RESUMEN
Genetically encoded voltage indicators (GEVIs) enable optical recording of electrical signals in the brain, providing subthreshold sensitivity and temporal resolution not possible with calcium indicators. However, one- and two-photon voltage imaging over prolonged periods with the same GEVI has not yet been demonstrated. Here, we report engineering of ASAP family GEVIs to enhance photostability by inversion of the fluorescence-voltage relationship. Two of the resulting GEVIs, ASAP4b and ASAP4e, respond to 100-mV depolarizations with ≥180% fluorescence increases, compared with the 50% fluorescence decrease of the parental ASAP3. With standard microscopy equipment, ASAP4e enables single-trial detection of spikes in mice over the course of minutes. Unlike GEVIs previously used for one-photon voltage recordings, ASAP4b and ASAP4e also perform well under two-photon illumination. By imaging voltage and calcium simultaneously, we show that ASAP4b and ASAP4e can identify place cells and detect voltage spikes with better temporal resolution than commonly used calcium indicators. Thus, ASAP4b and ASAP4e extend the capabilities of voltage imaging to standard one- and two-photon microscopes while improving the duration of voltage recordings.
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Encéfalo , Calcio , Animales , Ratones , Iluminación , Microscopía , FotonesRESUMEN
The development of the nervous system involves a coordinated succession of events including the migration of GABAergic (γ-aminobutyric-acid-releasing) neurons from ventral to dorsal forebrain and their integration into cortical circuits. However, these interregional interactions have not yet been modelled with human cells. Here we generate three-dimensional spheroids from human pluripotent stem cells that resemble either the dorsal or ventral forebrain and contain cortical glutamatergic or GABAergic neurons. These subdomain-specific forebrain spheroids can be assembled in vitro to recapitulate the saltatory migration of interneurons observed in the fetal forebrain. Using this system, we find that in Timothy syndrome-a neurodevelopmental disorder that is caused by mutations in the CaV1.2 calcium channel-interneurons display abnormal migratory saltations. We also show that after migration, interneurons functionally integrate with glutamatergic neurons to form a microphysiological system. We anticipate that this approach will be useful for studying neural development and disease, and for deriving spheroids that resemble other brain regions to assemble circuits in vitro.
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Neuronas/citología , Prosencéfalo/citología , Prosencéfalo/crecimiento & desarrollo , Esferoides Celulares/citología , Trastorno Autístico/genética , Trastorno Autístico/patología , Línea Celular , Movimiento Celular , Células Cultivadas , Femenino , Neuronas GABAérgicas/citología , Ácido Glutámico/metabolismo , Humanos , Interneuronas/citología , Interneuronas/patología , Síndrome de QT Prolongado/genética , Síndrome de QT Prolongado/patología , Masculino , Modelos Biológicos , Neurogénesis , Neuronas/patología , Células Madre Pluripotentes/citología , Prosencéfalo/anatomía & histología , Sinapsis/fisiología , Sindactilia/genética , Sindactilia/patologíaRESUMEN
The human cerebral cortex develops through an elaborate succession of cellular events that, when disrupted, can lead to neuropsychiatric disease. The ability to reprogram somatic cells into pluripotent cells that can be differentiated in vitro provides a unique opportunity to study normal and abnormal corticogenesis. Here, we present a simple and reproducible 3D culture approach for generating a laminated cerebral cortex-like structure, named human cortical spheroids (hCSs), from pluripotent stem cells. hCSs contain neurons from both deep and superficial cortical layers and map transcriptionally to in vivo fetal development. These neurons are electrophysiologically mature, display spontaneous activity, are surrounded by nonreactive astrocytes and form functional synapses. Experiments in acute hCS slices demonstrate that cortical neurons participate in network activity and produce complex synaptic events. These 3D cultures should allow a detailed interrogation of human cortical development, function and disease, and may prove a versatile platform for generating other neuronal and glial subtypes in vitro.
