Examining the fundamental biology of a novel population of directly reprogrammed human neural precursor cells.

Background: Cell reprogramming is a promising avenue for cell-based therapies as it allows for the generation of multipotent, unipotent, or mature somatic cells without going through a pluripotent state. While the use of autologous cells is considered ideal, key challenges for their clinical translation include the ability to reproducibly generate sufficient quantities of cells within a therapeutically relevant time window.

Methods: We performed transfection of three distinct human somatic starting populations of cells with a non-integrating synthetic plasmid expressing Musashi 1 (MSI1), Neurogenin 2 (NGN2), and Methyl-CpG-Binding Domain 2 (MBD2).

A Novel Approach for Studying the Physiology and Pathophysiology of Myelinated and Non-Myelinated Axons in the CNS White Matter.

Advances in brain connectomics set the need for detailed knowledge of functional properties of myelinated and non-myelinated (if present) axons in specific white matter pathways. The corpus callosum (CC), a major white matter structure interconnecting brain hemispheres, is extensively used for studying CNS axonal function. Unlike another widely used CNS white matter preparation, the optic nerve where all axons are myelinated, the CC contains also a large population of non-myelinated axons, making it particularly useful for studying both types of axons.

Contribution of fast and slow conducting myelinated axons to single-peak compound action potentials in rat spinal cord white matter preparations.

Unlike recordings derived from optic nerve or corpus callosum, compound action potentials (CAPs) recorded from rodent spinal cord white matter (WM) have a characteristic single-peak shape despite the heterogeneity of axonal populations. Using a double sucrose gap technique, we analyzed the CAPs recorded from dorsal, lateral, and ventral WM from mature rat spinal cord. The CAP decay was significantly prolonged with increasing stimulus intensities suggesting a recruitment of higher threshold, slower conducting axons.

Visualization of cytoplasmic diffusion within living myelin sheaths of CNS white matter axons using microinjection of the fluorescent dye Lucifer Yellow.

The compactness of myelin allows for efficient insulation defining rapid propagation of action potentials, but also raises questions about how cytoplasmic access to its membranes is achieved, which is critical for physiological activity. Understanding the organization of cytoplasmic ('water') spaces of myelin is also important for diffusion MRI studies of CNS white matter. Using longitudinal slices of mature rat spinal cord, we monitored the diffusion of the water-soluble fluorescent dye Lucifer Yellow injected into individual oligodendrocytes or internodal myelin.

Chronic in vitro ketosis is neuroprotective but not anti-convulsant.

The ketogenic diet (KD), used successfully to treat a variety of epilepsy syndromes in humans and to attenuate seizures in different animal models, also provides powerful neuroprotection in various CNS injury models. Yet, a direct role for ketone bodies in limiting seizure and neuronal damage remains poorly understood. Using organotypic hippocampal slice cultures, we established an in vitro model of chronic ketosis for parallel studies of its neuroprotective and anti-convulsant effects.

Modular double sucrose gap apparatus for improved recording of compound action potentials from rat and mouse spinal cord white matter preparations.

Compound action potential (CAP) recording is a powerful tool for studying the conduction properties and pharmacology of axons in multi-axonal preparations. The sucrose gap technique improves CAP recording by replacing the extracellular solution between the recording electrodes with a non-conductive sucrose solution to minimize extracellular shunting. The double sucrose gap (DSG), conferring similar advantages at the stimulation site, has been extensively used on guinea pig spinal cord white matter (WM) in vitro.

Hippocampal seizures alter the expression of the pannexin and connexin transcriptome.

Some forms of seizure activity can be stopped by gap junctional (GJ) blockade. Here, we found that GJ blockers attenuate hippocampal seizure activity induced by a novel seizuregenic protocol using Co(2+). We hypothesized that this activity may occur because of the altered expression of connexin (Cx) and/or pannexin (Panx) mRNAs and protein.

[Opsoclonus-myoclonus syndrome in children].

