ABSTRACT
A neuroprotective and nootropic drug Noopept increased the frequency of spontaneous calcium transients in neurons of CA1 radial layer in cultured rat hippocampal slices. In contrast, the drug exerted no significant effect on intracellular calcium concentration and its dynamics in neurons of hippocampal CA1 pyramidal layer.
Subject(s)
CA1 Region, Hippocampal/drug effects , Calcium/metabolism , Dipeptides/pharmacology , Neurons/drug effects , Neuroprotective Agents/pharmacology , Action Potentials/drug effects , Animals , Animals, Newborn , CA1 Region, Hippocampal/cytology , CA1 Region, Hippocampal/metabolism , Microtomy , Neurons/cytology , Neurons/metabolism , Nootropic Agents/pharmacology , Rats , Rats, Wistar , Tissue Culture TechniquesABSTRACT
iPSC-derived cells (from induced pluripotent stem cells) are a useful source that provide a powerful and widely accepted tool for the study of various types of human cells in vitro. Indeed, iPSC-derived cells from patients with hereditary diseases have been shown to reproduce the hallmarks of these diseases in vitro, phenotypes that can then also be manipulated in vitro. Quantitative reverse transcription PCR (qRT-PCR) is often used to characterize the progress of iPSC differentiation, validate mature cell types and to determine levels of pathological markers. Quantitative reverse transcription PCR (qRT-PCR) is used to quantify mRNA levels. This method requires some way of normalizing the data, typically by relating the obtained levels of gene expression to the levels of expression of a "house keeping gene", a gene whose expression is presumed not to change during manipulation of the cells. In the literature, typically only one such reference gene is used and its stability of expression during cell manipulation is not demonstrated. We are not aware of any study systematically looking at the expression of such genes in human iPSC or during their differentiation into neurons. Here we compare the expression of 16 reference genes in iPSC, neural stem cells (NSC) and neurons derived from iPSC. The applications GeNorm and NormFinder were used to identify the most suitable reference genes. We showed that ACTb, C1orf43, PSMB4, GAPDH and HMBS have the most stable expression. The use of these reference genes allows an accurate normalization of qRT-PCR results in all the cell types discussed above. We hope that this report will help to enable the performance of proper qRT-PCR results normalization in studies with iPSC-derived cells and in disease-modeling reports.
Subject(s)
Induced Pluripotent Stem Cells/metabolism , Real-Time Polymerase Chain Reaction/standards , Actins/genetics , Actins/metabolism , Cells, Cultured , Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)/genetics , Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)/metabolism , Humans , Hydroxymethylbilane Synthase/genetics , Hydroxymethylbilane Synthase/metabolism , Induced Pluripotent Stem Cells/cytology , Neurogenesis , Neurons/cytology , Neurons/metabolism , Proteasome Endopeptidase Complex/genetics , Proteasome Endopeptidase Complex/metabolism , Reference StandardsABSTRACT
In immature neurons activation of ionotropic GABA receptors induces depolarizing membrane responses due to a high intracellular Cl(-) concentration ([Cl(-)](i)). However, it is difficult to draw conclusions about the functional consequences of subthreshold GABAergic depolarizations, since GABAergic membrane shunting and additional effects on voltage-dependent ion channels or action potential threshold must be considered. To systematically investigate factors that determine the GABAergic effect on neuronal excitability we performed whole cell patch-clamp recordings from Cajal-Retzius cells in immature rat neocortex, using [Cl(-)](i) between 10 and 50 mM. The effect of focal GABA application was quantified by measuring various parameters of GABAergic responses including the shift in minimal threshold current (rheobase). The rheobase shift was correlated with other parameters of the GABAergic responses by multiple linear regression analyses with a set of simple mathematical models. Our experiments demonstrate that focal GABA application induces heterogeneous rheobase shifts in Cajal-Retzius cells that could not be predicted reliably from [Cl(-)](i) or the GABAergic membrane depolarization. Implementation of a linear mathematical model, which takes the GABAergic membrane conductance and the difference between action potential threshold and GABA reversal potential into account, resulted in a close correlation between calculated and experimentally obtained rheobase shifts. Addition of a linear term proportional to the GABAergic membrane depolarization improved the accuracy of correlation. The main advantage of using multiple linear regression with simple models is that direction and strength of GABAergic excitability shifts can be analyzed by using only measured parameters of GABAergic responses and with minimal a priori information about cellular parameters.
Subject(s)
Chlorides/pharmacology , GABAergic Neurons/physiology , Interneurons/physiology , Neocortex/cytology , Receptors, GABA-A/physiology , gamma-Aminobutyric Acid/physiology , Action Potentials/drug effects , Action Potentials/physiology , Animals , Animals, Newborn , Chlorides/metabolism , Differential Threshold , GABA Antagonists/pharmacology , GABAergic Neurons/drug effects , Interneurons/drug effects , Ion Channel Gating/drug effects , Ion Channel Gating/physiology , Membrane Potentials/drug effects , Membrane Potentials/physiology , Models, Neurological , Neocortex/growth & development , Patch-Clamp Techniques , Pyridazines/pharmacology , Rats , Rats, WistarABSTRACT
Lanthanum ions (10-1000 microM) potentiated GABA-activated currents and increased the sensitivity of GABA(A) receptors in isolated Purkinje cells from rat cerebellum. GABA receptors in Purkinje cells are comparable to receptors on other cells by their sensitivity lanthanum ions.