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Astrocitos/fisiología , Corteza Cerebral/fisiología , Células Madre Pluripotentes/citología , Astrocitos/citología , Células Cultivadas , Corteza Cerebral/citología , Humanos , Esferoides Celulares , Sinapsis/fisiologíaRESUMEN
SCN1A mutations are the main cause of the epilepsy disorders Dravet syndrome (DS) and genetic epilepsy with febrile seizures plus (GEFS+). Mutations that reduce the activity of the mouse Scn8a gene, in contrast, are found to confer seizure resistance and extend the lifespan of mouse models of DS and GEFS+. To investigate the mechanism by which reduced Scn8a expression confers seizure resistance, we induced interictal-like burst discharges in hippocampal slices of heterozygous Scn8a null mice (Scn8a(med/+)) with elevated extracellular potassium. Scn8a(med/+) mutants exhibited reduced epileptiform burst discharge activity after P20, indicating an age-dependent increased threshold for induction of epileptiform discharges. Scn8a deficiency also reduced the occurrence of burst discharges in a GEFS+ mouse model (Scn1a(R1648H/+)). There was no detectable change in the expression levels of Scn1a (Nav1.1) or Scn2a (Nav1.2) in the hippocampus of adult Scn8a(med/+) mutants. To determine whether the increased seizure resistance associated with reduced Scn8a expression was due to alterations that occurred during development, we examined the effect of deleting Scn8a in adult mice. Global Cre-mediated deletion of a heterozygous floxed Scn8a allele in adult mice was found to increase thresholds to chemically and electrically induced seizures. Finally, knockdown of Scn8a gene expression in the adult hippocampus via lentiviral Cre injection resulted in a reduction in the number of EEG-confirmed seizures following the administration of picrotoxin. Our results identify the hippocampus as an important structure in the mediation of Scn8a-dependent seizure protection and suggest that selective targeting of Scn8a activity might be efficacious in patients with epilepsy.
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Hipocampo/metabolismo , Canal de Sodio Activado por Voltaje NAV1.6/metabolismo , Convulsiones/metabolismo , Convulsiones/patología , Factores de Edad , Animales , Animales Recién Nacidos , Convulsivantes/toxicidad , Modelos Animales de Enfermedad , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Hipocampo/patología , Técnicas In Vitro , Ratones , Ratones Endogámicos C3H , Ratones Transgénicos , Mutación/genética , Canal de Sodio Activado por Voltaje NAV1.1/genética , Canal de Sodio Activado por Voltaje NAV1.6/genética , Neuronas/efectos de los fármacos , Neuronas/fisiología , Potasio/metabolismo , Desempeño Psicomotor , Tiempo de Reacción/genética , Tiempo de Reacción/fisiología , Convulsiones/etiología , Convulsiones/genéticaRESUMEN
The ability to optically stimulate and inhibit neurons has revolutionized neuroscience research. Here, we present a direct, potent, user-friendly chemical approach for optically silencing neurons. We have rendered saxitoxin (STX), a naturally occurring paralytic agent, transiently inert through chemical protection with a previously undisclosed nitrobenzyl-derived photocleavable group. Exposing the caged toxin, STX-bpc, to a brief (5 ms) pulse of light effects rapid release of a potent STX derivative and transient, spatially precise blockade of voltage-gated sodium channels (NaVs). We demonstrate the efficacy of STX-bpc for parametrically manipulating action potentials in mammalian neurons and brain slice. Additionally, we show the effectiveness of this reagent for silencing neural activity by dissecting sensory-evoked swimming in larval zebrafish. Photo-uncaging of STX-bpc is a straightforward method for non-invasive, reversible, spatiotemporally precise neural silencing without the need for genetic access, thus removing barriers for comparative research.
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Neuronas , Pez Cebra , Animales , Neuronas/metabolismo , Neuronas/efectos de los fármacos , Saxitoxina/farmacología , Saxitoxina/metabolismo , Saxitoxina/química , Potenciales de Acción/efectos de los fármacos , Humanos , Conducta Animal/efectos de los fármacos , Larva/efectos de los fármacos , Larva/metabolismo , Luz , RatonesRESUMEN
Fast electrical signaling in dendrites is central to neural computations that support adaptive behaviors. Conventional techniques lack temporal and spatial resolution and the ability to track underlying membrane potential dynamics present across the complex three-dimensional dendritic arbor in vivo. Here, we perform fast two-photon imaging of dendritic and somatic membrane potential dynamics in single pyramidal cells in the CA1 region of the mouse hippocampus during awake behavior. We study the dynamics of subthreshold membrane potential and suprathreshold dendritic events throughout the dendritic arbor in vivo by combining voltage imaging with simultaneous local field potential recording, post hoc morphological reconstruction, and a spatial navigation task. We systematically quantify the modulation of local event rates by locomotion in distinct dendritic regions, report an advancing gradient of dendritic theta phase along the basal-tuft axis, and describe a predominant hyperpolarization of the dendritic arbor during sharp-wave ripples. Finally, we find that spatial tuning of dendritic representations dynamically reorganizes following place field formation. Our data reveal how the organization of electrical signaling in dendrites maps onto the anatomy of the dendritic tree across behavior, oscillatory network, and functional cell states.
