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Whole Cell Recordings (whole + cell_recording)
Selected AbstractsRegulatory Mechanisms and Physiological Relevance of a Voltage-Gated H+ Channel in Murine Osteoclasts: Phorbol Myristate Acetate Induces Cell Acidosis and the Channel Activation,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 11 2003Hiroyuki Mori Abstract The voltage-gated H+ channel is a powerful H+ extruding mechanism of osteoclasts, but its functional roles and regulatory mechanisms remain unclear. Electrophysiological recordings revealed that the H+ channel operated on activation of protein kinase C together with cell acidosis. Introduction: H+ is a key signaling ion in bone resorption. In addition to H+ pumps and exchangers, osteoclasts are equipped with H+ conductive pathways to compensate rapidly for pH imbalance. The H+ channel is distinct in its strong H+ extrusion ability and voltage-dependent gatings. Methods: To investigate how and when the H+ channel is available in functional osteoclasts, the effects of phorbol 12-myristate 13-acetate (PMA), an activator for protein kinase C, on the H+ channel were examined in murine osteoclasts generated in the presence of soluble RANKL (sRANKL) and macrophage-colony stimulating factor (M-CSF). Results and Conclusions: Whole cell recordings clearly showed that the H+ current was enhanced by increasing the pH gradient across the plasma membrane (,pH), indicating that the H+ channel changed its activity by sensing ,pH. The reversal potential (Vrev) was a valuable tool for the real-time monitoring of ,pH in clamped cells. In the permeabilized patch, PMA (10 nM-1.6 ,M) increased the current density and the activation rate, slowed decay of tail currents, and shifted the threshold toward more negative voltages. In addition, PMA caused a negative shift of Vrev, suggesting that intracellular acidification occurred. The PMA-induced cell acidosis was confirmed using a fluorescent pH indicator (BCECF), which recovered quickly in a K+ -rich alkaline solution, probably through the activated H+ channel. Both cell acidosis and activation of the H+ channel by PMA were inhibited by staurosporine. In ,80% of cells, the PMA-induced augmentation in the current activity remained after compensating for the ,pH changes, implying that both ,pH-dependent and -independent mechanisms mediated the channel activation. Activation of the H+ channel shifted the membrane potential toward Vrev. These data suggest that the H+ channel may contribute to regulation of the pH environments and the membrane potential in osteoclasts activated by protein kinase C. [source] Tibolone Rapidly Attenuates the GABAB Response in Hypothalamic NeuronesJOURNAL OF NEUROENDOCRINOLOGY, Issue 12 2008J. Qiu Tibolone is primarily used for the treatment of climacteric symptoms. Tibolone is rapidly converted into three major metabolites: 3,- and 3,-hydroxy (OH)-tibolone, which have oestrogenic effects, and the ,4-isomer (,4-tibolone), which has progestogenic and androgenic effects. Because tibolone is effective in treating climacteric symptoms, the effects on the brain may be explained by the oestrogenic activity of tibolone. Using whole-cell patch clamp recording, we found previously that 17,-oestradiol (E2) rapidly altered ,-aminobutyric acid (GABA) neurotransmission in hypothalamic neurones through a membrane oestrogen receptor (mER). E2 reduced the potency of the GABAB receptor agonist baclofen to activate G-protein-coupled, inwardly rectifying K+ (GIRK) channels in hypothalamic neurones. Therefore, we hypothesised that tibolone may have some rapid effects through the mER and sought to elucidate the signalling pathway of tibolone's action using selective inhibitors and whole cell recording in ovariectomised female guinea pigs and mice. A sub-population of neurones was identified post hoc as pro-opiomelanocortin (POMC) neurones by immunocytochemical staining. Similar to E2, we have found that tibolone and its active metabolite 3,OH-tibolone rapidly reduced the potency of the GABAB receptor agonist baclofen to activate GIRK channels in POMC neurones. The effects were blocked by the ER antagonist ICI 182 780. Other metabolites of tibolone (3,OH-tibolone and ,4-tibolone) had no effect. Furthermore, tibolone (and 3,OH-tibolone) was fully efficacious in ER, knockout (KO) and ER,KO mice to attenuate GABAB responses. The effects of tibolone were blocked by phospholipase C inhibitor U73122. However, in contrast to E2, the effects of tibolone were not blocked by protein kinase C inhibitors or protein kinase A inhibitors. It appears that tibolone (and 3,OH-tibolone) activates phospholipase C leading to phosphatidylinositol bisphosphate metabolism and direct alteration of GIRK channel function. Therefore, tibolone may enhance synaptic efficacy through the Gq signalling pathways of mER in brain circuits that are critical for maintaining homeostatic functions. [source] Depression of retinogeniculate synaptic transmission by presynaptic D2 -like dopamine receptors in rat lateral geniculate nucleusEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 2 2006G. Govindaiah Abstract Extraretinal projections onto neurons in the dorsal lateral geniculate nucleus (dLGN) play an important role in modifying sensory information as