Influx of Calcium through L-type Calcium Channels in Early Postnatal Regulation of Chloride Transporters in the Rat Hippocampus

During the early postnatal period, GABAB receptor activation facilitates L-type calcium current in rat hippocampus. One developmental process that L-type current may regulate is the change in expression of the K+Cl− co-transporter (KCC2) and N+K+2Cl− co-transporter (NKCC1), which are involved in the maturation of the GABAergic system. The present study investigated the connection between L-type current, GABAB receptors, and expression of chloride transporters during development. The facilitation of L-type current by GABAB receptors is more prominent in the second week of development, with the highest percentage of cells exhibiting facilitation in cultures isolated from 7 day old rats (37.5%). The protein levels of KCC2 and NKCC1 were investigated to determine the developmental timecourse of expression as well as expression following treatment with an L-type channel antagonist and a GABAB receptor agonist. The time course of both chloride transporters in culture mimics that seen in hippocampal tissue isolated from various ages. KCC2 levels increased drastically in the first two postnatal weeks while NKCC1 remained relatively stable, suggesting that the ratio of the chloride transporters is important in mediating the developmental change in chloride reversal potential. Treatment of cultures with the L-type antagonist nimodipine did not affect protein levels of NKCC1, but significantly decreased the upregulation of KCC2 during the first postnatal week. In addition, calcium current facilitation occurs slightly before the large increase in KCC2 expression. These results suggest that the expression of KCC2 is regulated by calcium influx through L-type channels in the early postnatal period in hippocampal neurons

Recovery of succinic acid in fermentation broth via reactive LL extraction: effect of chemical kinetics and solvent choice

In this paper, a study of kinetics effects on the reactive liquid-liquid extraction column is proposed. In a first part, design parameters of reactive liquid-liquid extraction column are derived from a method proposed by Mizzi (2016). In a second part, using a kinetic model, the performances of the column are studied with different configuration and design parameters. This study allows a comparison of the performances of the column in terms of conversion rate, recovery rate and purity. For the chosen examples, the kinetic limitation is very strong. So the unit operations of reactive liquid-liquid extraction with a high retention capacity will be privileged: a cascade of decanter mixers. In conclusion, this article shows that the choice of solvent and the parameters of the column as the solvent flowrate, the number of theoretical stage, liquid hold up or kinetics of the reaction have an important influence on the performances of the column and sometimes on the feasibility of the separation

Intracellular blockade of GABAA receptors in the rat hippocampal neurons

The intracellular blockade of GABAA-receptor-mediated currents is a useful approach to suppress the GABAergic conductance in a single cell and to isolate the glutamatergic component of network-driven activities. Previously an approach has been described allowing intracellular blockade of GABA A receptors by means of intracellular dialysis of a neuron with the pipette-filling solution, in which fluoride ions that hardly pass through the GABAA receptor channels substitute for Cl- and in which Mg2+ and ATP are omitted to induce rundown of the GABAA receptors during whole-cell patch-clamp recordings. However, the kinetics of suppression of GABAergic conductance and the effect on the currents mediated by glutamate receptors remain unknown. Here, using whole-cell recordings with fluoride-based, Mg2+- and ATP-free solution on CA3 hippocampal neurons of neonatal rats, we show that after 1 h of such dialysis, both spontaneous and evoked GABAA-receptor-mediated synaptic currents and responses induced by the GABAA receptor agonist isoguvacine were completely suppressed. Inward GABAergic postsynaptic currents were suppressed prior to outward currents. Synaptic responses mediated by AMPA receptors were not affected by the dialysis, whereas the NMDA-receptor-mediated postsynaptic currents were reduced by approximately 20%. Dialysis with fluoride-based Mg 2+, ATP-free solution either fully blocked giant depolarizing potentials (GDPs) in CA3 pyramidal cells (n = 2) or reduced the charge crossing the membrane during GDPs and shifted the GDP reversal potential to more positive values (n = 5). The dialysis-resistant component of GDPs was mediated by glutamate receptors, since: (i) it reversed around 0 mV; (ii) it demonstrated a negative slope conductance at negative membrane voltages, which is characteristic of NMDA receptor-mediated responses; (iii) kinetics of the individual events composing the dialysis-resistant component of GDPs at negative voltages were very similar to those of AMPA receptor-mediated synaptic currents. Thus, this procedure can be useful to isolate the glutamate receptor-mediated component of neuronal network-driven activities. © 2014 Pleiades Publishing, Ltd

