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Whole-cell Patch-clamp Experiments (whole-cell + patch-clamp_experiment)
Selected AbstractsInvolvement of Calmodulin in Glucagon-Like Peptide 1(7-36) Amide-Induced Inhibition of the ATP-Sensitive K+ Channel in Mouse Pancreatic ,-CellsEXPERIMENTAL PHYSIOLOGY, Issue 3 2001W. G. Ding The present investigation was designed to examine whether calmodulin is involved in the inhibition of the ATP-sensitive K+ (KATP) channel by glucagon-like peptide 1(7-36) amide (GLP-1) in mouse pancreatic ,-cells. Membrane potential, single channel and whole-cell currents through the KATP channels, and intracellular free Ca2+ concentration ([Ca2+]i) were measured in single mouse pancreatic ,-cells. Whole-cell patch-clamp experiments with amphotericin-perforated patches revealed that membrane conductance at around the resting potential is predominantly supplied by the KATP channels in mouse pancreatic ,-cells. The addition of 20 nM GLP-1 in the presence of 5 mM glucose significantly reduced the membrane KATP conductance, accompanied by membrane depolarization and the generation of electrical activity. A calmodulin inhibitor N -(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide (W-7, 20 ,M) completely reversed the inhibitory actions of GLP-1 on the membrane KATP conductance and resultant membrane depolarization. Cell-attached patch recordings confirmed the inhibition of the KATP channel activity by 20 nM GLP-1 and its restoration by 20 ,M W-7 or 10 ,M calmidazolium at the single channel level. Bath application of 20 ,M W-7 also consistently abolished the GLP-1-evoked increase in [Ca2+]i in the presence of 5 mM glucose. These results strongly suggest that the mechanisms by which GLP-1 inhibits the KATP channel activity accompanied by the initiation of electrical activity in mouse pancreatic ,-cells include a calmodulin-dependent mechanism in addition to the well-documented activation of the cyclic AMP-protein kinase A system. [source] Cerebellar granule cells cultured from adolescent rats express functional NMDA receptors: an in vitro model for studying the developing cerebellumJOURNAL OF NEUROCHEMISTRY, Issue 2 2008R. Lisa Popp Abstract In the developing rat cerebellum functional NMDA receptors (NMDARs) expressing the NR2C subunit have been identified on or after postnatal day 19. We obtained primary cultured cells from 19- to 35-day-old rat cerebellum that expressed few oligodendrocytes or astrocytes. Cultured cells were immunoreactive for neuron-specific proteins thus indicating a neuronal population. The primary neuron present was the granule cell as indicated by immunofluorescence for the GABAA alpha 6 subunit. Whole-cell patch-clamp experiments indicated that functional NMDARs were present. Functional characteristics of NMDARs expressed in cerebellar granule cells (CGCs) obtained from adolescent animals were similar to those previously reported for NMDARs expressed in CGCs obtained from neonatal rats. Cultured CGCs obtained from older animals contained NMDARs that were inhibited by EtOH and were less sensitive to the NR2B subunit-specific antagonist Ro 25-6981. Furthermore, NMDA-induced currents were smaller than those observed in CGCs. Western blot analysis indicated the presence of the NMDA NR2A and NR2C subunits, but not the NR2B in cultures obtained from the adolescent rats. CGCs obtained from adolescent rats express functional NMDARs consistent with a developmental profile observed in vivo. [source] Low-threshold heat response antagonized by capsazepine in chick sensory neurons, which are capsaicin-insensitiveEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 10 2000Antonia Marín-Burgin Abstract The heat-transducing receptor VR1 cloned from rat sensory neurons can be activated by both noxious heat and capsaicin. As the response of sensory neurons to capsaicin is species dependent, it is conceivable that the responses to noxious heat and to capsaicin are transduced by distinct receptors across different species. Therefore, we investigated responses to noxious heat from a capsaicin-insensitive (chick) and a capsaicin-sensitive (rat) species. In chick, whole-cell patch-clamp experiments in isolated dorsal root ganglion neurons revealed two populations of neurons with different thresholds to noxious heat, activated at ,,43 °C and ,,53 °C. In cobalt uptake experiments, the proportion of neurons showing a heat-induced response increased with increasing heat stimuli. Application of capsaicin (1,10 ,m) did not result in inward currents or cobalt uptake. Rat neurons yielded comparable results in heat experiments, but were capsaicin-sensitive. Although chick neurons are insensitive to capsaicin, the competitive capsaicin antagonist capsazepine (1,10 ,m) was effective in blocking heat-induced responses, verified by patch-clamp and cobalt uptake methods. The noncompetitive capsaicin antagonist ruthenium red (10 ,m) reduced to almost nil the proportion of heat-responsive neurons identified with the cobalt uptake method. These