To study clinical peculiarities of parainfectious opsoclonus-myoclonus syndrome (OMS) in children and to elaborate approaches to its pharmacotherapeutic correction, 20 children, including 12 girls and 8 boys, have been examined using neurological, neurophysiological, immunological and virological methods along with magnetic resonance tomography (MRT). Age-at-disease-onset was from 8 months to 3 years old. The development of neurological symptoms was related to a virus infection (55% of cases) or vaccination (15%).

Connexin 43 mimetic peptides inhibit spontaneous epileptiform activity in organotypic hippocampal slice cultures.

Gap junctions are cytoplasmic channels connecting adjacent cells and mediating their electrical and metabolic coupling. Different cell types in the CNS express various gap junction forming proteins, the connexins, in a cell-specific manner. Using the general gap junctional blocker, carbenoxolone, and two synthetic connexin mimetic peptides, corresponding to amino acid sequences of segments within the second extracellular loop of connexin 43, we studied the role of gap junctions in the generation of epileptiform activity in rat organotypic hippocampal slice cultures.

Factors which abolish hypoglycemic seizures do not increase cerebral glycogen content in vitro.

The brain is heavily dependant on glucose for its function and survival. Hypoglycemia can have severe, irreversible consequences, including seizures, coma and death. However, the in vivo content of brain glycogen, the storage form of glucose, is meager and is a function of both neuronal activity and glucose concentration.

High frequency stimulation or elevated K+ depresses neuronal activity in the rat entopeduncular nucleus.

High frequency stimulation (HFS) is applied to many brain regions to treat a variety of neurological disorders/diseases, yet the mechanism(s) underlying its effects remains unclear. While some studies showed that HFS inhibits the stimulated nucleus, others report excitation. In this in vitro study, we stimulated the rat globus pallidus interna (entopeduncular nucleus, EP), a commonly stimulated area for Parkinson's disease, to investigate the effect of HFS-induced elevation of extracellular potassium (K(+)(e)) on rat EP neuronal activity.

Supramolecular structure of self-assembling alamethicin analog studied by ESR and PELDOR.

Three analogs of alamethicin F50/5, labelled with the TOAC (='2,2,6,6-tetramethylpiperidin-1-oxyl-4-amino-4-carboxylic acid') spin label at positions 1 (Alm1), 8 (Alm8), and 16 (Alm16), resp., were studied by Electron-Spin-Resonance (ESR) and Pulsed Electron-Electron Double-Resonance (PELDOR) techniques in solvents of different polarity to investigate the self-assembly of amphipathic helical peptides in membrane-mimicking environments. In polar solvents, alamethicin forms homogeneous solutions.

Hypoglycemic seizures during transient hypoglycemia exacerbate hippocampal dysfunction.

Severe hypoglycemia constitutes a medical emergency, involving seizures, coma and death. We hypothesized that seizures, during limited substrate availability, aggravate hypoglycemia-induced brain damage. Using immature isolated, intact hippocampi and frontal neocortical blocks subjected to low glucose perfusion, we characterized hypoglycemic (neuroglycopenic) seizures in vitro during transient hypoglycemia and their effects on synaptic transmission and glycogen content.

General anesthetics inhibit gap junction communication in cultured organotypic hippocampal slices.

Gap junctions are protein channels that directly connect the cytosol of neighboring cells, thus forming electrical synapses and promoting synchronous neuronal activities. Such activities lead to the initiation and propagation of electroencephalogram oscillations implicated in cognition and consciousness. In this study, we investigated the effects of propofol, thiopental, and halothane on gap junction communication in cultured organotypic hippocampal slices by recovery of fluorescence after photo bleaching (FRAP) technique and electrophysiological recordings.

Calcium chelation improves spatial learning and synaptic plasticity in aged rats.

Impaired regulation of intracellular calcium is thought to adversely affect synaptic plasticity and cognition in the aged brain. Comparing young (2-3 months) and aged (23-26 months) Fisher 344 rats, stratum radiatum-evoked CA1 field EPSPs were smaller and long-term potentiation (LTP) was diminished in aged hippocampal slices. Resting calcium, in presynaptic axonal terminals in the CA1 stratum radiatum area, was elevated in aged slices.

Model of frequent, recurrent, and spontaneous seizures in the intact mouse hippocampus.