Intracellular blockade of GABA<inf>A</inf> receptors in the rat hippocampal neurons

The intracellular blockade of GABAA-receptor-mediated currents is a useful approach to suppress the GABAergic conductance in a single cell and to isolate the glutamatergic component of network-driven activities. Previously an approach has been described allowing intracellular blockade of GABAA receptors by means of intracellular dialysis of a neuron with the pipette-filling solution, in which fluoride ions that hardly pass through the GABAA receptor channels substitute for Cl- and in which Mg2+ and ATP are omitted to induce rundown of the GABAA receptors during whole-cell patch-clamp recordings. However, the kinetics of suppression of GABAergic conductance and the effect on the currents mediated by glutamate receptors remain unknown. Here, using whole-cell recordings with fluoride-based, Mg2+- and ATP-free solution on CA3 hippocampal neurons of neonatal rats, we show that after 1 h of such dialysis, both spontaneous and evoked GABAA-receptor-mediated synaptic currents and responses induced by the GABAA receptor agonist isoguvacine were completely suppressed. Inward GABAergic postsynaptic currents were suppressed prior to outward currents. Synaptic responses mediated by AM PA receptors were not affected by the dialysis, whereas the NM DA-receptor-mediated postsynaptic currents were reduced by approximately 20%. Dialysis with fluoride-based Mg2+, ATP-free solution either fully blocked giant depolarizing potentials (G DPs) in CA3 pyramidal cells (n = 2) or reduced the charge crossing the membrane during G DPs and shifted the G DP reversal potential to more positive values (n = 5). The dialysis-resistant component of G DPs was mediated by glutamate receptors, since: (i) it reversed around 0 mV; (ii) it demonstrated a negative slope conductance at negative membrane voltages, which is characteristic of NM DA receptor-mediated responses; (iii) kinetics of the individual events composing the dialysis-resistant component of G DPs at negative voltages were very similar to those of AM PA receptor-mediated synaptic currents. Thus, this procedure can be useful to isolate the glutamate receptor-mediated component of neuronal network-driven activities

Insights into GABA receptor signalling in TM3 Leydig cells

gamma-Aminobutyric acid (GABA) is an emerging signalling molecule in endocrine organs, since it is produced by endocrine cells and acts via GABA(A) receptors in a paracrine/autocrine fashion. Testicular Leydig cells are producers and targets for GABA. These cells express GABA(A) receptor subunits and in the murine Leydig cell line TM3 pharmacological activation leads to increased proliferation. The signalling pathway of GABA in these cells is not known in this study. We therefore attempted to elucidate details of GABA(A) signalling in TM3 and adult mouse Leydig cells using several experimental approaches. TM3 cells not only express GABA(A) receptor subunits, but also bind the GABA agonist {[}H-3] muscimol with a binding affinity in the range reported for other endocrine cells (K-d = 2.740 +/- 0.721 nM). However, they exhibit a low B-max value of 28.08 fmol/mg protein. Typical GABA(A) receptor-associated events, including Cl- currents, changes in resting membrane potential, intracellular Ca2+ or cAMP, were not measurable with the methods employed in TM3 cells, or, as studied in part, in primary mouse Leydig cells. GABA or GABA(A) agonist isoguvacine treatment resulted in increased or decreased levels of several mRNAs, including transcription factors (c-fos, hsf-1, egr-1) and cell cycle-associated genes (Cdk2, cyclin D1). In an attempt to verify the cDNA array results and because egr-1 was recently implied in Leydig cell development, we further studied this factor. RT-PCR and Western blotting confirmed a time-dependent regulation of egr-1 in TM3. In the postnatal testis egr-1 was seen in cytoplasmic and nuclear locations of developing Leydig cells, which bear GABA(A) receptors and correspond well to TM3 cells. Thus, GABA acts via an untypical novel signalling pathway in TM3 cells. Further details of this pathway remain to be elucidated. Copyright (c) 2005 S. Karger AG, Base

General design methodology for reactive liquid–liquid extraction: application to dicarboxylic acid recovery in fermentation broth

A general design methodology for reactive liquid–liquid extraction is introduced in this paper. It is composed of three different steps: feasibility analysis, pre-design determination and simulation validation. This paper is focused on the first and the second step. This methodology leads to the design specifications of the units from the information concerning the physico-chemical behaviour of the studied system, exploiting the equilibrium and material balance equations. The results of this methodology are a good starting point for an optimization study or for an investment calculation process. This methodology has been applied to different case studies: two different strategies of extraction and several solvents to recover succinic acid in fermentation broth

Cell Rep.