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Región CA1 Hipocampal , Dendritas , Células Piramidales , Animales , Dendritas/fisiología , Dendritas/metabolismo , Células Piramidales/fisiología , Células Piramidales/metabolismo , Ratones , Región CA1 Hipocampal/fisiología , Región CA1 Hipocampal/citología , Potenciales de la Membrana/fisiología , Masculino , Ratones Endogámicos C57BL , Hipocampo/fisiología , Hipocampo/citología , Navegación Espacial/fisiología , Locomoción/fisiologíaRESUMEN
Fast electrical signaling in dendrites is central to neural computations that support adaptive behaviors. Conventional techniques lack temporal and spatial resolution and the ability to track underlying membrane potential dynamics present across the complex three-dimensional dendritic arbor in vivo. Here, we perform fast two-photon imaging of dendritic and somatic membrane potential dynamics in single pyramidal cells in the CA1 region of the mouse hippocampus during awake behavior. We study the dynamics of subthreshold membrane potential and suprathreshold dendritic events throughout the dendritic arbor in vivo by combining voltage imaging with simultaneous local field potential recording, post hoc morphological reconstruction, and a spatial navigation task. We systematically quantify the modulation of local event rates by locomotion in distinct dendritic regions and report an advancing gradient of dendritic theta phase along the basal-tuft axis, then describe a predominant hyperpolarization of the dendritic arbor during sharp-wave ripples. Finally, we find spatial tuning of dendritic representations dynamically reorganizes following place field formation. Our data reveal how the organization of electrical signaling in dendrites maps onto the anatomy of the dendritic tree across behavior, oscillatory network, and functional cell states.
RESUMEN
Voltage-gated sodium channels (VGSCs) are essential for the generation and propagation of action potentials in electrically excitable cells. Dominant mutations in SCN1A, which encodes the Nav1.1 VGSC α-subunit, underlie several forms of epilepsy, including Dravet syndrome (DS) and genetic epilepsy with febrile seizures plus (GEFS+). Electrophysiological analyses of DS and GEFS+ mouse models have led to the hypothesis that SCN1A mutations reduce the excitability of inhibitory cortical and hippocampal interneurons. To more directly examine the relative contribution of inhibitory interneurons and excitatory pyramidal cells to SCN1A-derived epilepsy, we first compared the expression of Nav1.1 in inhibitory parvalbumin (PV) interneurons and excitatory neurons from P22 mice using fluorescent immunohistochemistry. In the hippocampus and neocortex, 69% of Nav1.1 immunoreactive neurons were also positive for PV. In contrast, 13% and 5% of Nav1.1 positive cells in the hippocampus and neocortex, respectively, were found to co-localize with excitatory cells identified by CaMK2α immunoreactivity. Next, we reduced the expression of Scn1a in either a subset of interneurons (mainly PV interneurons) or excitatory cells by crossing mice heterozygous for a floxed Scn1a allele to either the Ppp1r2-Cre or EMX1-Cre transgenic lines, respectively. The inactivation of one Scn1a allele in interneurons of the neocortex and hippocampus was sufficient to reduce thresholds to flurothyl- and hyperthermia-induced seizures, whereas thresholds were unaltered following inactivation in excitatory cells. Reduced interneuron Scn1a expression also resulted in the generation of spontaneous seizures. These findings provide direct evidence for an important role of PV interneurons in the pathogenesis of Scn1a-derived epilepsies.