findings suggest that chick DRG neurons express a low-threshold heat-transducing receptor with a pharmacological profile distinct from the low-threshold heat receptor VR1 cloned from rat DRG neurons. The data support the idea that there might be heat receptor subtypes with differences in the capsaicin binding site. [source] Surface protein patterns govern morphology, proliferation, and expression of cellular markers but have no effect on physiological properties of cortical precursor cellsJOURNAL OF NEUROSCIENCE RESEARCH, Issue 11 2008Anna K. Magnusson Abstract The ability to differentiate and give rise to neurons, astrocytes, and oligodendrocytes is an inherent feature of neural stem cells, which raises hopes for cell-based therapies of neurodegenerative diseases. However, there are many hurdles to cross before such regimens can be applied clinically. A considerable challenge is to elucidate the factors that contribute to neural differentiation. In this study, we evaluated the possibility of steering neuronal maturation by growing cortical precursor cells on microscale surface patterns of extracellular matrix (ECM) proteins. When the cells were encouraged to extend processes along lines of ECM proteins, they displayed a much more mature morphology, less proliferation capacity, and greater expression of a neuronal marker in comparison with cells grown in clusters on ECM dots. This implied that the growth pattern alone could play a crucial role for neural differentiation. However, in spite of the strikingly different morphology, when performing whole-cell patch-clamp experiments, we never observed any differences in the functional properties between cells grown on the two patterns. These results clearly demonstrate that morphological appearances are not representative measures of the functional phenotype or grade of neuronal maturation, stressing the importance of complementary electrophysiological evidence. To develop successful transplantation therapies, increased cell survival is critical. Because process-bearing neurons are sensitive and break easily, it would be of clinical interest to explore further the differentiating capacity of the cells cultured on the ECM dot pattern, described in this article, which are devoid of processes but display the same functional properties as neurons with mature morphology. © 2008 Wiley-Liss, Inc. [source] Electrophysiological characterization of the SK channel blockers methyl-laudanosine and methyl-noscapine in cell lines and rat brain slicesBRITISH JOURNAL OF PHARMACOLOGY, Issue 6 2004Jacqueline Scuvée-Moreau We have recently shown that the alkaloid methyl-laudanosine blocks SK channel-mediated afterhyperpolarizations (AHPs) in midbrain dopaminergic neurones. However, the relative potency of the compound on the SK channel subtypes and its ability to block AHPs of other neurones were unknown. Using whole-cell patch-clamp experiments in transfected cell lines, we found that the compound blocks SK1, SK2 and SK3 currents with equal potency: its mean IC50s were 1.2, 0.8 and 1.8 ,M, respectively. IK currents were unaffected. In rat brain slices, methyl-laudanosine blocked apamin-sensitive AHPs in serotonergic neurones of the dorsal raphe and noradrenergic neurones of the locus coeruleus with IC50s of 21 and 19 ,M, as compared to 15 ,M in dopaminergic neurones. However, at 100 ,M, methyl-laudanosine elicited a constant hyperpolarization of serotonergic neurones of about 9 mV, which was inconsistently (i.e. not in a reproducible manner) antagonized by atropine and hence partly due to the activation of muscarinic receptors. While exploring the pharmacology of related compounds, we found that methyl-noscapine also blocked SK channels. In cell lines, methyl-noscapine blocked SK1, SK2 and SK3 currents with mean IC50s of 5.9, 5.6 and 3.9 ,M, respectively. It also did not block IK currents. Methyl-noscapine was slightly less potent than methyl-laudanosine in blocking AHPs in brain slices, its IC50s being 42, 37 and 29 ,M in dopaminergic, serotonergic and noradrenergic neurones, respectively. Interestingly, no significant non-SK effects were observed with methyl-noscapine in slices. At a concentration of 300 ,M, methyl-noscapine elicited the same changes in excitability in the three neuronal types than did a supramaximal concentration of apamin (300 nM). Methyl-laudanosine and methyl-noscapine produced a rapidly reversible blockade of SK channels as compared with apamin. The difference between the IC50s of apamin (0.45 nM) and methyl-laudanosine (1.8 ,M) in SK3 cells was essentially due to a major difference in their k,1 (0.028 s,1 for apamin and 20 s,1 for methyl-laudanosine). These experiments demonstrate that both methyl-laudanosine and methyl-noscapine are medium potency, quickly dissociating, SK channel blockers with a similar potency on the three SK subtypes. Methyl-noscapine may be superior in terms of specificity for the SK channels. British Journal of Pharmacology (2004) 143, 753,764. doi:10.1038/sj.bjp.0705979 [source] | ||||||||||