Dravet syndrome caused by SCN1A variants is a severe developmental epileptic encephalopathy (DEE) characterized by pharmaco-resistant epileptic seizures and progressive neurodevelopmental decline with cognitive impairment and autism-spectrum-traits. Numerous preceding studies indicate that the initial pathophysiology due to impaired Na V 1.1 function mainly derives from reduced interneuron firing leading to a network hyperexcitability (Bender et al. 2012). However, little is known how epileptogenesis and generally disease pathogenesis progress from the inborn molecular defect to infantile seizure onset. We address this question in a Dravet mouse model by comprehensive single-cell RNA sequencing and selected downstream analysis via single-cell electrophysiology, histology, live cell imaging and electron microscopy. Our data reveal a continuum of early primary (preseizure) and secondary (post-seizure onset) transcriptomic changes in various cell populations in the hippocampus. Focusing on cornu ammonis , we find a number of transcriptional pathways that are dysregulated including synaptic transmembrane adhesion molecules of the neurexin superfamily and voltage-gated ion channels. Further investigations support an ultrastructural and functional axonopathy and synaptopathy of parvalbumin interneurons. These processes precede somatic firing impairment and seizures suggesting they underlie fundamental early-phase disease mechanisms. Taken together we provide a cellularly resolved transcriptomic resource of early disease phases of Dravet syndrome and demonstrate epileptogenesis beyond Na V 1.1 loss-of-function during an early developmental time window of CNS maturation. Altogether these data establish proof-of principle that the concept of epileptogenesis, originally devised for acquired forms of epilepsy, similarly applies to genetic epilepsies and DEEs.

Dravet syndrome (DS) is a developmental epileptic encephalopathy mainly caused by functional NaV1.1 haploinsufficiency in inhibitory interneurons. Recently, a new conditional mouse model expressing the recurrent human p.(Ala1783Val) missense variant has become available. In this study, we provided an electrophysiological characterization of this variant in tsA201 cells, revealing both altered voltage-dependence of activation and slow inactivation without reduced sodium peak current density. Based on these data, simulated interneuron (IN) firing properties in a conductance-based single-compartment model suggested surprisingly similar firing deficits for NaV1.1A1783V and full haploinsufficiency as caused by heterozygous truncation variants. Impaired NaV1.1A1783V channel activation was predicted to have a significantly larger impact on channel function than altered slow inactivation and is therefore proposed as the main mechanism underlying IN dysfunction. The computational model was validated in cortical organotypic slice cultures derived from conditional Scn1aA1783V mice. Pan-neuronal activation of the p.Ala1783V in vitro confirmed a predicted IN firing deficit and revealed an accompanying reduction of interneuronal input resistance while demonstrating normal excitability of pyramidal neurons. Altered input resistance was fed back into the model for further refinement. Taken together these data demonstrate that primary loss of function (LOF) gating properties accompanied by altered membrane characteristics may match effects of full haploinsufficiency on the neuronal level despite maintaining physiological peak current density, thereby causing DS.

Lamotrigine and other sodium‐channel blocking agents are among the most commonly used antiepileptic drugs (AEDs). Because other sodium channel blockers, such as riluzole, can severely alter respiratory rhythm generation during hypoxia, we wanted to investigate if AEDs can have similar effects. This is especially important in the context of sudden unexpected death in epilepsy (SUDEP), the major cause of death in patients suffering from therapy‐resistant epilepsy. Although the mechanism of action is not entirely understood, respiratory dysfunction after generalized tonic‐clonic seizures seems to play a major role.

Human cerebrospinal fluid (hCSF) have proven advantageous over conventional medium when culturing both rodent and human brain tissue. Increased excitability and synchronicity, similar to the active state exclusively recorded in vivo , reported in rodent slice and cell-cultures with hCSF as recording medium, indicates properties of the hCSF not matched by the artificial cerebrospinal fluid (aCSF) commonly used for electrophysiological recording. To evaluate the possible importance of using hCSF as electrophysiological recording medium of human brain tissue, we compared the general excitability in ex vivo human brain tissue slice cultures during perfusion with hCSF and aCSF. For measuring the general activity from a majority of neurons within neocortical and hippocampal human ex vivo slices we used a microelectrode array (MEA) recording technique with 252 electrodes covering an area of 3.2 x 3.2 mm 2 and a second CMOS-based MEA with 4225 electrodes on a 2 x 2 mm 2 area for detailed mapping of action potential waveforms. We found that hCSF increase the number of active neurons and the firing rate of the neurons in the slices as well as increasing the numbers of bursts while leaving the duration of the bursts unchanged. Interestingly, not only an increase in the overall activity in the slices was observed, but a reconfiguration of the network functionality could be detected with specific activation and inactivation of subpopulations of neuronal ensembles. In conclusion, hCSF is an important component to consider for future human tissue studies, especially for experiments designed to mimic the in vivo situation.

