Modulation of Hyperpolarization-Activated Cation Currents (Ih) by Ethanol in Rat Hippocampal CA3 Pyramidal Neurons

It is well established that ethanol (EtOH), through the interaction with several membrane proteins, as well as intracellular pathways, is capable to modulate many neuronal function. Recent reports show that EtOH increases the firing rate of hippocampal GABAergic interneurons through the positive modulation of the hyperpolarization-activated cyclic nucleotide-gated (HCN) cation channels. This effect might be consistent with the increase of GABA release from presynaptic terminals observed in both CA1 and CA3 inhibitory synapses that leads the enhancement of the GABAergic system induced by EtOH. The activation of HCN produced an inward currents that are commonly called Ih. Ih play an important role for generating specific neuronal activities in different brain regions, including specific sub-regions of the hippocampal formation, such as CA1 and CA3 pyramidal neurons and hippocampal GABAergic interneurons. The main physiologic effect mediated by HCN-induced Ih is directed to the control of the neuronal resting membrane potential and action potential (AP) discharge as well as dampen synaptic integration. Since robust Ih are also present in CA3 glutamatergic neurons, I here investigated whether the action of EtOH in the control of CA3 excitability can be correlated with its possible direct interaction with these cation channels. For this purpose, patch-clamp experiments were performed in CA3 pyramidal neurons from hippocampal coronal slices obtained from male Sprague-Dawley rats. The data obtained demonstrated that EtOH is able to modulate Ih in biphasic manner depending on the concentrations used. Low EtOH concentrations enhanced Ih amplitude, while high reversibly reduced them. This biphasic action induced by EtOH reflects on firing rate and synaptic integration. In addition, in this reports it has been shown that EtOH modulates the function of HCN channels through interfering with the cAMP/AC/PKA intracellular pathways, an effect that is mimicked also by other endogenous compounds such as dopamine through D1 receptors activation. These data suggest that the HCN-mediated Ih currents in CA3 pyramidal neurons are sensitive to EtOH action, which at low or relevant concentrations is able to increase or reduce their function respectively. Altogether these data suggest a potential new mechanism of EtOH actions on hippocampal formation and may help to better understand the depressant central activity showed by this drug of abus

Bi-directional Modulation of Hyperpolarization-Activated Cation Currents (Ih) by Ethanol in Rat Hippocampal CA3 Pyramidal Neurons

It is widely acknowledged that ethanol (EtOH) can alter many neuronal functions, including synaptic signaling, firing discharge, and membrane excitability, through its interaction with multiple membrane proteins and intracellular pathways. Previous work has demonstrated that EtOH enhances the firing rate of hippocampal GABAergic interneurons and thus the presynaptic GABA release at CA1 and CA3 inhibitory synapses through a positive modulation of the hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channels. Activation of HCN channels produce an inward current, commonly called Ih, which plays an essential role in generating/regulating specific neuronal activities in GABAergic interneurons and principal glutamatergic pyramidal neurons such as those in the CA3 subregion. Since the direct effect of EtOH on HCN channels expressed in CA3 pyramidal neurons was not thoroughly elucidated, we investigated the possible interaction between EtOH and HCN channels and the impact on excitability and postsynaptic integration of these neurons. Patch-clamp recordings were performed in single CA3 pyramidal neurons from acute male rat coronal hippocampal slices. Our results show that EtOH modulates HCN-mediated Ih in a concentration-dependent and bi-directional manner, with a positive modulation at lower (20 mM) and an inhibitory action at higher (60-80 mM) concentrations. The modulation of Ih by EtOH was mimicked by forskolin, antagonized by different drugs that selectively interfere with the AC/cAMP/PKA intracellular pathway, as well as by the selective HCN inhibitor ZD7288. Altogether, these data further support the evidence that HCN channels may represent an important molecular target through which EtOH may regulate neuronal activity

Isolation rearing reduces neuronal excitability in dentate gyrus granule cells of adolescent C57BL/6J mice: role of gabaergic tonic currents and neurosteroids

