Sodium current in rat and cat thalamocortical neurons:role of a non-inactivating component in tonic and burst firing

The properties of the Na+ current present in thalamocortical neurons of the dorsal lateral geniculate nucleus were investigated in dissociated neonate rat and cat neurons and in neurons from slices of neonate and adult rats using patch and sharp electrode recordings. The steady-state activation and inactivation of the transient Na+ current (INa) was well fitted with a Boltzmann curve (voltage of half-maximal activation and inactivation, V1/2, -29.84 mV and -70.04 mV, respectively). Steady-state activation and inactivation curves showed a small region of overlap, indicating the occurrence of a / Na window current.  / Na decay could be fitted with a single exponential function, consistent with the presence of only one channel type. Voltage ramp and step protocols showed the presence of a noninactivating component of the Na+ current (/ NaP) that activated at potentials more negative (V1/2 = -56.93 mV) than those of INa. The maximal amplitude of / NaP was approximately 2.5% of INa, thus significantly greater than the calculated contribution (0.2%) of the I Na window component. Comparison of results from dissociated neurons and neurons in slices suggested a dendritic as well as a somatic localization of I NaP. Inclusion of papain in the patch electrode removed the fast inactivation of / Na and induced a current with voltage-dependence (V1/2 = -56.92) and activation parameters similar to those of I NaP. Current-clamp recordings with sharp electrodes showed that I NaP contributed to depolarizations evoked from potentials of approximately -60 mV and unexpectedly to the amplitude and latency of low-threshold Ca2+ potentials, suggesting that this noninactivating component of the Na+ channel population plays an important role in the integrative properties of thalamocortical neurons during both tonic and burst-firing patterns

Multiple components of Ca2+ channel facilitation in cerebellar granule cells:expression of facilitation during development in culture

The contribution of pharmacologically distinct Ca2+ channels to prepulse-induced facilitation was studied in mouse cerebellar granule cells. Ca2+ channel facilitation was measured as the percentage increase in the whole-cell current recorded during a test pulse before and after it was paired with a positive prepulse. The amount of facilitation was small in recordings made during the first few days in tissue culture but increased substantially after 1 week. L-type channels accounted for the largest proportion of facilitation in 1-week-old cells (60-70%), whereas N-type channels contributed very little (approximately 3%). The toxins omega-agatoxin IVa or omega-conotoxin MVIIC (after block of N-, L-, and P-type channels) each blocked a small percentage of facilitation (approximately 12 and 14%, respectively). Perfusion of cells with GTP-gamma-S enhanced the facilitation of N-type channels, whereas it inhibited of L-type channels. During development in vitro, the contribution of L-type channels to the whole-cell current decreased. Single-channel recordings showed the presence of 10 and 15 pS L-type Ca2+ channels in 1-d-old cells. After 1 week in culture, a approximately 25 pS L-type channel dominated recordings from cell-attached patches. Positive prepulses increased the activity of the 25 pS channel but not of the smaller conductance channels. The expression of Ca(2+) channel facilitation during development may contribute to changes in excitability that allow frequency-dependent Ca(2+) influx during the period of active synaptogenesi

Calibration of thickness-dependent k-factors for germanium X-ray lines to improve energy-dispersive X-ray spectroscopy of SiGe layers in analytical transmission electron microscopy

We show that the accuracy of energy-dispersive X-ray spectroscopy can be improved by analysing and comparing multiple lines from the same element. For each line, an effective k-factor can be defined that varies as a function of the intensity ratio of multiple lines (e.g. K/L) from the same element. This basically performs an internal self-consistency check in the quantification using differently absorbed X-ray lines, which is in principle equivalent to an absorption correction as a function of specimen thickness but has the practical advantage that the specimen thickness itself does not actually need to be measured

Glial plasticity

Editoria

Astrocyte-Mediated Neuronal Synchronization Properties Revealed by False Gliotransmitter Release

