Differential expression of voltage-gated K+ currents in medial septum/diagonal band complex neurons exhibiting distinct firing phenotypes

The medial septum/diagonal band complex (MSDB) controls hippocampal excitability, rhythms and plastic processes. Medial septal neuronal populations display heterogeneous firing patterns. In addition, some of these populations degenerate during age-related disorders (e.g. cholinergic neurons). Thus, it is particularly important to examine the intrinsic properties of theses neurons in order to create new agents that effectively modulate hippocampal excitability and enhance memory processes. Here, we have examined the properties of voltage-gated, K+ currents in electrophysiologically-identified neurons. These neurons were taken from young rat brain slices containing the MS/DB complex. Whole-cell, patch recordings of outward currents were obtained from slow firing, fast-spiking, regular-firing and burst-firing neurons. Slow firing neurons showed depolarization-activated K+ current peaks and densities larger than in other neuronal subtypes. Slow firing total current exhibited an inactivating A-type current component that activates at subthreshold depolarization and was reliably blocked by high concentrations of 4-AP. In addition, slow firing neurons expressed a low-threshold delayed rectifier K+ current component with slow inactivation and intermediate sensitivity to tetraethylamonium. Fast-spiking neurons exhibited the smaller IK and IA current densities. Burst and regular firing neurons displayed an intermediate firing phenotype with IK and IA current densities that were larger than the ones observed in fastspiking neurons but smaller than the ones observed in slow-firing neurons. In addition, the prevalence of each current differed among electrophysiological groups with slow firing and regular firing neurons expressing mostly IA and fast spiking and bursting neurons exhibiting mostly delayer rectifier K+ currents with only minimal contributions of the IA. The pharmacological or genetic modulations of these currents constitute an important target for the treatment of age-related disorders

Differential changes in mGlu2 and mGlu3 gene expression following pilocarpine-induced status epilepticus: A comparative real-time PCR analysis

Group II metabotropic glutamate (mGlu II) receptors subtype 2 and 3 (mGlu2 and mGlu3) are subtle regulators of neuronal excitability and synaptic plasticity in the hippocampus. In recent years, researchers have investigated the potential neuroprotective and anticonvulsant effects of compounds acting on mGlu II receptors. However, abnormal expression and function of mGlu2 and mGlu3 have been reported in temporal lobe epilepsy, a phenomena that may limit the therapeutic effectiveness of these potentially new antiepileptic drugs. Here, we investigated seizure-induced changes in mGlu2 and mGlu3 mRNA following pilocarpine-inducted status epilepticus (SE) and subsequent epileptogenesis. Relative changes in gene expression were assessed by comparative analysis of quantitative real-time PCR (qrtPCR) by the delta–delta CT method. Pilocarpine-treated and control rats were sacrificed at different periods (24 h, 10 days, one month and more than two months) following SE. Total RNA was isolated from microdissected dentate gyrus and processed for RT-PCR and qrtPCR using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an endogenous control gene. Analysis of relative quantification (RQ) ratios of mGlu2 and mGlu3 mRNA expression revealed a significant down-regulation of both targets at 24 h after SE. Gene expression partially recovered at 10 days following SE reaching control levels at one month after SE. Two month after SE, mGlu2 mRNA expression was significantly down-regulated to ~ 41% of control expression whereas mGlu3 mRNA was comparable to control levels. Our data indicate that mGlu2 and mGlu3 expression is dynamically down-regulated or selectively enhanced during critical periods of epileptogenesis. Seizure-induced differential dysregulation of mGlu2 and mGlu3 receptors may affect the availability of these molecular targets for therapeutic compounds in epilepsy