Early-life exposure to stress, by impacting on a brain still under development, is considered a critical factor for the increased vulnerability to psychiatric disorders and abuse of psychotropic substances during adulthood. As previously reported, rearing C57BL/6J weanling mice in social isolation (SI) from their peers for several weeks, a model of prolonged stress, is associated with a decreased plasma and brain levels of neuroactive steroids such as 3α,5α-THP, with a parallel up-regulation of extrasynaptic GABAA receptors (GABAAR) in dentate gyrus (DG) granule cells compared to group-housed (GH) mice. In the present study, together with the SI-induced decrease in plasma concentration of both progesterone and 3a,5a-THP, and an increase in THIP-stimulated GABAergic tonic currents, patch-clamp analysis of DG granule cells revealed a significant decrease in membrane input resistance and action potential (AP) firing rate, in SI compared to GH mice, suggesting that SI exerts an inhibitory action on neuronal excitability of these neurons. Voltage-clamp recordings of glutamatergic spontaneous excitatory postsynaptic currents (sEPSCs) revealed a SI-associated decrease in frequency as well as a shift from paired-pulse (PP) depression to PP facilitation (PPF) of evoked EPSCs, indicative of a reduced probability of glutamate release. Daily administration of progesterone during isolation reverted the changes in plasma 3α,5α-THP as well as in GABAergic tonic currents and neuronal excitability caused by SI, but it had only a limited effect on the changes in the probability of presynaptic glutamate release. Overall, the results obtained in this work, together with those previously published, indicate that exposure of mice to SI during adolescence reduces neuronal excitability of DG granule cells, an effect that may be linked to the increased GABAergic tonic currents as a consequence of the sustained decrease in plasma and hippocampal levels of neurosteroids. All these changes may be consistent with cognitive deficits observed in animals exposed to such type of prolonged stres

Neuregulin1/ErbB4-induced migration in ST14A striatal progenitors: calcium-dependent mechanisms and modulation by NMDA receptor activation

<p>Abstract</p> <p>Background</p> <p>A number of studies have separately shown that the neuregulin1 (NRG1)/ErbB4 system and NMDA-type glutamate receptors (NMDARs) are involved in several aspects of neuronal migration. In addition, intracellular calcium fluctuations play central roles in neuronal motility. Stable expression of the tyrosine kinase receptor ErbB4 promotes migratory activity in the neural progenitor cell line ST14A upon NRG1 stimulation. In this work we analyzed the potential interactions between the NRG1/ErbB4 system and NMDARs in the ST14A migratory process as well as its calcium dependence.</p> <p>Results</p> <p>RT-PCR studies have shown that both native ST14A cells (non-expressing ErbB4), as well as ErbB4-transfected cells express low levels of a restricted number of NMDAR subunits: NR1, NR2C, NR2D and NR3B. The resulting NMDAR would form Ca²⁺channels characterized by low Mg²⁺-sensitivity and low Ca²⁺-permeability, generating small, long-lasting currents. Ca²⁺-imaging experiments showed slow [Ca²⁺]_iincreases in 45% of the cells following 8 μM NMDA stimulation. Basal migration of ErbB4-transfected ST14A cells was unaffected by 18 hrs NMDA incubation. However, over the same incubation time, NMDA was able to significantly enhance NRG1-induced migration. Pre-incubation with the intracellular calcium chelator BAPTA-AM reduced both NRG1- and NRG1/NMDA-stimulated migration, suggesting the involvement of Ca²⁺in these processes. NRG1 stimulation of ErbB4-transfected ST14A cells induced a sustained, long-lasting increase in [Ca²⁺]_i, in 99% of the cells. These intracellular Ca²⁺signals could be ascribed to both release from intracellular stores and influx from the extracellular medium trough a mechanism of store-operated calcium entry (SOCE). Short-time co-incubation of NMDA and NRG1 did not substantially modify the NRG1-induced intracellular calcium signals.</p> <p>Conclusions</p> <p>In summary, NRG1 stimulation of the ErbB4 receptor exerts a sustained [Ca²⁺]_iincrease in ST14A neural progenitors; NRG1-induced migration is Ca²⁺-dependent and can be positively modulated by activation of the NMDA receptor.</p

Docker4Circ: A Framework for the Reproducible Characterization of circRNAs from RNA-Seq Data