Astrocytes spontaneously release glutamate (Glut) as a gliotransmitter (GT), resulting in the generation of extrasynaptic NMDAR-mediated slow inward currents (SICs) in neighboring neurons, which can increase local neuronal excitability. However, there is a deficit in our knowledge of the factors that control spontaneous astrocyte GT release and the extent of its influence. We found that, in rat brain slices, increasing the supply of the physiological transmitter Glut increased the frequency and signaling charge of SICs over an extended period. This phenomenon was replicated by exogenous preexposure to the amino acid D-aspartate (D-Asp). Using D-Asp as a "false" GT, we determined the extent of local neuron excitation by GT release in ventrobasal thalamus, CA1 hippocampus, and somatosensory cortex. By analyzing synchronized neuronal NMDAR-mediated excitation, we found that the properties of the excitation were conserved in different brain areas. In the three areas, astrocyte-derived GT release synchronized groups of neurons at distances of >;200 μm. Individual neurons participated in more than one synchronized population, indicating that individual neurons can be excited by more than one astrocyte and that individual astrocytes may determine a neuron's synchronized network. The results confirm that astrocytes can act as excitatory nodes that can influence neurons over a significant range in a number of brain regions. Our findings further suggest that chronic elevation of ambient Glut levels can lead to increased GT Glut release, which may be relevant in some pathological states. Astrocytes spontaneously release glutamate (Glut) and other gliotransmitters (GTs) that can modify neuronal activity. Exposing brain slices to Glut and D-aspartate (D-Asp) before recording resulted in an increase in frequency of GT-mediated astrocyte-neuron signaling. Using D-Asp, it was possible to investigate the effects of specific GT release at neuronal NMDARs. Calcium imaging showed synchronized activity in groups of neurons in cortex, hippocampus, and thalamus. The size of these populations was similar in all areas and some neurons were involved in more than one synchronous group. The findings show that GT release is supply dependent and that the properties of the signaling and activated networks are largely conserved between different brain areas

In vitro Models for Seizure-Liability Testing Using Induced Pluripotent Stem Cells

The brain is the most complex organ in the body, controlling our highest functions, as well as regulating myriad processes which incorporate the entire physiological system. The effects of prospective therapeutic entities on the brain and central nervous system (CNS) may potentially cause significant injury, hence, CNS toxicity testing forms part of the “core battery” of safety pharmacology studies. Drug-induced seizure is a major reason for compound attrition during drug development. Currently, the rat ex vivo hippocampal slice assay is the standard option for seizure-liability studies, followed by primary rodent cultures. These models can respond to diverse agents and predict seizure outcome, yet controversy over the relevance, efficacy, and cost of these animal-based methods has led to interest in the development of human-derived models. Existing platforms often utilize rodents, and so lack human receptors and other drug targets, which may produce misleading data, with difficulties in inter-species extrapolation. Current electrophysiological approaches are typically used in a low-throughput capacity and network function may be overlooked. Human-derived induced pluripotent stem cells (iPSCs) are a promising avenue for neurotoxicity testing, increasingly utilized in drug screening and disease modeling. Furthermore, the combination of iPSC-derived models with functional techniques such as multi-electrode array (MEA) analysis can provide information on neuronal network function, with increased sensitivity to neurotoxic effects which disrupt different pathways. The use of an in vitro human iPSC-derived neural model for neurotoxicity studies, combined with high-throughput techniques such as MEA recordings, could be a suitable addition to existing pre-clinical seizure-liability testing strategies