Deficit of Kcnma1 mRNA expression in the dentate gyrus of epileptic rats

Epileptogenesis in mesial temporal lobe epilepsy is determined by several factors including abnormalities in the expression and function of ion channels. Here, we report a long-lasting deficit in gene expression of Kcnma1 coding for the large-conductance calcium-activated potassium (BK, MaxiK) channel alpha-subunits after pilocarpine-induced status epilepticus. By using comparative real-time PCR, Taqman gene expression assays, and the delta-delta comparative threshold method we detected a significant reduction in Kcnma1 expression in microdissected dentate gyrus at different intervals after status epilepticus (24 h, 10 days, 1 month, and more than 2 months). BK channels are key regulators of neuronal excitability and transmitter release. Hence, defective Kcnma1 expression may play a critical role in the pathogenesis of mesial temporal lobe epilepsy

Deficit of Kcnma1 mRNA expression in the dentate gyrus of epileptic rats

Epileptogenesis in mesial temporal lobe epilepsy is determined by several factors including abnormalities in the expression and function of ion channels. Here, we report a long-lasting deficit in gene expression of Kcnma1 coding for the large-conductance calcium-activated potassium (BK, MaxiK) channel α-subunits after pilocarpine-induced status epilepticus. By using comparative realtime PCR, Taqman gene expression assays, and the delta-delta comparative threshold method we detected a significant reduction in Kcnma1 expression in microdissected dentate gyrus at different intervals after status epilepticus (24 h, 10 days, 1 month, and more than 2 months). BK channels are key regulators of neuronal excitability and transmitter release. Hence, defective Kcnma1 expression may play a critical role in the pathogenesis of mesial temporal lobe epilepsy

“Upregulation of STREX splice variant of the large conductance Ca2+-activated potassium (BK) channel in a rat model of mesial temporal lobe epilepsy”

Functional properties of large conductance Ca2+ activated potassium (BK) channels are determined by complex alternative splicing of the Kcnma1 gene encoding the alpha pore-forming subunit. Inclusion of the STREX exon in a C-terminal splice site is dynamically regulated and confers enhanced Ca2+ sensitivity and channel inhibition via cAMP-dependent phosphorylation. Here, we describe a real time quantitative PCR (qPCR) approach to investigate relative changes in the expression of STREX and ZERO splice variants using a newly designed set of probes and primers for TaqMan-based qPCR analysis of cDNA from the rat dentate gyrus at different time points following pilocarpine-induced status epilepticus. Reduction in Kcnma1 gene expression is associated with a relative increase of STREX splice variant. Relative expression of STREX variant mRNA was increased at 10 days and at more than 1 month following status epilepticus. The biological consequences of seizure-related changes in alternative splicing of Kcnma1 deserve additional investigation

Characterization of Glutamatergic Phenotypes in Hybrid Septal Neuroblastoma (SN56) cells

Septal Neuroblastoma (SN 56) cells are hybrid cells made through cell fusions between quiescent medial septum neurons (cholinergic) and tumoral neuroblastoma cells. Cholinergic cells synthesize and release the neurotransmitter acetylcholine. Preliminary studies in our laboratory revealed that SN 56 neurons also express the vesicular glutamate transporter type 1 (VGluT1), a protein that is normally produced by glutamatergic neurons. This discovery prompted us to hypothesize that SN 56 neurons may also co-express a glutamatergic phenotype which is important because glutamatergic neurons have been associated to the pathogenesis of neurological disorders such as Alzheimer’s disease. To assess whether SN 56 neurons express in fact both phenotypes, we conducted experiments in differentiated and no differentiated SN 56 cell, to confirm the expression of glutamatergic phenotype, by qPCR, western blotting and Immunocytochemistry assay. The cells are cultured in an incubator gassed with 5% CO2 at 37°C. After differentiation for 3-5 days with cAMP and retinoic acid, SN 56 cells were prepared for qPCR, western blotting and immunocytochemistry. Cells were separated by each experiment, primary antibodies or primers against NMDA glutamate receptor subunit NR2B, VG luT1 and vesicular cholinergic transport (ChAT) how positive control were used to confirm our hypothesis,. Expression of these markers will indicate a glutamatergic phenotype. After secondary detection with appropriate fluorescently-labeled antibodies we confirmed that differentiated SN 56 neurons express glutamate NR2B receptor subtype and the VGluT1 transporter in both post-synaptic and presynaptic structures respectively. Hence, these findings support our hypothesis that SN 56 neurons can co-express both cholinergic and glutamatergic phenotype