Recent improvements in cost-effectiveness of high-throughput technologies has allowed RNA sequencing of total transcriptomes suitable for evaluating the expression and regulation of circRNAs, a relatively novel class of transcript isoforms with suggested roles in transcriptional and post-transcriptional gene expression regulation, as well as their possible use as biomarkers, due to their deregulation in various human diseases. A limited number of integrated workflows exists for prediction, characterization, and differential expression analysis of circRNAs, none of them complying with computational reproducibility requirements. We developed Docker4Circ for the complete analysis of circRNAs from RNA-Seq data. Docker4Circ runs a comprehensive analysis of circRNAs in human and model organisms, including: circRNAs prediction; classification and annotation using six public databases; back-splice sequence reconstruction; internal alternative splicing of circularizing exons; alignment-free circRNAs quantification from RNA-Seq reads; and differential expression analysis. Docker4Circ makes circRNAs analysis easier and more accessible thanks to: (i) its R interface; (ii) encapsulation of computational tasks into docker images; (iii) user-friendly Java GUI Interface availability; and (iv) no need of advanced bash scripting skills for correct use. Furthermore, Docker4Circ ensures a reproducible analysis since all its tasks are embedded into a docker image following the guidelines provided by Reproducible Bioinformatics Project

Sex-dependent changes of hippocampal synaptic plasticity and cognitive performance in C57BL/6J mice exposed to neonatal repeated maternal separation

The repeated maternal separation (RMS) is a useful experimental model in rodents to study the long-term influence of early-life stress on brain neurophysiology. We here investigated the influence of RMS exposure on hippocampal inhibitory and excitatory synaptic transmission, long-term synaptic plasticity and the related potential alterations in learning and memory performance in adult male and female C57Bl/6J mice. Mice were separated daily from their dam for 360 min, from postnatal day 2 (PND2) to PND17, and experiments were performed at PND60. Patch-clamp recordings in hippocampal CA1 pyramidal neurons revealed a significant enhancement of GABAergic miniature IPSC (mIPSC) frequency and a decrease in the amplitude of glutamatergic mEPSCs in male mice exposed to RMS. Only a slight but significant reduction in the amplitude of GABAergic mIPSCs was observed in females exposed to RMS compared to the relative controls. A marked increase in long-term depression (LTD) at CA3-CA1 glutamatergic synapses and in the response to the CB1r agonist win55,212 were detected in RMS male but not female mice. An impaired spatial memory and a reduced preference for novelty were observed in males exposed to RMS but not in females. A single injection of -ethynyl estradiol at PND2 prevented the changes observed in RMS male mice, suggesting that estrogens may play a protective role early in life against the exposure to stressful conditions. Our findings strengthen the idea of a sex-dependent influence of RMS on long-lasting modifications in synaptic transmission, effects that may be relevant to cognitive performance

Docker4Circ: A Framework for the Reproducible Characterization of circRNAs from RNA-Seq Data.

Recent improvements in cost-effectiveness of high-throughput technologies has allowed RNA sequencing of total transcriptomes suitable for evaluating the expression and regulation of circRNAs, a relatively novel class of transcript isoforms with suggested roles in transcriptional and post-transcriptional gene expression regulation, as well as their possible use as biomarkers, due to their deregulation in various human diseases. A limited number of integrated workflows exists for prediction, characterization, and differential expression analysis of circRNAs, none of them complying with computational reproducibility requirements. We developed Docker4Circ for the complete analysis of circRNAs from RNA-Seq data. Docker4Circ runs a comprehensive analysis of circRNAs in human and model organisms, including: circRNAs prediction; classification and annotation using six public databases; back-splice sequence reconstruction; internal alternative splicing of circularizing exons; alignment-free circRNAs quantification from RNA-Seq reads; and differential expression analysis. Docker4Circ makes circRNAs analysis easier and more accessible thanks to: (i) its R interface; (ii) encapsulation of computational tasks into docker images; (iii) user-friendly Java GUI Interface availability; and (iv) no need of advanced bash scripting skills for correct use. Furthermore, Docker4Circ ensures a reproducible analysis since all its tasks are embedded into a docker image following the guidelines provided by Reproducible Bioinformatics Project