On the action of the anti-absence drug ethosuximide in the rat and cat thalamus

The action of ethosuximide (ETX) on Na+, K+, and Ca2+ currents and on tonic and burst-firing patterns was investigated in rat and cat thalamic neurons in vitro by using patch and sharp microelectrode recordings. In thalamocortical (TC) neurons of the rat dorsal lateral geniculate nucleus (LGN), ETX (0.75-1 mM) decreased the noninactivating Na+ current, INaP, by 60% but had no effect on the transient Na+ current. In TC neurons of the rat and cat LGN, the whole-cell transient outward current was not affected by ETX (up to 1 mM), but the sustained outward current was decreased by 39% at 20 mV in the presence of ETX (0.25-0.5 mM): this reduction was not observed in a low Ca2+ (0.5 mM) and high Mg2+ (8 mM) medium or in the presence of Ni2+ (1 mM) and Cd2+ (100 µm). In addition, ETX (up to 1 mM) had no effect on the low-threshold Ca2+ current, I T, of TC neurons of the rat ventrobasal (VB) thalamus and LGN and in neurons of the rat nucleus reticularis thalami nor on the high-threshold Ca2+ current in TC neurons of the rat LGN. Sharp microelectrode recordings in TC neurons of the rat and cat LGN and VB showed that ETX did not change the resting membrane potential but increased the apparent input resistance at potentials greater than -60 mV, resulting in an increase in tonic firing. In contrast, ETX decreased the number of action potentials in the burst evoked by a low-threshold Ca2+ potential. The frequency of the remaining action potentials in a burst also was decreased, whereas the latency of the first action potential was increased. Similar effects were observed on the burst firing evoked during intrinsic δ oscillations. These results indicate an action of ETX on / NaP and on the Ca2+-activated K+ current, which explains the decrease in burst firing and the increase in tonic firing, and, together with the lack of action on low- and high-threshold Ca2+ currents, the results cast doubts on the hypothesis that a reduction of / τ in thalamic neurons underlies the therapeutic action of this anti-absence medicine

GABAB receptor-mediated activation of astrocytes by gamma-hydroxybutyric acid

The gamma-aminobutyric acid (GABA) metabolite gamma-hydroxybutyric acid (GHB) shows a variety of behavioural effects when administered to animals and humans, including reward/addiction properties and absence seizures. At the cellular level, these actions of GHB are mediated by activation of neuronal GABAB receptors (GABABRs) where it acts as a weak agonist. Because astrocytes respond to endogenous and exogenously applied GABA by activation of both GABAA and GABABRs, here we investigated the action of GHB on astrocytes on the ventral tegmental area (VTA) and the ventrobasal (VB) thalamic nucleus, two brain areas involved in the reward and proepileptic action of GHB, respectively, and compared it with that of the potent GABABR agonist baclofen. We found that GHB and baclofen elicited dose-dependent (ED50: 1.6 mM and 1.3 µM, respectively) transient increases in intracellular Ca2+ in VTA and VB astrocytes of young mice and rats, which were accounted for by activation of their GABABRs and mediated by Ca2+ release from intracellular store release. In contrast, prolonged GHB and baclofen exposure caused a reduction in spontaneous astrocyte activity and glutamate release from VTA astrocytes. These findings have key (patho)physiological implications for our understanding of the addictive and proepileptic actions of GHB

Functional astrocyte-neuron lactate shuttle in a human stem cell-derived neuronal network

The NT2.D1 cell line is one of the most well-documented embryocarcinoma cell lines, and can be differentiated into neurons and astrocytes. Great focus has also been placed on defining the electrophysiological properties of the neuronal cells, and more recently we have investigated the functional properties of their associated astrocytes. We now show for the first time that human stem cell-derived astrocytes produce glycogen and that co-cultures of these cells demonstrate a functional astrocyte-neuron lactate shuttle (ANLS). The ANLS hypothesis proposes that during neuronal activity, glutamate released into the synaptic cleft is taken up by astrocytes and triggers glucose uptake, which is converted into lactate and released via monocarboxylate transporters for neuronal use. Using mixed cultures of NT2-derived neurons and astrocytes, we have shown that these cells modulate their glucose uptake in response to glutamate. Additionally, we demonstrate that in response to increased neuronal activity and under hypoglycaemic conditions, co-cultures modulate glycogen turnover and increase lactate production. Similar results were also shown after treatment with glutamate, potassium, isoproterenol, and dbcAMP. Together, these results demonstrate for the first time a functional ANLS in a human stem cell-derived co-culture. © 2013 ISCBFM