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Whole-cell Patch Clamp Recordings (whole-cell + patch_clamp_recording)
Selected AbstractsContribution of Kir3.1, Kir3.2A and Kir3.2C subunits to native G protein-gated inwardly rectifying potassium currents in cultured hippocampal neuronsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 8 2003Joanne L. Leaney Abstract G protein-gated inwardly rectifying potassium (GIRK) channels are found in neurons, atrial myocytes and neuroendocrine cells. A characteristic feature is their activation by stimulation of Gi/o -coupled receptors. In central neurons, for example, they are activated by adenosine and GABA and, as such, they play an important role in neurotransmitter-mediated regulation of membrane excitability. The channels are tetrameric assemblies of Kir3.x subunits (Kir3.1,3.4 plus splice variants). In this study I have attempted to identify the channel subunits which contribute to the native GIRK current recorded from primary cultured rat hippocampal pyramidal neurons. Reverse transcriptase,polymerase chain reaction revealed the expression of mRNA for Kir3.1, 3.2A, 3.2C and 3.3 subunits and confocal immunofluorescence microscopy was used to investigate their expression patterns. Diffuse staining was observed on both cell somata and dendrites for Kir3.1 and Kir3.2A yet that for Kir3.2C was weaker and punctate. Whole-cell patch clamp recordings were used to record GIRK currents from hippocampal pyramidal neurons which were identified on the basis of inward rectification, dependence of reversal potential on external potassium concentration and sensitivity to tertiapin. The GIRK currents were enhanced by the stimulation of a number of Gi/o -coupled receptors and were inhibited by pertussis toxin. In order to ascertain which Kir3.x subunits were responsible for the native GIRK current I compared the properties with those of the cloned Kir3.1 + 3.2A and Kir3.1 + 3.2C channels heterologously expressed in HEK293 cells. [source] Ca2+ - and thromboxane-dependent distribution of MaxiK channels in cultured astrocytes: From microtubules to the plasma membraneGLIA, Issue 12 2009J. W. Ou Abstract Large-conductance, voltage- and Ca2+ -activated K+ channels (MaxiK) are broadly expressed ion channels minimally assembled by four pore-forming ,-subunits (MaxiK,) and typically observed as plasma membrane proteins in various cell types. In murine astrocyte primary cultures, we show that MaxiK, is predominantly confined to the microtubule network. Distinct microtubule distribution of MaxiK, was visualized by three independent labeling approaches: (1) MaxiK,-specific antibodies, (2) expressed EGFP-labeled MaxiK,, and (3) fluorophore-conjugated iberiotoxin, a specific MaxiK pore-blocker. This MaxiK, association with microtubules was further confirmed by in vitro His-tag pulldown, co-immunoprecipitation from brain lysates, and microtubule depolymerization experiments. Changes in intracellular Ca2+ elicited by general pharmacological agents, caffeine or thapsigargin, resulted in increased MaxiK, labeling at the plasma membrane. More notably, U46619, an analog of thromboxane A2 (TXA2), which triggers Ca2+ -release pathways and whose levels increase during cerebral hemorrhage/trauma, also elicits a similar increase in MaxiK, surface labeling. Whole-cell patch clamp recordings of U46619-stimulated cells develop a ,3-fold increase in current amplitude indicating that TXA2 stimulation results in the recruitment of additional, functional MaxiK channels to the surface membrane. While microtubules are largely absent in mature astrocytes, immunohistochemistry results in brain slices show that cortical astrocytes in the newborn mouse (P1) exhibit a robust expression of microtubules that significantly colocalize with MaxiK,. The results of this study provide the novel insight that suggests that Ca2+ released from intracellular stores may play a key role in regulating the traffic of intracellular, microtubule-associated MaxiK, stores to the plasma membrane of developing murine astrocytes. © 2009 Wiley-Liss, Inc. [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] Altered functional properties of satellite glial cells in compressed spinal gangliaGLIA, Issue 15 2009Haijun Zhang Abstract The cell bodies of sensory neurons in the dorsal root ganglion (DRG) are enveloped by satellite glial cells (SGCs). In an animal model of intervertebral foraminal stenosis and low-back pain, a chronic compression of the DRG (CCD) increases the excitability of neuronal cell bodies in the compressed ganglion. The morphological and electrophysiological properties of SGCs were investigated in both CCD and uninjured, control lumbar DRGs. SGCs responded within 12 h of the onset of CCD as indicated by an increased expression of glial fibrillary acidic protein (GFAP) in the compressed DRG but to lesser extent in neighboring or contralateral DRGs. Within 1 week, coupling through gap junctions between SGCs was significantly enhanced in the compressed ganglion. Under whole-cell patch clamp recordings, inward and outward potassium currents, but not sodium currents, were detected in individual SGCs. SGCs enveloping differently sized neurons had similar electrophysiological properties. SGCs in the compressed vs. control DRG exhibited significantly reduced inwardly rectifying potassium currents (Kir), increased input resistances and positively shifted resting membrane potentials. The reduction in Kir was greater for nociceptive medium-sized neurons compared to non-nociceptive neurons. Kir currents of SGCs around spontaneously active neurons were significantly reduced 1 day after compression but recovered by 7 days. These data demonstrate rapid alterations in glial membrane currents and GFAP expression in close temporal association with the development of neuronal hyperexcitability in the CCD model of neuropathic pain. However, these alterations are not fully sustained and suggest other mechanisms for the maintenance of the hyperexcitable state. © 2009 Wiley-Liss, Inc. [source] Alpha-synuclein overexpression in mice alters synaptic communication in the corticostriatal pathwayJOURNAL OF NEUROSCIENCE RESEARCH, Issue 8 2010Nanping Wu Abstract ,-Synuclein (,-Syn) is a presynaptic protein implicated in Parkinson's disease (PD). Mice overexpressing human wildtype (WT) ,-Syn under the Thy1 promoter show high levels of ,-Syn in cortical and subcortical regions, exhibit progressive sensorimotor anomalies, as well as non-motor abnormalities and are considered models of pre-manifest PD as there is little evidence of early loss of dopaminergic (DA) neurons. We used whole-cell patch clamp recordings from visually identified striatal medium-sized spiny neurons (MSSNs) in slices from ,-Syn and WT littermate control mice at 35, 90 and 300 days of age to examine corticostriatal synaptic function. MSSNs displayed significant decreases in the frequency of spontaneous excitatory postsynaptic currents (EPSCs) in ,-Syn mice at all ages. This difference persisted in the presence of tetrodotoxin, indicating it was independent of action potentials. Stimulation thresholds for evoking EPSCs were significantly higher and responses were smaller in ,-Syn mice. These data suggest a decrease in neurotransmitter release at the corticostriatal synapse. At 90 days the frequency of spontaneous GABAA receptor-mediated synaptic currents was decreased in MSSNs but increased in cortical pyramidal neurons. These observations indicate that high levels of expression of ,-Syn alter corticostriatal synaptic function early and they provide evidence for early synaptic dysfunction in a pre-manifest model of PD. Of importance, these changes are opposite to those found in DA-depletion models, suggesting that before degeneration of DA neurons in the substantia nigra synaptic adaptations occur at the corticostriatal synapse that may initiate subtle preclinical manifestations. © 2009 Wiley-Liss, Inc. [source] | ||||||||||