This study presents a model of chronic, recurrent, spontaneous seizures in the intact isolated hippocampal preparation from mice aged P8-P25. Field activity from the CA1 pyramidal cell layer was recorded and recurrent, spontaneous seizure-like events (SLEs) were observed in the presence of low Mg2+ (0.25 mM) artificial cerebrospinal fluid (ACSF).

Epileptiform activity in hippocampal slice cultures exposed chronically to bicuculline: increased gap junctional function and expression.

Chronic (18 h) exposure of cultured hippocampal slices to the type-A GABA receptor blocker, bicuculline methiodide (BMI) 10 micro m increased the levels of connexin 43 (Cx43) and connexin 32 (Cx32) mRNAs, but not connexin 26 and connexin 36, as demonstrated by RNase protection assays. The levels of Cx43 and Cx32 proteins in membrane fractions detected by western blotting were also significantly increased. Immunoblotting indicated that BMI also promoted a significant expression of the transcription protein c-fos.

Studies of the structure of glutamate receptor ion channels and the mechanisms of their blockade by organic cations.

The structural determinants for blockade of the AMPA and NMDA subtypes of glutamate receptors were studied by analysis of structural-functional relationships in a series of mono- and dicationic compounds. The results showed that the hydrophobic and nucleophilic components of the blocker binding sites are located close to each other in the channel of the NMDA receptor, while they are spatially distant in the channel of the AMPA receptor. Molecular mechanical methods were used to construct models of these channels satisfying these topographic criteria and providing adequate descriptions of the binding of the channel blockers.

Calcium signaling components of oscillating invertebrate neurons in vitro.

We have studied the Ca(2+) dynamics of bursting-spiking neurons in the lobster stomatogastric ganglion. Neurons in this ganglion undergo spontaneous oscillations in membrane voltage with a period of 1-10 s in situ. We found that neurons isolated from the ganglion and filled with the fluorescent calcium indicator Fluo-4 show simultaneous changes of membrane potential and cytoplasmic Ca(2+) concentration ([Ca(2+)](I)).

Structural characteristics of ionotropic glutamate receptors as identified by channel blockade.

The channels of four types of ionotropic glutamate receptor (NMDA receptors and Ca-permeable AMPA receptors of rat brain neurons, and cation-selective receptors from mollusk neurons and insect postsynaptic muscle membranes) and two subtypes of nicotinic cholinoreceptor (from frog neuromuscular junctions and cat sympathetic ganglia) were studied. The structural characteristics of channels determining their susceptibility to blockade by organic mono- and dications were identified. These studies used homologous series of adamantane and phenylcyclohexyl derivatives.

[Structure of glutamate receptor ion channels and mechanisms of their blockade by organic cations].

Structural determinants of blocking the glutamate receptors of AMPA and NMDA subtypes, were studied. Close location of hydrophobic and ammonium groups is necessary for affective blocking of the NMDA receptor channels, whereas blockers of the AMPA receptor channels have a distance of about 10 angstroms between these two groups. Models of the channels meeting these topographic data have been devised using a molecular mechanics approach.

[Structural characteristics of ionotropic glutamate receptors revealed by channel blockade].

The topography of the channel binding site in glutamate receptors (AMPA and NMDA types of rat brain neurons, receptors of molluscan neurons and insect muscle), and in two subtypes of nicotinic cholinoreceptors (in frog muscle and cat sympathetic ganglion), has been investigated by comparison of the blocking effects of mono- and dicationic derivatives of adamantane and phenylcyclohexyl. The channels studied can be divided into two groups. The first one includes AMPA receptor and glutamate receptors of mollusc and insect, and is characterised by the absence of activity of monocationic drugs and the strong dependence of dicationic once on the internitrogen distance in the drug molecule.

Voltage-dependent block of native AMPA receptor channels by dicationic compounds.

1. The kinetics of open channel block of GluR2-containing and GluR2-lacking AMPA receptors (AMPAR) by dicationic compounds (IEM-1460, IEM-1754, and IEM-1925) have been studied in rat hippocampal neurones using whole-cell patch clamp recording and concentration-jump techniques. Neurones were isolated from hippocampal slices by vibrodissociation.