Voltage-gated K + channels of the K V 7 (KCNQ) family have been identified in the last 10â15 years by discovering the causative genes for three autosomal dominant
diseases: cardiac arrhythmia (long ...QT syndrome) with or without congenital deafness ( KCNQ1 ), a neonatal epilepsy ( KCNQ2 and KCNQ3 ) and progressive deafness alone ( KCNQ4 ). A fifth member of this gene family ( KCNQ5 ) is not affected in a disease so far. Four genes ( KCNQ2â5 ) are expressed in the nervous system. This review is focused on recent findings on the neuronal K V 7 channelopathies, in particular on benign familial neonatal seizures (BFNS) and peripheral nerve hyperexcitability (PNH,
neuromyotonia, myokymia) caused by KCNQ2 mutations. The phenotypic spectrum associated with KCNQ2 mutations is probably broader than initially thought, as patients with severe epilepsies and developmental delay, or with
Rolando epilepsy have been described. With regard to the underlying molecular pathophysiology, it has been shown that mutations
with very subtle changes restricted to subthreshold voltages can cause BFNS thereby proving in a human disease model that
this is the relevant voltage range for these channels to modulate neuronal firing. The two mutations associated with PNH induce
much more severe channel dysfunction with a dominant negative effect on wild type (WT) channels. Finally, K V 7 channels present interesting targets for new therapeutic approaches to diseases caused by neuronal hyperexcitability, such
as epilepsy, neuropathic pain, and migraine. The molecular mechanism of K V 7 activation by retigabine, which is in phase III clinical testing to treat pharmacoresistant focal epilepsies, has been recently
elucidated as a stabilization of the open conformation by binding to the pore region.
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
. 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.
Cortical spreading depression (CSD), a wave of depolarization followed by depression of cortical activity, is a pathophysiological process implicated in migraine with aura and various other brain ...pathologies, such as ischemic stroke and traumatic brain injury. To gain insight into the pathophysiology of CSD, we generated a mouse model for a severe monogenic subtype of migraine with aura, familial hemiplegic migraine type 3 (FHM3). FHM3 is caused by mutations in SCN1A, encoding the voltage-gated Na+ channel NaV1.1 predominantly expressed in inhibitory interneurons. Homozygous Scn1aL1649Q knock-in mice died prematurely, whereas heterozygous mice had a normal lifespan. Heterozygous Scn1aL1649Q knock-in mice compared with WT mice displayed a significantly enhanced susceptibility to CSD. We found L1649Q to cause a gain-of-function effect with an impaired Na+-channel inactivation and increased ramp Na+ currents leading to hyperactivity of fast-spiking inhibitory interneurons. Brain slice recordings using K+-sensitive electrodes revealed an increase in extracellular K+ in the early phase of CSD in heterozygous mice, likely representing the mechanistic link between interneuron hyperactivity and CSD initiation. The neuronal phenotype and premature death of homozygous Scn1aL1649Q knock-in mice was partially rescued by GS967, a blocker of persistent Na+ currents. Collectively, our findings identify interneuron hyperactivity as a mechanism to trigger CSD.
Objective
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.
Methods
We used transverse brainstem slice preparations from neonatal and juvenile mice containing the pre‐Bötzinger complex (PreBötC) and measured population as well as intracellular activity of the rhythm‐generating network under normoxia and hypoxia in the presence or absence of AEDs.
Results
We found a substantial inhibition of the gasping response induced by the application of sodium channel blockers (lamotrigine and carbamazepine). In contrast, levetiracetam, an AED‐modulating synaptic function, had a much smaller effect. The inhibition of gasping by lamotrigine was accompanied by a significant reduction of the persistent sodium current (INap) in PreBötC neurons. Surprisingly, the suppression of persistent sodium currents by lamotrigine did not affect the voltage‐dependent bursting activity in PreBötC pacemaker neurons, but led to a hypoxia‐dependent shift of the action potential rheobase in all measured PreBötC neurons.
