RESUMEN
Changes in the shape and size of the dendritic spines are critical for synaptic transmission. These morphological changes depend on dynamic assembly of the actin cytoskeleton and occur differently in various types of neurons. However, how the actin dynamics are regulated in a neuronal cell type-specific manner remains largely unknown. We show that Fhod3, a member of the formin family proteins that mediate F-actin assembly, controls the dendritic spine morphogenesis of specific subpopulations of cerebrocortical pyramidal neurons. Fhod3 is expressed specifically in excitatory pyramidal neurons within layers II/III and V of restricted areas of the mouse cerebral cortex. Immunohistochemical and biochemical analyses revealed the accumulation of Fhod3 in postsynaptic spines. Although targeted deletion of Fhod3 in the brain did not lead to any defects in the gross or histological appearance of the brain, the dendritic spines in pyramidal neurons within presumptive Fhod3-positive areas were morphologically abnormal. In primary cultures prepared from the Fhod3-depleted cortex, defects in spine morphology were only detected in Fhod3 promoter-active cells, a small population of pyramidal neurons, and not in Fhod3 promoter-negative pyramidal neurons. Thus, Fhod3 plays a crucial role in dendritic spine morphogenesis only in a specific population of pyramidal neurons in a cell type-specific manner.
Asunto(s)
Corteza Cerebral/metabolismo , Espinas Dendríticas/metabolismo , Forminas/biosíntesis , Células Piramidales/metabolismo , Animales , Células Cultivadas , Corteza Cerebral/ultraestructura , Espinas Dendríticas/genética , Espinas Dendríticas/ultraestructura , Forminas/genética , Células HEK293 , Humanos , Ratones , Ratones Transgénicos , Células Piramidales/ultraestructuraRESUMEN
Water adsorption onto a material surface is known to change macroscopic surface properties such as wettability and friction coefficient. While the role of the adsorbed water has been discussed for a long time, the interfacial structure of the adsorbed water has not been fully recognized in many cases. In this study, the hydration structure of water adsorbed on a vapor/silica interface at room temperature was studied via heterodyne-detected vibrational sum-frequency generation spectroscopy. The vibrational spectra of the interfacial molecules obtained here were different from those estimated via conventional sum-frequency generation spectroscopy. Interestingly, our results suggest that, at low humidity, the adsorbed water on silica forms nanodroplets instead of a uniform film. Because no silanol group was found to be hydrogen-bonding free, it was concluded that water molecules gather around the silanol group to form strongly hydrogen-bonded droplets. At high humidity, while the adsorbed water partially behaves like a bulk liquid, deprotonation of the silanol was not observed, unlike the case of silica surfaces in contact with bulk liquid water.
RESUMEN
OBJECTIVE: Mutations of the KCNQ2 gene, which encodes the Kv 7.2 subunit of voltage-gated M-type potassium channels, have been associated with epilepsy in the neonatal period. This developmental stage is unique in that the neurotransmitter gamma aminobutyric acid (GABA), which is inhibitory in adults, triggers excitatory action due to a reversed chloride gradient. METHODS: To examine whether KCNQ2-related neuronal hyperexcitability involves neonatally excitatory GABA, we examined 1-week-old knockin mice expressing the Kv 7.2 variant p.Tyr284Cys (Y284C). RESULTS: Brain slice electrophysiology revealed elevated CA1 hippocampal GABAergic interneuron activity with respect to presynaptic firing and postsynaptic current frequency. Blockade with the GABAA receptor antagonist bicuculline decreased ictal-like bursting in brain slices with lowered divalent ion concentration, which is consistent with GABA mediating an excitatory function that contributes to the hyperexcitability observed in mutant animals. SIGNIFICANCE: We conclude that excitatory GABA contributes to the phenotype in these animals, which raises the question of whether this special type of neurotransmission has broader importance in neonatal epilepsy than is currently recognized.