it is relayed from the visual thalamus to neocortex. The dLGN receives dopaminergic innervation from the ventral tegmental area; however, the role of dopamine in synaptic transmission in dLGN has not been explored. In the present study, whole cell recordings were obtained to examine the actions of dopamine on glutamatergic synaptic transmission. Dopamine (2,100 µm) strongly suppressed excitatory synaptic transmission in dLGN relay neurons that was evoked by optic tract stimulation and mediated by both N -methyl- d -aspartate and non -N -methyl- d -aspartate glutamate receptors. In contrast, dopamine did not alter inhibitory synaptic transmission arising from either dLGN interneurons or thalamic reticular nucleus neurons. The suppressive action of dopamine on excitatory synaptic transmission was mimicked by the D2 -like dopamine receptor agonist bromocriptine (2,25 µm) but not by the D1 -like receptor agonist SKF38393 (10,25 µm). In addition, the dopamine-mediated suppression was antagonized by the D2 -like receptor antagonist sulpiride (10,20 µm) but not by the D1 -like receptor antagonist SCH23390 (5,25 µm). The dopamine-mediated decrease in evoked excitatory postsynaptic current amplitude was accompanied by an increase in the magnitude of paired-pulse depression. Furthermore, dopamine also reduced the frequency but not the amplitude of miniature excitatory postsynaptic currents. Taken together, these data suggest that dopamine may act presynaptically to regulate the release of glutamate at the retinogeniculate synapse and modify transmission of visual information in the dLGN. [source] Modulation of inhibitory neurotransmission in brainstem vagal circuits by NPY and PYY is controlled by cAMP levelsNEUROGASTROENTEROLOGY & MOTILITY, Issue 12 2009K. N. Browning Abstract, Pancreatic polypeptides such as neuropeptide Y (NPY) and peptide YY (PYY) exert profound, vagally mediated effects on gastrointestinal (GI) motility. Vagal efferent outflow to the GI tract is determined principally by tonic GABAergic synaptic inputs onto dorsal motor nucleus of the vagus (DMV) neurons, yet neither peptide modulates GABAergic transmission. We showed recently that opioid peptides appear similarly ineffective because of the low resting cAMP levels. Using whole cell recordings from identified DMV neurons, we aimed to correlate the influence of brainstem cAMP levels with the ability of pancreatic polypeptides to modulate GABAergic synaptic transmission. Neither NPY, PYY, nor the Y1 or Y2 receptor selective agonists [Leu,Pro]NPY or NPY(3-36) respectively, inhibited evoked inhibitory postsynaptic current (eIPSC) amplitude unless cAMP levels were elevated by forskolin or 8-bromo-cAMP, by exposure to adenylate cyclase-coupled modulators such as cholecystokinin octapeptide (sulfated) (CCK-8s) or thyrotropin releasing hormone (TRH), or by vagal deafferentation. The inhibition of eIPSC amplitude by [Leu,Pro]NPY or NPY(3-36) was stable for approximately 30 min following the initial increase in cAMP levels. Thereafter, the inhibition declined gradually until the agonists were again ineffective after 60 min. Analysis of spontaneous and miniature currents revealed that such inhibitory effects were due to actions at presynaptic Y1 and Y2 receptors. These results suggest that, similar to opioid peptides, the effects of pancreatic polypeptides on GABAergic transmission depend upon the levels of cAMP within gastric inhibitory vagal circuits. [source] Glycinergic input of widefield, displaced amacrine cells of the mouse retinaTHE JOURNAL OF PHYSIOLOGY, Issue 15 2009Sriparna Majumdar Glycine receptors (GlyRs) of displaced amacrine cells of the mouse retina were analysed using whole cell recordings and immunocytochemical staining with subunit-specific antibodies. During the recordings the cells were filled with a fluorescent tracer and 11 different morphological types could be identified. The studies were performed in wild-type mice and in mutant mice deficient in the GlyR,1 (Glra1spd-ot, ,oscillator' mouse), the GlyR,2 (Glra2,/,) and the GlyR,3 subunit (Glra3,/,). Based on their responses to the application of exogenous glycine in the retinas of wild-type and mutant mice, the cells were grouped into three major classes: group I cells (comprising the morphological types MA-S5, MA-S1, MA-S1/S5, A17, PA-S1, PA-S5 and WA-S1), group II cells (comprising the morphological types PA-S4, WA-S3 and WA-multi) and ON-starburst cells. For further analysis, spontaneous inhibitory postsynaptic currents (sIPSCs) were measured both in wild-type and mutant mouse retinas. Glycinergic sIPSCs and glycine induced currents of group I cells remained unaltered across wild-type and the three mutant mice (mean decay time constant of sIPSCs, ,,25 ms). Group II cells showed glycinergic sIPSCs and glycine induced currents in wild-type, Glra1spd-ot and Glra3,/, mice (,,25 ms); however, glycinergic currents were absent in group II cells of Glra2,/, mice. Glycine induced currents and sIPSCs recorded from ON-starburst amacrine cells did not differ significantly between wild-type and the mutant mouse retinas (,,50,70 ms). We propose that GlyRs of group II cells are dominated by the ,2 subunit; GlyRs of ON-starburst amacrine cells appear to be dominated by the ,4 subunit. [source] | |||||||||||||