Downregulation of BK channel expression in the pilocarpine model of temporal lobe epilepsy

In the hippocampus, BK channels are preferentially localized in presynaptic glutamatergic terminals including mossy fibers where they are thought to play an important role regulating excessive glutamate release during hyperactive states. Large conductance calcium-activated potassium channels (BK, MaxiK, Slo) have recently been implicated in the pathogenesis of genetic epilepsy. However, the role of BK channels in acquired mesial temporal lobe epilepsy (MTLE) remains unknown. Here we used immunohistochemistry, laser scanning confocal microscopy (LSCM), western immunoblotting and RT-PCR to investigate the expression pattern of the alpha-pore forming subunit of BK channels in the hippocampus and cortex of chronically epileptic rats obtained by the pilocarpine model of MTLE. All epileptic rats experiencing recurrent spontaneous seizures exhibited a significant down-regulation of BK channel immunostaining in the mossy fibers at the hilus and stratum lucidum of the CA3 area. Quantitative analysis of immunofluorescence signals by LSCM revealed a significant 47% reduction in BK channel in epileptic rats when compared to age-matched non-epileptic control rats. These data correlate with a similar reduction in BK channel protein levels and transcripts in the cortex and hippocampus. Our data indicate a seizure-related down-regulation of BK channels in chronically epileptic rats. Further functional assays are necessary to determine whether altered BK channel expression is an acquired channelopathy or a compensatory mechanism affecting the network excitability in MTLE. Moreover, seizure-mediated BK down-regulation may disturb neuronal excitability and presynaptic control at glutamatergic terminals triggering exaggerated glutamate release and seizures

Measurement of the t-channel single top quark production cross section in pp collisions at √s =7 TeV

Peer reviewe

Calibration of the CMS Drift Tube Chambers and Measurement of the Drift Velocity with Cosmic Rays

Peer reviewe

Novel genes and sex differences in COVID-19 severity

[EN] Here, we describe the results of a genome-wide study conducted in 11 939 coronavirus disease 2019 (COVID-19) positive cases with an extensive clinical information that were recruited from 34 hospitals across Spain (SCOURGE consortium). In sex-disaggregated genome-wide association studies for COVID-19 hospitalization, genome-wide significance (P < 5 × 10−8) was crossed for variants in 3p21.31 and 21q22.11 loci only among males (P = 1.3 × 10−22 and P = 8.1 × 10−12, respectively), and for variants in 9q21.32 near TLE1 only among females (P = 4.4 × 10−8). In a second phase, results were combined with an independent Spanish cohort (1598 COVID-19 cases and 1068 population controls), revealing in the overall analysis two novel risk loci in 9p13.3 and 19q13.12, with fine-mapping prioritized variants functionally associated with AQP3 (P = 2.7 × 10−8) and ARHGAP33 (P = 1.3 × 10−8), respectively. The meta-analysis of both phases with four European studies stratified by sex from the Host Genetics Initiative (HGI) confirmed the association of the 3p21.31 and 21q22.11 loci predominantly in males and replicated a recently reported variant in 11p13 (ELF5, P = 4.1 × 10−8). Six of the COVID-19 HGI discovered loci were replicated and an HGI-based genetic risk score predicted the severity strata in SCOURGE. We also found more SNP-heritability and larger heritability differences by age (<60 or ≥60 years) among males than among females. Parallel genome-wide screening of inbreeding depression in SCOURGE also showed an effect of homozygosity in COVID-19 hospitalization and severity and this effect was stronger among older males. In summary, new candidate genes for COVID-19 severity and evidence supporting genetic disparities among sexes are provided.S