous generalized tonic-clonic seizures. Hcn1G380D/+ animals had an overall more severe phenotype, with pronounced alterations in the levels and distribution of HCN1 protein, including disrupted targeting to the axon terminals of basket cell interneurons. In line with clinical reports from HCN1 patients, administration of the antiepileptic Na+ channel antagonists lamotrigine and phenytoin resulted in the paradoxical induction of seizures in both lines, consistent with an effect to further impair inhibitory neuron function. We also show that these variants can render HCN1 channels unresponsive to classic antagonists, indicating the need to screen mutated channels to identify novel compounds with diverse mechanism of action. Our results underscore the need to tailor effective therapies for specific channel gene variants, and how strongly validated animal models may provide an invaluable tool towards reaching this objective.

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terning during regeneration. One possible regulator is the H,K-ATPase, for which we have identified several potential candidate genes.Inactivating H,K-ATPase activity with the highly potent and specific inhibitor, SCH-28080 (SCH), is sufficient to block regeneration of visible anterior structures in cut fragments. Marker analysis confirms a lack of anterior development, but without any duplication of posterior patterning, suggesting H,K-ATPase activity plays an important role in the anterior patterning of new tissues. Physiologically, membrane-voltage-sensitive dyes reveal that SCH treatment hyperpolarizes regenerating fragments. Our investigations have shown treatment with SCH blocks anterior polarity only in posterior fragments, and that the presence of the cephalic ganglia (brain) in any fragment is sufficient to rescue head development. Interestingly, there must be sufficient length along the A/P axis of a fragment for SCH to be effective, because fragments with significantly small A/P distances do not respond to SCH and are indistinguishable from controls. These data suggest that non-local A/P polarity information may be transmitted through ion flows along the A/P axis, providing information about the original A/P patterning of existing tissues that in turn regulates blastema morphology. Neural stem cells (NSCs) of the Axolotl (Ambystoma mexicanum) have a unique ability to regenerate the fully functional spinal cord upon the tail amputation. The NSCs isolated from Axolotl spinal cord can be cultured as primary neurospheres in suspension. These neurospheres can be re-implanted into the tail spinal cord and are able to regenerate the fully functional spinal cord with the entire diversity of neuronal and glial derivatives. But the open question is: whether a single neural stem cell is able to regenerate a fully functional spinal cord? To address this issue, we have developed the axolotl NSC lines (from transgenic fluorescent animals) that are propagated and maintained under adherent conditions in the neural rosette formation state. Using various fluorescent cell lines, we were able to propagate axolotl NSCs clonally and re-implant them into the regenerating spinal cord. Some vertebrates such as salamanders and teleost fish have a remarkable capacity to regenerate injured body parts. Zebrafish can regenerate their tail fins in less than two weeks after amputation which is accompanied by growth of fin tissues such as bone, mesenchyme and epidermis. Fin regeneration includes the formation of a blastema, a mass of undifferentiated cells accumulating at the amputation plane, which are generally believed to represent the progenitors of the regenerating structures. However, while proliferation in the blastema has been thoroughly studied, little is known about proliferation patterns in the stump of the regenerating fin. Here we show that amputation of the tail fin leads to a strong increase of proliferation in the stump of the fin. Interestingly, bone forming cells (osteoblasts) in a region up to 1 mm (3 fin ray segmen...