Significance
Our results contribute to the understanding of the effects of AEDs on the vital respiratory functions of the central nervous system. Moreover, our study adds further insight into sodium‐dependent changes occurring during hypoxia and the contribution of cellular properties to the respiratory rhythm generation in the pre‐Bötzinger complex. It raises the question of whether sodium channel blocking AEDs could, in conditions of extreme hypoxia, contribute to SUDEP, an important issue that warrants further studies.
Abstract Background Key functions of Ca 2+ signaling in rodent microglia include monitoring the brain state as well as the surrounding neuronal activity and sensing the danger or damage in their ...vicinity. Microglial Ca 2+ dyshomeostasis is a disease hallmark in many mouse models of neurological disorders but the Ca 2+ signal properties of human microglia remain unknown. Methods We developed a novel genetically-encoded ratiometric Ca 2+ indicator, targeting microglial cells in the freshly resected human tissue, organotypically cultured tissue slices and analyzed in situ ongoing Ca 2+ signaling of decades-old microglia dwelling in their native microenvironment. Results The data revealed marked compartmentalization of Ca 2+ signals, with signal properties differing across the compartments and resident morphotypes. The basal Ca 2+ levels were low in ramified and high in ameboid microglia. The fraction of cells with ongoing Ca 2+ signaling, the fraction and the amplitude of process Ca 2+ signals and the duration of somatic Ca 2+ signals decreased when moving from ramified via hypertrophic to ameboid microglia. In contrast, the size of active compartments, the fraction and amplitude of somatic Ca 2+ signals and the duration of process Ca 2+ signals increased along this pathway.
Objective
Antiepileptic treatment of brain tumor patients mainly depends on the individual physician's choice rather than on well‐defined predictive factors. We investigated the predictive value of ...defined clinical parameters to formulate a model of risk estimations for subpopulations of brain tumor patients.
Methods
We enclosed 650 patients > 18 years of age who underwent brain tumor surgery and included a number of clinical data. Logistic regressions were performed to determine the effect sizes of seizure‐related risk factors and to develop prognostic scores for the occurrence of preoperative and early postoperative seizures.
Results
A total of 492 patients (334 gliomas) were eligible for logistic regression for preoperative seizures, and 338 patients for early postoperative seizures. Age ≤ 60 years (odds ratio OR = 1.66, p = 0.020), grades I and II glioma (OR = 4.00, p = 0.0002), total tumor/edema volume ≤ 64cm3 (OR = 2.18, p = 0.0003), and frontal location (OR = 2.28, p = 0.034) demonstrated an increased risk for preoperative seizures. Isocitrate–dehydrogenase mutations (OR = 2.52, p = 0.026) were an independent risk factor in the glioma subgroup. Age ≥ 60 years (OR = 3.32, p = 0.041), total tumor/edema volume ≤ 64cm3 (OR = 3.17, p = 0.034), complete resection (OR = 15.50, p = 0.0009), diencephalic location (OR = 12.2, p = 0.013), and high‐grade tumors (OR = 5.67, p = 0.013) were significant risk factors for surgery‐related seizures. Antiepileptics (OR = 1.20, p = 0.60) did not affect seizure occurrence. For seizure occurrence, patients could be stratified into 3 prognostic preoperative and into 2 prognostic early postoperative groups.
Interpretation
Based on the developed prognostic scores, seizure prophylaxis should be considered in high‐risk patients and patient stratification for prospective studies may be feasible in the future. Ann Neurol 2015;78:917–928
Proteopathic brain lesions are a hallmark of many age-related neurodegenerative diseases including synucleinopathies and develop at least a decade before the onset of clinical symptoms. Thus, ...understanding of the initiation and propagation of such lesions is key for developing therapeutics to delay or halt disease progression.
Alpha-synuclein (αS) inclusions were induced in long-term murine and human slice cultures by seeded aggregation. An αS seed-recognizing human antibody was tested for blocking seeding and/or spreading of the αS lesions. Release of neurofilament light chain (NfL) into the culture medium was assessed.