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Interneuronas/fisiología , Canal de Sodio Activado por Voltaje NAV1.1/deficiencia , Parvalbúminas/metabolismo , Convulsiones Febriles/fisiopatología , Convulsiones/fisiopatología , Animales , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Susceptibilidad a Enfermedades/metabolismo , Fiebre , Flurotilo , Hipocampo/fisiopatología , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Transgénicos , Canal de Sodio Activado por Voltaje NAV1.1/genética , Neocórtex/fisiopatología , Inhibición Neural/fisiología , Células Piramidales/fisiopatologíaRESUMEN
PURPOSE: Mutations in the voltage-gated sodium channel (VGSC) gene SCN1A are responsible for a number of epilepsy disorders, including genetic epilepsy with febrile seizures plus (GEFS+) and Dravet syndrome. In addition to seizures, patients with SCN1A mutations often experience sleep abnormalities, suggesting that SCN1A may also play a role in the neuronal pathways involved in the regulation of sleep. However, to date, a role for SCN1A in the regulation of sleep architecture has not been directly examined. To fill this gap, we tested the hypothesis that SCN1A contributes to the regulation of sleep architecture, and by extension, that SCN1A dysfunction contributes to the sleep abnormalities observed in patients with SCN1A mutations. METHODS: Using immunohistochemistry we first examined the expression of mouse Scn1a in regions of the mouse brain that are known to be involved in seizure generation and sleep regulation. Next, we performed detailed analysis of sleep and wake electroencephalography (EEG) patterns during 48 continuous hours of baseline recordings in a knock-in mouse line that expresses the human SCN1A GEFS+ mutation R1648H (RH mutants). We also characterized the sleep-wake pattern following 6 h of sleep deprivation. KEY FINDINGS: Immunohistochemistry revealed broad expression of Scn1a in the neocortex, hippocampus, hypothalamus, thalamic reticular nuclei, dorsal raphe nuclei, pedunculopontine, and laterodorsal tegmental nuclei. Co-localization between Scn1a immunoreactivity and critical cell types within these regions was also observed. EEG analysis under baseline conditions revealed increased wakefulness and reduced non-rapid eye movement (NREM) and rapid eye movement (REM) sleep amounts during the dark phase in the RH mutants, suggesting a sleep deficit. Nevertheless, the mutants exhibited levels of NREM and REM sleep that were generally similar to wild-type littermates during the recovery period following 6 h of sleep deprivation. SIGNIFICANCE: These results establish a direct role for SCN1A in the regulation of sleep and suggest that patients with SCN1A mutations may experience chronic alterations in sleep, potentially leading to negative outcomes over time. In addition, the expression of Scn1a in specific cell types/brain regions that are known to play critical roles in seizure generation and sleep now provides a mechanistic basis for the clinical features (seizures and sleep abnormalities) associated with human SCN1A mutations.
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Epilepsia/genética , Epilepsia/fisiopatología , Canal de Sodio Activado por Voltaje NAV1.1/genética , Convulsiones Febriles/genética , Convulsiones Febriles/fisiopatología , Sueño/genética , Sueño/fisiología , Análisis de Varianza , Animales , Ritmo Delta , Electroencefalografía , Electromiografía , Genotipo , Hipocampo/metabolismo , Hipocampo/fisiopatología , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Mutación/fisiología , Canal de Sodio Activado por Voltaje NAV1.1/biosíntesis , Privación de Sueño/fisiopatología , Sueño REM/fisiología , Vigilia/fisiologíaRESUMEN
De novo mutations in GNB1, encoding the Gß1 subunit of G proteins, cause a neurodevelopmental disorder with global developmental delay and epilepsy, GNB1 encephalopathy. Here, we show that mice carrying a pathogenic mutation, K78R, recapitulate aspects of the disorder, including developmental delay and generalized seizures. Cultured mutant cortical neurons also display aberrant bursting activity on multi-electrode arrays. Strikingly, the antiepileptic drug ethosuximide (ETX) restores normal neuronal network behavior in vitro and suppresses spike-and-wave discharges (SWD) in vivo. ETX is a known blocker of T-type voltage-gated Ca2+ channels and G protein-coupled potassium (GIRK) channels. Accordingly, we present evidence that K78R results in a gain-of-function (GoF) effect by increasing the activation of GIRK channels in cultured neurons and a heterologous model (Xenopus oocytes)-an effect we show can be potently inhibited by ETX. This work implicates a GoF mechanism for GIRK channels in epilepsy, identifies a new mechanism of action for ETX in preventing seizures, and establishes this mouse model as a pre-clinical tool for translational research with predicative value for GNB1 encephalopathy.
