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Patch-clamp Electrophysiology (patch-clamp + electrophysiology)
Selected AbstractsOrexin B/hypocretin 2 increases glutamatergic transmission to ventral tegmental area neuronsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 8 2008S. L. Borgland Abstract The orexins (hypocretins) play a crucial role in arousal, feeding and reward. Highly relevant to these functions, orexin-containing neurons from the lateral hypothalamus project densely to the ventral tegmental area (VTA), which is the origin of dopamine projections implicated in motivation and reward. Orexin A/hypocretin 1 (oxA/hcrt-1) can enable long-term changes associated with drugs of abuse; however, the effects of orexin B/hypocretin 2 (oxB/hcrt-2) on excitatory synaptic transmission in the VTA are unknown. We used whole-cell patch-clamp electrophysiology in rat horizontal midbrain slices to examine the effects of oxB/hcrt-2 on excitatory synaptic transmission. We observed that oxB/hcrt-2 has distinct effects from oxA/hcrt-1 in the VTA. oxB/Hcrt-2 (100 nm) increased presynaptic glutamate release in addition to a postsynaptic potentiation of NMDA receptors (NMDARs). The oxB/hcrt-2-mediated postsynaptic potentiation of NMDARs was mediated via activation of orexin/hypocretin 2 (OX2/Hcrt-2) receptors and protein kinase C (PKC). Furthermore, the increase in transmitter release probability was also PKC-dependent, but not through activation of orexin/hypocretin 1 (OX1/Hcrt-1) or OX2/Hcrt-2 receptors. Finally, oxB/hcrt-2 or the selective OX2/Hcrt-2 receptor agonist ala11 - d -leu15 -orexin B, significantly reduced spike-timing-induced long-term potentiation. Taken together, these results support a dual role for oxB/hcrt-2 in mediating enhanced glutamatergic transmission in the VTA, and suggest that oxA/hcrt-1 and oxB/hcrt-2 exert different functional roles in modulating the enhancement of the motivational components of arousal and feeding. [source] Effects of Ethanol on Persistent Activity and Up-States in Excitatory and Inhibitory Neurons in Prefrontal CortexALCOHOLISM, Issue 12 2009John J. Woodward Background:, Elucidating mechanisms that underlie the neural actions of ethanol is critical for understanding how this drug affects behavior. Increasing evidence suggests that, in addition to mid-brain dopaminergic regions, higher cortical structures play an important role in the pathophysiology associated with alcohol abuse. Previous studies from this laboratory used a novel slice co-culture system to demonstrate that ethanol reduces network-dependent patterns of activity in excitatory pyramidal neurons of the prefrontal cortex (PFC). In the present study, we examine the effect of ethanol on the activity of fast-spiking (FS) interneurons, a sub-population of neurons critically involved in shaping cortical activity. Methods:, Slices containing the dorsolateral PFC were prepared from neonatal C57 mice and maintained in culture. After 2 to 3 weeks in vitro, whole-cell patch-clamp electrophysiology was used to monitor spontaneous episodes of persistent activity in prelimbic PFC neurons. In some experiments, the use-dependent NMDA receptor blocker, MK801, was included in the pipette recording solution to assess the contribution of NMDA receptors to up-states. Results:, MK801 reduced up-state amplitude and revealed underlying fast EPSPs in excitatory pyramidal neurons while having little effect on these parameters in FS interneurons. Despite this difference, ethanol (44 mM), significantly reduced up-state duration and up-state area in both pyramidal and FS interneurons. Conclusions:, These results suggest that ethanol reduces the activity of FS interneurons due to disruption of network-dependent activity. This would be expected to further impair the ability of PFC networks to carry out their normal function and may contribute to the adverse effects of ethanol on PFC-dependent behaviors. [source] Plant extracellular ATP signalling by plasma membrane NADPH oxidase and Ca2+ channelsTHE PLANT JOURNAL, Issue 6 2009Vadim Demidchik Summary Extracellular ATP regulates higher plant growth and adaptation. The signalling events may be unique to higher plants, as they lack animal purinoceptor homologues. Although it is known that plant cytosolic free Ca2+ can be elevated by extracellular ATP, the mechanism is unknown. Here, we have studied roots of Arabidopsis thaliana to determine the events that lead to the transcriptional stress response evoked by extracellular ATP. Root cell protoplasts were used to demonstrate that signalling to elevate cytosolic free Ca2+ is determined by ATP perception at the plasma membrane, and not at the cell wall. Imaging revealed that extracellular ATP causes the production of reactive oxygen species in intact roots, with the plasma membrane NADPH oxidase AtRBOHC being the major contributor. This resulted in the stimulation of plasma membrane Ca2+ -permeable channels (determined using patch-clamp electrophysiology), which contribute to the elevation of cytosolic free Ca2+. Disruption of this pathway in the AtrbohC mutant impaired the extracellular ATP-induced increase in reactive oxygen species (ROS), the activation