7 research outputs found
A Commentary on Comparison of spike parameters from optically identified GABAergic and glutamatergic neurons in sparse cortical cultures
No full textAgonist and antagonist effects of tobacco-related nitrosamines on human α4β2 nicotinic acetylcholine receptors.
No full textRegulation of the ‘neuronal’ nicotinic acetylcholine receptors (nAChRs) is implicated in both tobacco addiction and smoking-dependent tumor promotion. Some of these effects are caused by the tobacco-derived N-nitrosamines, which are carcinogenic compounds that avidly bind to nAChRs. However, the functional effects of these drugs on specific nAChR subtypes are largely unknown. By using patch-clamp methods, we tested 4-(methylnitrosamine)-1-(3-pyridyl)-1-butanone (NNK) and N’-nitrosonornicotine (NNN) on human α4β2 nAChRs. These latter are widely distributed in the mammalian brain and are also frequently expressed outside the nervous system. NNK behaved as a partial agonist, with an apparent EC50 of 16.7 μM. At 100 μM, it activated 16 % of the maximal current activated by nicotine. When NNK was co-applied with nicotine, it potentiated the currents elicited by nicotine concentrations ≤ 100 nM. At higher concentrations of nicotine, NNK always inhibited the α4β2 nAChR. In contrast, NNN was a pure inhibitor of this nAChR subtype, with IC50 of approximately 1 nM in the presence of 10 μM nicotine. The effects of both NNK and NNN were mainly competitive and largely independent of Vm. The different actions of NNN and NNK must be taken into account when interpreting their biological effects in vitro and in vivo
Exact distinction of excitatory and inhibitory neurons in neural networks: a study with GAD67-GFP neurons optically and electrophysiologically recognized on multi-electrode arrays
No full textDistinguishing excitatory from inhibitory neurons with multielectrode array (MEA) recordings is a serious experimental challenge. The current methods, developed in vitro, mostly rely on spike waveform analysis. These however often display poor resolution and may produce errors caused by the variability of spike amplitudes and neuron shapes. Recent recordings in human brain suggest that the spike waveform features correlate with time-domain statistics such as spiking rate, autocorrelation and coefficient of variation. However, no precise criteria are available to exactly assign identified units to specific neuronal types, either in vivo or in vitro. To solve this problem, we combined MEA recording with fluorescence imaging of neocortical cultures from mice expressing green fluorescent protein (GFP) in GABAergic cells. In this way, we could sort out ‘authentic excitatory neurons’ (AENs) and ‘authentic inhibitory neurons’ (AINs). We thus characterized 1275 units (from 405 electrodes, n=10 experiments), based on autocorrelation, burst length, spike number, spiking rate, squared coefficient of variation and Fano factor (the ratio between spike-count variance and mean). These metrics differed by about one order of magnitude between AINs and AENs. In particular, the Fano factor turned out to provide a firing code which exactly (no overlap) recognizes excitatory and inhibitory units. The difference in Fano factor between all of the identified AEN and AIN groups was highly significant (p < 10-8, ANOVA post-hoc Tukey test). Our results indicate a statistical metric-based approach to distinguish excitatory from inhibitory neurons independently from the spike width
Multi-electrode array study of neuronal cultures expressing nicotinic β2-V287L subunits, linked to autosomal dominant nocturnal frontal lobe epilepsy. An in vitro model of spontaneous epilepsy.