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Understanding the neural mechanisms underlying sleep state transitions is a fundamental goal of neurobiology and important for the development of new treatments for insomnia and other sleep disorders. Yet, brain circuits controlling this process remain poorly understood. Here we identify a population of sleep-active glutamatergic neurons in the ventrolateral medulla (VLM) that project to the preoptic area (POA), a prominent sleep-promoting region, in mice. Microendoscopic calcium imaging demonstrate that these VLM glutamatergic neurons display increased activity during the transitions from wakefulness to Non-Rapid Eye Movement (NREM) sleep. Chemogenetic silencing of POA-projecting VLM neurons suppresses NREM sleep, whereas chemogenetic activation of these neurons promotes NREM sleep. Moreover, we show that optogenetic activation of VLM glutamatergic neurons or their projections in the POA initiates NREM sleep in awake mice. Together, our findings uncover an excitatory brainstem-hypothalamic circuit that controls the wake-sleep transitions.
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Área Preóptica , Vigilia , Animales , Bulbo Raquídeo , Ratones , Neuronas/fisiología , Área Preóptica/fisiología , Sueño/fisiología , Vigilia/fisiologíaRESUMEN
Here we report the pharmacologic blockade of voltage-gated sodium ion channels (NaVs) by a synthetic saxitoxin derivative affixed to a photocleavable protecting group. We demonstrate that a functionalized saxitoxin (STX-eac) enables exquisite spatiotemporal control of NaVs to interrupt action potentials in dissociated neurons and nerve fiber bundles. The photo-uncaged inhibitor (STX-ea) is a nanomolar potent, reversible binder of NaVs. We use STX-eac to reveal differential susceptibility of myelinated and unmyelinated axons in the corpus callosum to NaV-dependent alterations in action potential propagation, with unmyelinated axons preferentially showing reduced action potential fidelity under conditions of partial NaV block. These results validate STX-eac as a high precision tool for robust photocontrol of neuronal excitability and action potential generation.
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Potenciales de Acción/efectos de los fármacos , Canal de Sodio Activado por Voltaje NAV1.2/metabolismo , Saxitoxina/farmacología , Bloqueadores del Canal de Sodio Activado por Voltaje/farmacología , Animales , Axones/efectos de los fármacos , Axones/metabolismo , Células CHO , Células Cultivadas , Cuerpo Calloso/citología , Cuerpo Calloso/efectos de los fármacos , Cuerpo Calloso/metabolismo , Cricetulus , Embrión de Mamíferos , Femenino , Hipocampo/citología , Masculino , Ratones , Canal de Sodio Activado por Voltaje NAV1.2/genética , Técnicas de Placa-Clamp , Cultivo Primario de Células , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saxitoxina/análogos & derivados , Saxitoxina/efectos de la radiación , Análisis de la Célula Individual , Análisis Espacio-Temporal , Rayos Ultravioleta , Bloqueadores del Canal de Sodio Activado por Voltaje/efectos de la radiaciónRESUMEN
Human stem-cell-derived models provide the promise of accelerating our understanding of brain disorders, but not knowing whether they possess the ability to mature beyond mid- to late-fetal stages potentially limits their utility. We leveraged a directed differentiation protocol to comprehensively assess maturation in vitro. Based on genome-wide analysis of the epigenetic clock and transcriptomics, as well as RNA editing, we observe that three-dimensional human cortical organoids reach postnatal stages between 250 and 300 days, a timeline paralleling in vivo development. We demonstrate the presence of several known developmental milestones, including switches in the histone deacetylase complex and NMDA receptor subunits, which we confirm at the protein and physiological levels. These results suggest that important components of an intrinsic in vivo developmental program persist in vitro. We further map neurodevelopmental and neurodegenerative disease risk genes onto in vitro gene expression trajectories to provide a resource and webtool (Gene Expression in Cortical Organoids, GECO) to guide disease modeling.