The hypothesis that reversed, excitatory GABA may be involved in various brain pathologies, including epileptogenesis, is appealing but controversial because of the technical difficulty of probing endogenous GABAergic synaptic function in vivo. We overcome this challenge by non-invasive extracellular recording of neuronal firing responses to optogenetically evoked and spontaneously occurring inhibitory perisomatic GABAergic field potentials, generated by individual parvalbumin interneurons on their target pyramidal cells. Our direct probing of GABAergic transmission suggests a rather anecdotal participation of excitatory GABA in two specific models of epileptogenesis in the mouse CA3 circuit in vivo, even though this does not preclude its expression in other brain areas or pathological conditions. Our approach allows the detection of distinct alterations of inhibition during spontaneous activity in vivo, with high sensitivity. It represents a promising tool for the investigation of excitatory GABA in different pathological conditions that may affect the hippocampal circuit

Novel Patient Cell-Based HTS Assay for Identification of Small Molecules for a Lysosomal Storage Disease

Small molecules have been identified as potential therapeutic agents for lysosomal storage diseases (LSDs), inherited metabolic disorders caused by defects in proteins that result in lysosome dysfunctional. Some small molecules function assisting the folding of mutant misfolded lysosomal enzymes that are otherwise degraded in ER-associated degradation. The ultimate result is the enhancement of the residual enzymatic activity of the deficient enzyme. Most of the high throughput screening (HTS) assays developed to identify these molecules are single-target biochemical assays. Here we describe a cell-based assay using patient cell lines to identify small molecules that enhance the residual arylsulfatase A (ASA) activity found in patients with metachromatic leukodystrophy (MLD), a progressive neurodegenerative LSD. In order to generate sufficient cell lines for a large scale HTS, primary cultured fibroblasts from MLD patients were transformed using SV40 large T antigen. These SV40 transformed (SV40t) cells showed to conserve biochemical characteristics of the primary cells. Using a specific colorimetric substrate para-nitrocatechol sulfate (pNCS), detectable ASA residual activity were observed in primary and SV40t fibroblasts from a MLD patient (ASA-I179S) cultured in multi-well plates. A robust fluorescence ASA assay was developed in high-density 1,536-well plates using the traditional colorimetric pNCS substrate, whose product (pNC) acts as “plate fluorescence quencher” in white solid-bottom plates. The quantitative cell-based HTS assay for ASA generated strong statistical parameters when tested against a diverse small molecule collection. This cell-based assay approach can be used for several other LSDs and genetic disorders, especially those that rely on colorimetric substrates which traditionally present low sensitivity for assay-miniaturization. In addition, the quantitative cell-based HTS assay here developed using patient cells creates an opportunity to identify therapeutic small molecules in a disease-cellular environment where potentially disrupted pathways are exposed and available as targets

Functional Integration of Grafted Neural Stem Cell-Derived Dopaminergic Neurons Monitored by Optogenetics in an In Vitro Parkinson Model

Intrastriatal grafts of stem cell-derived dopamine (DA) neurons induce behavioral recovery in animal models of Parkinson's disease (PD), but how they functionally integrate in host neural circuitries is poorly understood. Here, Wnt5a-overexpressing neural stem cells derived from embryonic ventral mesencephalon of tyrosine hydroxylase-GFP transgenic mice were expanded as neurospheres and transplanted into organotypic cultures of wild type mouse striatum. Differentiated GFP-labeled DA neurons in the grafts exhibited mature neuronal properties, including spontaneous firing of action potentials, presence of post-synaptic currents, and functional expression of DA D2 autoreceptors. These properties resembled those recorded from identical cells in acute slices of intrastriatal grafts in the 6-hydroxy-DA-induced mouse PD model and from DA neurons in intact substantia nigra. Optogenetic activation or inhibition of grafted cells and host neurons using channelrhodopsin-2 (ChR2) and halorhodopsin (NpHR), respectively, revealed complex, bi-directional synaptic interactions between grafted cells and host neurons and extensive synaptic connectivity within the graft. Our data demonstrate for the first time using optogenetics that ectopically grafted stem cell-derived DA neurons become functionally integrated in the DA-denervated striatum. Further optogenetic dissection of the synaptic wiring between grafted and host neurons will be crucial to clarify the cellular and synaptic mechanisms underlying behavioral recovery as well as adverse effects following stem cell-based DA cell replacement strategies in PD