To study initial stages of α-synucleinopathies, we induced αS inclusions in murine hippocampal slice cultures by seeded aggregation. Induction of αS inclusions in neurons was apparent as early as 1week post-seeding, followed by the occurrence of microglial inclusions in vicinity of the neuronal lesions at 2-3 weeks. The amount of αS inclusions was dependent on the type of αS seed and on the culture's genetic background (wildtype vs A53T-αS genotype). Formation of αS inclusions could be monitored by neurofilament light chain protein release into the culture medium, a fluid biomarker of neurodegeneration commonly used in clinical settings. Local microinjection of αS seeds resulted in spreading of αS inclusions to neuronally connected hippocampal subregions, and seeding and spreading could be inhibited by an αS seed-recognizing human antibody. We then applied parameters of the murine cultures to surgical resection-derived adult human long-term neocortical slice cultures from 22 to 61-year-old donors. Similarly, in these human slice cultures, proof-of-principle induction of αS lesions was achieved at 1week post-seeding in combination with viral A53T-αS expressions.
The successful translation of these brain cultures from mouse to human with the first reported induction of human αS lesions in a true adult human brain environment underlines the potential of this model to study proteopathic lesions in intact mouse and now even aged human brain environments.
Paroxysmal dyskinesias are episodic movement disorders that can be inherited or are sporadic in nature. The pathophysiology underlying these disorders remains largely unknown but may involve ...disrupted ion homeostasis due to defects in cell-surface channels or nutrient transporters. In this study, we describe a family with paroxysmal exertion-induced dyskinesia (PED) over 3 generations. Their PED was accompanied by epilepsy, mild developmental delay, reduced CSF glucose levels, hemolytic anemia with echinocytosis, and altered erythrocyte ion concentrations. Using a candidate gene approach, we identified a causative deletion of 4 highly conserved amino acids (Q282_S285del) in the pore region of the glucose transporter 1 (GLUT1). Functional studies in Xenopus oocytes and human erythrocytes revealed that this mutation decreased glucose transport and caused a cation leak that alters intracellular concentrations of sodium, potassium, and calcium. We screened 4 additional families, in which PED is combined with epilepsy, developmental delay, or migraine, but not with hemolysis or echinocytosis, and identified 2 additional GLUT1 mutations (A275T, G314S) that decreased glucose transport but did not affect cation permeability. Combining these data with brain imaging studies, we propose that the dyskinesias result from an exertion-induced energy deficit that may cause episodic dysfunction of the basal ganglia, and that the hemolysis with echinocytosis may result from alterations in intracellular electrolytes caused by a cation leak through mutant GLUT1.
Human cerebrospinal fluid (hCSF) has proven advantageous over conventional medium for culturing both rodent and human brain tissue. In addition, increased activity and synchrony, closer to the ...dynamic states exclusively recorded
, were reported in rodent slices and cell cultures switching from artificial cerebrospinal fluid (aCSF) to hCSF. This indicates that hCSF possesses properties that are not matched by the aCSF, which is generally used for most electrophysiological recordings. To evaluate the possible significance of using hCSF as an electrophysiological recording medium, also for human brain tissue, we compared the network and single-cell firing properties of human brain slice cultures during perfusion with hCSF and aCSF. For measuring the overall activity from a majority of neurons within neocortical and hippocampal human slices, we used a microelectrode array (MEA) recording technique with 252 electrodes covering an area of 3.2 × 3.2 mm
. A second CMOS-based MEA with 4225 sensors on a 2 × 2 mm
area was used for detailed mapping of action potential waveforms and cell identification. We found that hCSF increased the number of active electrodes and neurons and the firing rate of the neurons in the slices and induced an increase in the numbers of single channel and population bursts. Interestingly, not only an increase in the overall activity in the slices was observed, but a reconfiguration of the network could also be detected with specific activation and inactivation of subpopulations of neuronal ensembles. In conclusion, hCSF is an important component to consider for future human brain slice studies, especially for experiments designed to mimic parts of physiology and disease observed
.
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