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Diferenciación Celular/fisiología , Metilación de ADN/fisiología , Células Madre Pluripotentes Inducidas/citología , Organoides/citología , Redes Reguladoras de Genes , Humanos , Técnicas In Vitro , Enfermedades Neurodegenerativas/genéticaRESUMEN
Tactile co-activation, i.e., synchronous stimulation of a region of skin, has been reported to improve tactile spatial acuity and expand the corresponding somatosensory cortical representation. The current study aimed to clarify the nature of the changes resulting from tactile co-activation, using three measures of tactile sensitivity obtained with controlled mechanical stimulation. One was the grating orientation (GR/OR) discrimination task, where acuity is indexed by the threshold groove width required for 75% correct discrimination between two orthogonal orientations of a grating on the fingerpad. Since this task may be susceptible to intensity cues due to tactile anisotropy, another acuity measure, the 3-dot task, was also used. In this task, the acuity threshold corresponds to 75% correct discrimination of the direction of offset of the central dot in a 3-dot array. In Experiment 1, co-activation failed to induce significant improvement in acuity with either of these measures. Experiment 2 employed both the GR/OR task, and a third measure based on discriminating a grooved from a smooth surface (SM/GV). While the former task demands detailed spatial resolution, the latter requires only that spatial modulation in the afferent population be detected. This experiment also included a control group. GR/OR performance did not significantly improve for either the control or experimental groups. There was, however, a significant improvement in SM/GV performance following co-activation for the experimental but not the control group. These findings indicate that the SM/GV task may be better suited than the GR/OR or 3-dot tasks for measuring changes in tactile sensitivity following co-activation.
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Percepción Espacial , Percepción del Tacto , Tacto , Análisis de Varianza , Anisotropía , Femenino , Dedos , Humanos , Juicio , Masculino , Estimulación Física , Psicofísica , Umbral Sensorial , Análisis y Desempeño de TareasRESUMEN
We previously found that genetic mutants with reduced expression or activity of Scn8a are resistant to induced seizures and that co-segregation of a mutant Scn8a allele can increase survival and seizure resistance of Scn1a mutant mice. In contrast, Scn8a expression is increased in the hippocampus following status epilepticus and amygdala kindling. These findings point to Scn8a as a promising therapeutic target for epilepsy and raise the possibility that aberrant overexpression of Scn8a in limbic structures may contribute to some epilepsies, including temporal lobe epilepsy. Using a small-hairpin-interfering RNA directed against the Scn8a gene, we selectively reduced Scn8a expression in the hippocampus of the intrahippocampal kainic acid (KA) mouse model of mesial temporal lobe epilepsy. We found that Scn8a knockdown prevented the development of spontaneous seizures in 9/10 mice, ameliorated KA-induced hyperactivity, and reduced reactive gliosis. These results support the potential of selectively targeting Scn8a for the treatment of refractory epilepsy.
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Epilepsia del Lóbulo Temporal/genética , Epilepsia del Lóbulo Temporal/metabolismo , Canal de Sodio Activado por Voltaje NAV1.6/genética , Canal de Sodio Activado por Voltaje NAV1.6/metabolismo , Convulsiones/genética , Convulsiones/metabolismo , Animales , Modelos Animales de Enfermedad , Electroencefalografía , Epilepsia del Lóbulo Temporal/diagnóstico , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Predisposición Genética a la Enfermedad , Hipocampo/metabolismo , Inmunohistoquímica , Masculino , Ratones , ARN Interferente Pequeño/genética , Convulsiones/diagnósticoRESUMEN
Voltage-gated sodium channel (VGSC) mutations cause severe epilepsies marked by intermittent, pathological hypersynchronous brain states. Here we present two mechanisms that help to explain how mutations in one VGSC gene, Scn8a, contribute to two distinct seizure phenotypes: (1) hypoexcitation of cortical circuits leading to convulsive seizure resistance, and (2) hyperexcitation of thalamocortical circuits leading to non-convulsive absence epilepsy. We found that loss of Scn8a leads to altered RT cell intrinsic excitability and a failure in recurrent RT synaptic inhibition. We propose that these deficits cooperate to enhance thalamocortical network synchrony and generate pathological oscillations. To our knowledge, this finding is the first clear demonstration of a pathological state tied to disruption of the RT-RT synapse. Our observation that loss of a single gene in the thalamus of an adult wild-type animal is sufficient to cause spike-wave discharges is striking and represents an example of absence epilepsy of thalamic origin.
