A high intracellular chloride concentration in immature neurons leads to a depolarizing action of GABA that is thought to shape the developing neuronal network. We show that GABA-triggered depolarization and Ca 2ϩ transients were attenuated in mice deficient for the Na-K-2Cl cotransporter NKCC1. Correlated Ca 2ϩ transients and giant depolarizing potentials (GDPs) were drastically reduced and the maturation of the glutamatergic and GABAergic transmission in CA1 delayed. Brain morphology, synaptic density, and expression levels of certain developmental marker genes were unchanged. The expression of lynx1, a protein known to dampen network activity, was decreased. In mice deficient for the neuronal Cl Ϫ /HCO 3 Ϫ exchanger AE3, GDPs were also diminished. These data show that NKCC1-mediated Cl Ϫ accumulation contributes to GABAergic excitation and network activity during early postnatal development and thus facilitates the maturation of excitatory and inhibitory synapses.
The function of voltage-gated chloride channels in neurons is essentially unknown. The voltage-gated chloride channel ClC-2 mediates a chloride current in pyramidal cells of the hippocampus. We directly show that ClC-2 assists chloride extrusion after high chloride load. Furthermore, the loss of this chloride channel leads to a dramatic increase of the input resistance of CA1 pyramidal cells, making these cells more excitable. Surprisingly, basal synaptic transmission, as judged from recordings of field EPSPs, was decreased. This difference was eliminated when GABAergic inhibition was blocked. Recordings from hippocampal interneurons revealed ClC-2-mediated currents in a subset of these cells. An observed increase in GABAergic inhibition could thus be explained by an increase in the excitability of interneurons, caused by the loss of ClC-2. Together, we suggest a dual role for ClC-2 in neurons, providing an additional efflux pathway for chloride and constituting a substantial part of the background conductance, which regulates excitability. In ClC-2 knock-out mice, an increased inhibition seemingly balances the hyperexcitability of the network and thereby prevents epilepsy.
The use of transgenic mice in which subtypes of neurons are labeled with a fluorescent protein has greatly facilitated modern neuroscience research. GAD65-GFP mice, which have GABAergic interneurons labeled with GFP, are widely used in many research laboratories, although the properties of the labeled cells have not been studied in detail. Here we investigate these cells in the hippocampal area CA1 and show that they constitute ∼20% of interneurons in this area. The majority of them expresses either reelin (70±2%) or vasoactive intestinal peptide (VIP; 15±2%), while expression of parvalbumin and somatostatin is virtually absent. This strongly suggests they originate from the caudal, and not the medial, ganglionic eminence. GFP-labeled interneurons can be subdivided according to the (partially overlapping) expression of neuropeptide Y (42±3%), cholecystokinin (25±3%), calbindin (20±2%) or calretinin (20±2%). Most of these subtypes (with the exception of calretinin-expressing interneurons) target the dendrites of CA1 pyramidal cells. GFP-labeled interneurons mostly show delayed onset of firing around threshold, and regular firing with moderate frequency adaptation at more depolarized potentials.
Milenkovic I, Rinke I, Witte M, Dietz B, Rü bsamen R. P2 receptor-mediated signaling in spherical bushy cells of the mammalian cochlear nucleus.
We have developed an automated patch clamp module for high-throughput ion channel screening, recording from 384 cells simultaneously. The module is incorporated into a laboratory pipetting robot and uses a 384-channel pipettor head for application of cells and compounds. The module contains 384 amplifier channels for fully parallel recordings using a digital amplifier. Success rates for completed experiments (1- to 4-point concentration-response curves for cells satisfying defined quality control parameters) of greater than 85% have been routinely achieved with, for example, HEK, CHO, and RBL cell lines expressing hNa1.7, hERG, Kir2.1, GABA, or glutamate receptors. Pharmacology experiments are recorded and analyzed using specialized software, and the pharmacology of hNa1.7 and hERG is described. Fast external solution exchange rates of <50 ms are demonstrated using Kir2.1. Short exposure times are achieved by stacking the external solutions inside the pipette of the robot to minimize exposure of the ligand on the receptor. This ensures that ligand-gated ion channels, for example, GABA and glutamate described in this report, can be reproducibly recorded. Stem cell-derived cardiomyocytes have also been used with success rates of 52% for cells that have a seal resistance of >200 MΩ, and recordings of voltage-gated Na and Ca are shown.
Automated patch clamp devices are now commonly used for studying ion channels. A useful modification of this approach is the replacement of the glass pipet with a thin planar glass layer with a small hole in the middle. Planar patch clamp devices, such as the three described in this unit, are overtaking glass pipets in popularity because they increase throughput, are easier to use, provide for the acquisition of high-quality and information-rich data, and allow for rapid perfusion and temperature control. Covered in this unit are two challenging targets in drug discovery: voltage-gated sodium subtype 1.7 (Na(V)1.7) and nicotinic acetylcholine α7 receptors (nAChα7R). Provided herein are protocols for recording activation and inactivation kinetics of Na(V)1.7, and activation and allosteric modulation of nAChα7R.
were performed on HEK-293 cells expressing wild-type or mutant hERG channels. Whole-cell patch clamp measurements of I hERG were made at 37 C and data are presented as mean 5 SEM, nR5. Commercially synthesized Cavalli-2 suppressed hERG tail currents (I hERG) with a half-maximal inhibitory concentration (IC 50) of 35.6 5 1.0 nM. Aromatic residues on the S6 helix are known to be structural determinants for high affinity hERG channel block. Mutation of Y652 residues to alanine markedly reduced the affinity of Cavalli-2 for hERG (100 nM Cavalli-2 caused 61.5 5 5.0% block of WT I hERG but only 23.0 5 4.1% of Y652A I hERG). The pore helical residues T623 and S624 have been reported to make important interactions with polar aromatic para-substituents of drugs. Despite the absence of polar para-substituents in the minimal hERG blocker, T623A I hERG (which required measuring inward tail currents in high [K þ ] at À120 mV), was blocked 47.5 5 4.2% by 100 nM Cavalli-2 compared to 73.8 5 7.1% of inward WT I hERG and S624A I hERG was blocked by 29.1 5 3.8% (compared to 61.5 5 5.0% WT I hERG). Combined with computational modelling of interactions of Cavalli-2 with the hERG pore, these results support binding modes in which S624 stabilises the protonated amino group of Cavalli-2 near the internal K binding site and T623 may promote favourable pore geometries for drug binding, possibly by stabilising optimal conformations of Y652.
In recent years, human stem cell-derived cardiomyocytes have proven to recapitulate key features of human cardiac electrophysiology in vitro. Furthermore, it has become apparent that the intact ensemble of cardiac ion channels is necessary to determine proarrhythmic effects reliably. Hence, due to their increasing availability, stem cell-derived cardiomyocytes have become the preferred choice of cardiac cells. This poster summarizes the promises and challenges of combining iPS derived cardiac myocytes with automated patch clamp. Features like high throughput, temperature control, easy internal solution exchange and full automation make planar patch clamp a desired method for characterizing iPS derived cardiomyocytes. One of the biggest challenges of planar patch clamp in this context is the fact that individual cells cannot be chosen, but cells will be selected randomly. In addition cells have to be harvested before the application to the patch clamp chip and cannot be patched as adherent cells. With this poster, we show our progress on these challenges. Two automated patch clamp platforms, the Patchliner, as a medium throughput, and the SyncroPatch 384PE, as a high throughput device, were used for this study. Pharmacological measurements in voltage clamp as well as current clamp will be shown also under physiological temperatures and perforated patch.
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