No full textAutosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) is a partial sleep-related epilepsy which can be caused by mutant neuronal nicotinic acetylcholine receptors (nAChR). We applied multi-electrode array (MEA) recording methods to study the spontaneous firing activity of neocortical cultures obtained from mice expressing or not (WT) an ADNFLE-linked nAChR subunit (β2-V287L).More than 100,000 up-states were recorded during experiments sampling from several thousand neurons. Data were analyzed by using a fast sliding-window procedure which computes histograms of the up-state durations. Differently from the WT, cultures expressing β2-V287L displayed long (10-32 s) synaptic-induced up-state firing events. The occurrence of such long up-states was prevented by both negative (gabazine, penicillin G) and positive (benzodiazepines) modulators of GABAA receptors. Carbamazepine (CBZ), a drug of choice in ADNFLE patients, also inhibited the long up-states at micromolar concentrations. In cultures expressing β2-V287L, no significant effect was observed on the action potential waveform either in the absence or in the presence of pharmacological treatment.Our results show that some aspects of the spontaneous hyperexcitability displayed by a murine model of a human channelopathy can be reproduced in neuronal cultures. In particular, our cultures represent an in vitro chronic model of spontaneous epileptiform activity, i.e. not requiring pre-treatment with convulsants. This opens the way to the study in vitro of the role of β2-V287L on synaptic formation. Moreover, our neocortical cultures on MEA platforms allow to determine the effects of prolonged pharmacological treatment on spontaneous network hyperexcitability (which is impossible in the short-living brain slices). Methods such as the one we illustrate in the present paper should also considerably facilitate the preliminary screening of antiepileptic drugs, thereby reducing the number of in vivo experiments
The role of nicotinic acetylcholine receptors in autosomal dominant nocturnal frontal lobe epilepsy.
No full textAutosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) is a focal epilepsy with attacks typically arising in the frontal lobe during non rapid eye movement (NREM) sleep. It is characterized by clusters of complex and stereotyped hypermotor seizures, frequently accompanied by sudden arousals. Cognitive and psychiatric symptoms may be also observed. Approximately 12% of the ADNFLE families carry mutations on genes coding for subunits of the heteromeric neuronal nicotinic receptors (nAChRs). This is consistent with the widespread expression of these receptors, particularly the α4β2* subtype, in the neocortex and thalamus. However, understanding how mutant nAChRs lead to partial frontal epilepsy is far from being straightforward because of the complexity of the cholinergic regulation in both developing and mature brains. The relation with the sleep-waking cycle must be also explained. We discuss some possible pathogenetic mechanisms in the light of recent advances about the nAChR role in prefrontal regions as well as the studies carried out in murine models of ADNFLE. Functional evidence points to alterations in prefrontal GABA release, and the synaptic unbalance probably arises during the cortical circuit maturation. Although most of the available functional evidence concerns mutations on nAChR subunit genes, other genes have been recently implicated in the disease, such as KCNT1 (coding for a Na+-dependent K+ channel), DEPD5 (Dishevelled, Egl-10 and Pleckstrin Domain-containing protein 5), and CRH (Corticotropin-Releasing Hormone). Overall, the uncertainties about both the etiology and the pathogenesis of ADNFLE point to the current gaps in our knowledge the regulation of neuronal networks in the cerebral cortex
Deregulation of ion channel and transporter encoding genes in paediatric gliomas
No full textBrain tumours, including the majority gliomas, are the leading cause of cancer-related death in children. World Health Organization (WHO) has divided paediatric brain tumours into different grades and, based upon cDNA microarray data identifying gene expression profiles (GEPs), it has become evident in the last decade that the various grades involve different types of genetic alterations. However, it is not known whether ion channel and transporter genes, intimately involved in brain functioning, are associated with such GEPs.We determined the GEPs in an available cohort of ten paediatric brain tumours initially by comparing the data obtained from four primary tumour samples and corresponding short-term cultures. The correspondence between the two types of samples was statistically significant. We then performed bioinformatic analyses on those samples (a total of nine) which corresponded to tumors of glial origin, either tissues or cell cultures, depending on the best RNA integrity number (RIN). We used R software to evaluate the genes which were differentially expressed (DE) in gliomas compared with normal brain. Applying a p-value below 0.01 and fold change = 4, led to identification of 2284 DE genes. Through a Functional Annotation Analysis (FAA) using the NIH-DAVID software, the DE genes turned out to be associated mainly with: immune/inflammatory response, cell proliferation and survival, cell adhesion and motility, neuronal phenotype and ion transport.We have shown that GEPs of paediatric brain tumours can be studied using either primary tumour samples or short-term cultures with similar results. From FAA, we concluded that, among DE genes, paediatric gliomas show a strong deregulation of genes related to ion channels and transporters