Asunto(s)
Epilepsia/genética , Epilepsia/metabolismo , Canal de Potasio KCNQ2/genética , Mutación/genética , Proteínas del Tejido Nervioso/genética , Transmisión Sináptica/genética , Ácido gamma-Aminobutírico/metabolismo , Animales , Animales Recién Nacidos , Biofisica , Región CA1 Hipocampal/citología , Región CA1 Hipocampal/metabolismo , Modelos Animales de Enfermedad , Estimulación Eléctrica , GABAérgicos/farmacología , Técnicas In Vitro , Potenciales Postsinápticos Inhibidores/efectos de los fármacos , Potenciales Postsinápticos Inhibidores/genética , Interneuronas/efectos de los fármacos , Interneuronas/fisiología , Magnesio/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Técnicas de Placa-Clamp , Transmisión Sináptica/efectos de los fármacos , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
The hetero-tetrameric voltage-gated potassium channel Kv7.2/Kv7.3, which is encoded by KCNQ2 and KCNQ3, plays an important role in limiting network excitability in the neonatal brain. Kv7.2/Kv7.3 dysfunction resulting from KCNQ2 mutations predominantly causes self-limited or benign epilepsy in neonates, but also causes early onset epileptic encephalopathy. Retigabine (RTG), a Kv7.2/ Kv7.3-channel opener, seems to be a rational antiepileptic drug for epilepsies caused by KCNQ2 mutations. We therefore evaluated the effects of RTG on seizures in two strains of knock-in mice harboring different Kcnq2 mutations, in comparison to the effects of phenobarbital (PB), which is the first-line antiepileptic drug for seizures in neonates. The subjects were heterozygous knock-in mice (Kcnq2Y284C/+ and Kcnq2A306T/+) bearing the Y284C or A306T Kcnq2 mutation, respectively, and their wild-type (WT) littermates, at 63-100 days of age. Seizures induced by intraperitoneal injection of kainic acid (KA, 12mg/kg) were recorded using a video-electroencephalography (EEG) monitoring system. Effects of RTG on KA-induced seizures of both strains of knock-in mice were assessed using seizure scores from a modified Racine's scale and compared with those of PB. The number and total duration of spike bursts on EEG and behaviors monitored by video recording were also used to evaluate the effects of RTG and PB. Both Kcnq2Y284C/+ and Kcnq2A306T/+ mice showed significantly more KA-induced seizures than WT mice. RTG significantly attenuated KA-induced seizure activities in both Kcnq2Y284C/+ and Kcnq2A306T/+ mice, and more markedly than PB. This is the first reported evidence of RTG ameliorating KA-induced seizures in knock-in mice bearing mutations of Kcnq2, with more marked effects than those observed with PB. RTG or other Kv7.2-channel openers may be considered as first-line antiepileptic treatments for epilepsies resulting from KCNQ2 mutations.
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Carbamatos/farmacología , Activación del Canal Iónico/efectos de los fármacos , Canal de Potasio KCNQ2/genética , Canal de Potasio KCNQ2/metabolismo , Canal de Potasio KCNQ3/metabolismo , Mutación , Fenilendiaminas/farmacología , Convulsiones/tratamiento farmacológico , Animales , Carbamatos/uso terapéutico , Técnicas de Sustitución del Gen , Humanos , Canal de Potasio KCNQ2/química , Ácido Kaínico/farmacología , Ratones , Modelos Moleculares , Fenilendiaminas/uso terapéutico , Estructura Terciaria de Proteína , Convulsiones/inducido químicamente , Convulsiones/genética , Convulsiones/metabolismoRESUMEN
Knock-in mouse models have contributed tremendously to our understanding of human disorders. However, generation of knock-in animals requires a significant investment of time and effort. We addressed this problem by developing a novel knock-in system that circumvents several traditional challenges by establishing stem cells with acceptor elements enveloping a particular genomic target. Once established, these acceptor embryonic stem (ES) cells are efficient at directionally incorporating mutated target DNA using modified Cre/lox technology. This is advantageous, because knock-ins are not restricted to one a priori selected variation. Rather, it is possible to generate several mutant animal lines harboring desired alterations in the targeted area. Acceptor ES cell generation is the rate-limiting step, lasting approximately 2 months. Subsequent manipulations toward animal production require an additional 8 weeks, but this delimits the full period from conception of the genetic alteration to its animal incorporation. We call this system a "kick-in" to emphasize its unique characteristics of speed and convenience. To demonstrate the functionality of the kick-in methodology, we generated two mouse lines with separate mutant versions of the voltage-dependent potassium channel Kv7.2 (Kcnq2): p.Tyr284Cys (Y284C) and p.Ala306Thr (A306T); both variations have been associated with benign familial neonatal epilepsy. Adult mice homozygous for Y284C, heretofore unexamined in animals, presented with spontaneous seizures, whereas A306T homozygotes died early. Heterozygous mice of both lines showed increased sensitivity to pentylenetetrazole, possibly due to a reduction in M-current in CA1 hippocampal pyramidal neurons. Our observations for the A306T animals match those obtained with traditional knock-in technology, demonstrating that the kick-in system can readily generate mice bearing various mutations, making it a suitable feeder technology toward streamlined phenotyping.
Asunto(s)
Técnicas de Sustitución del Gen/métodos , Canal de Potasio KCNQ2/genética , Animales , Conducta Animal , Células Madre Embrionarias/metabolismo , Epilepsia Benigna Neonatal/inducido químicamente , Epilepsia Benigna Neonatal/genética , Epilepsia Benigna Neonatal/metabolismo , Femenino , Regulación de la Expresión Génica , Humanos , Masculino , Ratones , Mutación , Pentilenotetrazol/efectos adversos , Embarazo , Proteínas Proto-Oncogénicas c-fos/metabolismo , Factores de TiempoRESUMEN
BACKGROUND: Dravet syndrome is a devastating infantile-onset epilepsy syndrome with cognitive deficits and autistic traits caused by genetic alterations in SCN1A gene encoding the α-subunit of the voltage-gated sodium channel Na(v)1.1. Disease modeling using patient-derived induced pluripotent stem cells (iPSCs) can be a powerful tool to reproduce this syndrome's human pathology. However, no such effort has been reported to date. We here report a cellular model for DS that utilizes patient-derived iPSCs. RESULTS: We generated iPSCs from a Dravet syndrome patient with a c.4933C>T substitution in SCN1A, which is predicted to result in truncation in the fourth homologous domain of the protein (p.R1645*). Neurons derived from these iPSCs were primarily GABAergic (>50%), although glutamatergic neurons were observed as a minor population (<1%). Current-clamp analyses revealed significant impairment in action potential generation when strong depolarizing currents were injected. CONCLUSIONS: Our results indicate a functional decline in Dravet neurons, especially in the GABAergic subtype, which supports previous findings in murine disease models, where loss-of-function in GABAergic inhibition appears to be a main driver in epileptogenesis. Our data indicate that patient-derived iPSCs may serve as a new and powerful research platform for genetic disorders, including the epilepsies.