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Canal de Sodio Activado por Voltaje NAV1.6/genética , Canal de Sodio Activado por Voltaje NAV1.6/metabolismo , Red Nerviosa/metabolismo , Sinapsis/metabolismo , Tálamo/metabolismo , Animales , Modelos Animales de Enfermedad , Electroencefalografía/métodos , Epilepsia Tipo Ausencia/genética , Epilepsia Tipo Ausencia/metabolismo , Ratones , Fenotipo , Convulsiones/genética , Convulsiones/metabolismoRESUMEN
Understanding the role of SCN8A in epilepsy and behavior is critical in light of recently identified human SCN8A epilepsy mutations. We have previously demonstrated that Scn8a(med) and Scn8a(med-jo) mice carrying mutations in the Scn8a gene display increased resistance to flurothyl and kainic acid-induced seizures; however, they also exhibit spontaneous absence seizures. To further investigate the relationship between altered SCN8A function and epilepsy, we introduced the SCN1A-R1648H mutation, identified in a family with generalized epilepsy with febrile seizures plus (GEFS+), into the corresponding position (R1627H) of the mouse Scn8a gene. Heterozygous R1627H mice exhibited increased resistance to some forms of pharmacologically and electrically induced seizures and the mutant Scn8a allele ameliorated the phenotype of Scn1a-R1648H mutants. Hippocampal slices from heterozygous R1627H mice displayed decreased bursting behavior compared to wild-type littermates. Paradoxically, at the homozygous level, R1627H mice did not display increased seizure resistance and were susceptible to audiogenic seizures. We furthermore observed increased hippocampal pyramidal cell excitability in heterozygous and homozygous Scn8a-R1627H mutants, and decreased interneuron excitability in heterozygous Scn8a-R1627H mutants. These results expand the phenotypes associated with disruption of the Scn8a gene and demonstrate that an Scn8a mutation can both confer seizure protection and increase seizure susceptibility.
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Hipocampo/fisiopatología , Interneuronas/metabolismo , Mutación , Canal de Sodio Activado por Voltaje NAV1.6/genética , Células Piramidales/metabolismo , Convulsiones/genética , Estimulación Acústica , Animales , Tronco Encefálico/metabolismo , Tronco Encefálico/fisiopatología , Susceptibilidad a Enfermedades , Hipocampo/metabolismo , Masculino , Ratones , Ratones Noqueados , Canal de Sodio Activado por Voltaje NAV1.6/metabolismo , Convulsiones/metabolismo , Convulsiones/fisiopatologíaRESUMEN
RATIONALE: High doses of cocaine can elicit seizures in humans and in laboratory animals. Several mechanisms have been proposed for the induction of seizures by cocaine, including enhanced monoaminergic signaling, blockade of ion channels, and alterations in GABA and glutamate transmission. Mutations in the SCN1A gene, which encodes the central nervous system (CNS) voltage-gated sodium channel (VGSC) Nav1.1, are responsible for several human epilepsy disorders including Dravet syndrome and genetic (generalized) epilepsy with febrile seizures plus (GEFS+). Mice heterozygous for the R1648H GEFS+ mutation (RH mice) exhibit reduced interneuron excitability, spontaneous seizures, and lower thresholds to flurothyl- and hyperthermia-induced seizures. However, it is unknown whether impaired CNS VGSC function or a genetic predisposition to epilepsy increases susceptibility to cocaine-induced seizures. OBJECTIVES: Our primary goal was to determine whether Scn1a dysfunction caused by the RH mutation alters sensitivity to cocaine-induced behavioral and electrographic (EEG) seizures. We also tested novelty- and cocaine-induced locomotor activity and assessed the expression of Nav1.1 in midbrain dopaminergic neurons. RESULTS: We found that RH mice had a profound increase in cocaine-induced behavioral seizure susceptibility compared to wild-type (WT) controls, which was confirmed with cortical EEG recordings. By contrast, although the RH mice were hyperactive in novel environments, cocaine-induced locomotor activity was comparable between the mutants and WT littermates. Finally, immunofluorescence experiments revealed a lack of Nav1.1 immunoreactivity in dopaminergic neurons. CONCLUSION: These data indicate that a disease-causing CNS VGSC mutation confers susceptibility to the proconvulsant, but not motoric, effects of cocaine.