Most of our knowledge on human CNS circuitry and related disorders originates from model organisms. How well such data translate to the human CNS remains largely to be determined. Human brain slice cultures derived from neurosurgical resections may offer novel avenues to approach this translational gap. We now demonstrate robust preservation of the complex neuronal cytoarchitecture and electrophysiological properties of human pyramidal neurons in long-term brain slice cultures. Further experiments delineate the optimal conditions for efficient viral transduction of cultures, enabling 'high throughput' fluorescence-mediated 3D reconstruction of genetically targeted neurons at comparable quality to state-of-the-art biocytin fillings, and demonstrate feasibility of long term live cell imaging of human cells in vitro. This model system has implications toward a broad spectrum of translational studies, regarding the validation of data obtained in non-human model systems, for therapeutic screening and genetic dissection of human CNS circuitry.

We have shown previously that genetic or pharmacological deletion of KATP channels protect against beta cell dysfunction induced by reactive oxygen species (ROS). Since it is assumed that glucolipotoxicity (GLTx) causes ROS production, we aimed to evaluate whether suppression of KATP channel activity can also prevent beta cell damage evoked by GLTx. We used an in vitro model of GLTx and measured distinct parameters of stimulus-secretion coupling. GLTx gradually induced disturbances of Ca2+ oscillations over 3 days. This impairment in Ca2+ dynamics was partially reversed in beta cells without functional KATP channels (SUR1−/−) and by the sulfonylurea gliclazide but not by tolbutamide. By contrast, the GLTx-induced suppression of glucose-induced insulin secretion could not be rescued by decreased KATP channel activity pointing to a direct interaction of GLTx with the secretory capacity. Accordingly, GLTx also suppressed KCl-induced insulin secretion. GLTx was not accompanied by decisively increased ROS production or enhanced apoptosis. Insulin content of beta cells was markedly reduced by GLTx, an effect not prevented by gliclazide. Since GLTx markedly diminished the mitochondrial membrane potential and cellular ATP content, lack of ATP is assumed to decrease insulin biosynthesis. The deleterious effect of GLTx is therefore caused by direct interference with the secretory capacity whereby reduction of insulin content is one important parameter. These findings deepen our understanding how GLTx damages beta cells and reveal that GLTx is disconnected from ROS formation, a notion important for targeting beta cells in the treatment of diabetes. Overall, GLTx-induced energy depletion may be a primary step in the cascade of events leading to loss of beta cell function in type-2 diabetes mellitus.

Locus coeruleus-noradrenergic system dysfunction is known to contribute to the progression of Alzheimer’s disease (AD). Besides a variety of reports showing the involvement of norepinephrine and its receptor systems in cognition, amyloid β (Aβ) metabolism, neuroinflammation, and neurogenesis, little is known about the contribution of the specific receptors to these actions. Here, we investigated the neurogenic and neuroprotective properties of a new α2 adrenoblocker, mesedin, in astroglial primary cultures (APC) from C57BL/6 and 3×Tg-AD mice. Our results demonstrate that mesedin rescues neuronal precursors and young neurons, and reduces the lactate dehydrogenase (LDH) release from astroglia under hypoxic and normoxic conditions. Mesedin also increased choline acetyltransferase, postsynaptic density marker 95 (PSD95), and Aβ-degrading enzyme neprilysin in the wild type APC, while in the 3×Tg-AD APC exposed to glutamate, it decreased the intracellular content of Aβ and enhanced the survival of synaptophysin-positive astroglia and neurons. These effects in APC can at least partially be attributed to the mesedin’s ability of increasing the expression of Interleukine(IL)-10, which is a potent anti-inflammatory, neuroprotective neurogenic, and Aβ metabolism enhancing factor. In summary, our data identify the neurogenic, neuroprotective, and anti-amyloidogenic action of mesedin in APC. Further in vivo studies are needed to estimate the therapeutic value of mesedin for AD.