Asunto(s)
Epilepsias Mioclónicas/patología , Células Madre Pluripotentes Inducidas/patología , Modelos Biológicos , Potenciales de Acción , Adulto , Diferenciación Celular , Epilepsias Mioclónicas/fisiopatología , Femenino , Genes Reporteros/genética , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Lactante , Lentivirus/metabolismo , Canal de Sodio Activado por Voltaje NAV1.1/metabolismo , Neuronas/metabolismo , Neuronas/patología , Parvalbúminas/genética , Parvalbúminas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Adulto JovenRESUMEN
Both down-regulation of glutamate decarboxylase 67 (GAD67) and maternal exposure to severe stress during pregnancy can increase the risk of schizophrenia and related psychotic disorders in the offspring. To investigate a gene-environment interaction, we performed the restraint-and-light stress to pregnant GAD67-GFP knock-in (GAD67(+/GFP)) and wild-type (GAD67(+/+)) mice three times a day for 45 min per session during gestational day (G) 15.0-17.5. The stress hormone (corticosterone) level of pregnant GAD67(+/GFP) mice (the overall GABA content is reduced because of the destruction of one allele of the endogenous GAD67 gene) was higher than that of GAD67(+/+), even without stress. The fetal body weights (GAD67(+/+)) in the GAD67(+/GFP) mothers were lower than those in the GAD67(+/+) mothers. GAD67(+/GFP) fetuses exhibited higher corticosterone (CORT) levels than GAD67(+/+) fetuses, even in non-stressed GAD67(+/+) mothers. Fetal body weight-decreases and CORT-increases by maternal stress (GAD67(+/+) mother) were significantly more in the GAD67(+/GFP) fetuses than the GAD67(+/+) fetuses. These results indicate that a GAD67 heterozygous deletion itself enhances vulnerability by many aspects, e.g., maternal stress, maternity, and being in utero. Thus, an abnormality in GAD67 could interact with environmental risk factors of psychiatric disorders, including schizophrenia.
Asunto(s)
Glutamato Descarboxilasa/genética , Pérdida de Heterocigocidad , Efectos Tardíos de la Exposición Prenatal , Esquizofrenia/etiología , Estrés Psicológico/complicaciones , Animales , Corticosterona/sangre , Femenino , Feto , Técnicas de Sustitución del Gen , Glutamato Descarboxilasa/deficiencia , Ratones , Ratones Endogámicos C57BL , Embarazo , Factores de Riesgo , Estrés Psicológico/fisiopatologíaRESUMEN
Cortical GABAergic interneurons originate from ganglionic eminences and tangentially migrate into the cortical plate at early developmental stages. To elucidate the characteristics of this migration of GABAergic interneurons in living animals, we established an experimental design specialized for in vivo time-lapse imaging of the neocortex of neonate mice with two-photon laser-scanning microscopy. In vesicular GABA/glycine transporter (VGAT)-Venus transgenic mice from birth (P0) through P3, we observed multidirectional tangential migration of genetically-defined GABAergic interneurons in the neocortical marginal zone. The properties of this migration, such as the motility rate (distance/hr), the direction moved, and the proportion of migrating neurons to stationary neurons, did not change through P0 to P3, although the density of GABAergic neurons at the marginal zone decreased with age. Thus, the characteristics of the tangential motility of individual GABAergic neurons remained constant in development. Pharmacological block of GABA(A) receptors and of the Naâº-Kâº-Clâ» cotransporters, and chelating intracellular Ca²âº, all significantly reduced the motility rate in vivo. The motility rate and GABA content within the cortex of neonatal VGAT-Venus transgenic mice were significantly greater than those of GAD67-GFP knock-in mice, suggesting that extracellular GABA concentration could facilitate the multidirectional tangential migration. Indeed, diazepam applied to GAD67-GFP mice increased the motility rate substantially. In an in vitro neocortical slice preparation, we confirmed that GABA induced a NKCC sensitive depolarization of GABAergic interneurons in VGAT-Venus mice at P0-P3. Thus, activation of GABA(A)R by ambient GABA depolarizes GABAergic interneurons, leading to an acceleration of their multidirectional motility in vivo.