of Ca2+ channels, and the transcription of the MAP kinase3 gene that is known to be involved in stress responses. This study shows that higher plants, although bereft of purinoceptor homologues, could have evolved a distinct mechanism to transduce the ATP signal at the plasma membrane. [source] A Drosophila melanogaster cell line (S2) facilitates post-genome functional analysis of receptors and ion channelsBIOESSAYS, Issue 11 2002Paula R. Towers The complete sequencing of the genome of the fruit fly Drosophila melanogaster offers the prospect of detailed functional analysis of the extensive gene families in this genetic model organism. Comprehensive functional analysis of family members is facilitated by access to a robust, stable and inducible expression system in a fly cell line. Here we show how the Schneider S2 cell line, derived from the Drosophila embryo, provides such an expression system, with the bonus that radioligand binding studies, second messenger assays, ion imaging, patch-clamp electrophysiology and gene silencing can readily be applied. Drosophila is also ideal for the study of new control strategies for insect pests since the receptors and ion channels that many new animal health drugs and crop protection chemicals target can be expressed in this cell line. In addition, many useful orthologues of human disease genes are emerging from the Drosophila genome and the study of their functions and interactions is another area for postgenome applications of S2 cell lines. BioEssays 24:1066,1073, 2002. © 2002 Wiley-Periodicals, Inc. [source] Identification of domains influencing assembly and ion channel properties in ,7 nicotinic receptor and 5-HT3 receptor subunit chimaerasBRITISH JOURNAL OF PHARMACOLOGY, Issue 4 2007V J Gee Background and purpose: Nicotinic acetylcholine receptors (nAChRs) and 5-hydroxytryptamine type 3 receptors (5-HT3Rs) are members of the superfamily of neurotransmitter-gated ion channels. Both contain five subunits which assemble to form either homomeric or heteromeric subunit complexes. With the aim of identifying the influence of subunit domains upon receptor assembly and function, a series of chimaeras have been constructed containing regions of the neuronal nAChR ,7 subunit and the 5-HT3 receptor 3A subunit. Experimental approach: A series of subunit chimaeras containing ,7 and 5-HT3A subunit domains have been constructed and expressed in cultured mammalian cells. Properties of the expressed receptors have been examined by means of radioligand binding, agonist-induced changes in intracellular calcium and patch-clamp electrophysiology. Key results: Subunit domains which influence properties such as rectification, desensitization and conductance have been identified. In addition, the influence of subunit domains upon subunit folding, receptor assembly and cell-surface expression has been identified. Co-expression studies with the nAChR-associated protein RIC-3 revealed that, in contrast to the potentiating effect of RIC-3 on ,7 nAChRs, RIC-3 caused reduced levels of cell-surface expression of some ,7/5-HT3A chimaeras. Conclusions and implications: Evidence has been obtained which demonstrates that subunit transmembrane domains are critical for efficient subunit folding and assembly. In addition, functional characterization of subunit chimaeras revealed that both extracellular and cytoplasmic domains exert a dramatic and significant influence upon single-channel conductance. These data support a role for regions other than hydrophobic transmembrane domains in determining ion channel properties. British Journal of Pharmacology (2007) 152, 501,512; doi:10.1038/sj.bjp.0707429; published online 27 August 2007 [source] KMUP-1 activates BKCa channels in basilar artery myocytes via cyclic nucleotide-dependent protein kinasesBRITISH JOURNAL OF PHARMACOLOGY, Issue 6 2005Bin-Nan Wu This study investigated whether KMUP-1, a synthetic xanthine-based derivative, augments the delayed-rectifier potassium (KDR)- or large-conductance Ca2+ -activated potassium (BKCa) channel activity in rat basilar arteries through protein kinase-dependent and -independent mechanisms. Cerebral smooth muscle cells were enzymatically dissociated from rat basilar arteries. Conventional whole cell, perforated and inside-out patch-clamp electrophysiology was used to monitor K+ - and Ca2+ channel activities. KMUP-1 (1 ,M) had no effect on the KDR current but dramatically enhanced BKCa channel activity. This increased BKCa current activity was abolished by charybdotoxin (100 nM) and iberiotoxin (100 nM). Like KMUP-1, the membrane-permeable analogs of cGMP (8-Br-cGMP) and cAMP (8-Br-cAMP) enhanced the BKCa current. BKCa current activation by KMUP-1 was markedly inhibited by a soluble guanylate cyclase inhibitor (ODQ 10 ,M), an adenylate cyclase inhibitor (SQ 22536 10 ,M), competitive antagonists of cGMP and cAMP (Rp-cGMP, 100 ,M and Rp-cAMP, 100 ,M), and cGMP- and cAMP-dependent protein kinase inhibitors (KT5823, 300 nM and KT5720, 300 nM). Voltage-dependent L-type Ca2+ current was significantly suppressed by KMUP-1 (1 ,M), and nearly abolished by a calcium channel blocker (nifedipine, 1 ,M). In conclusion, KMUP-1 stimulates BKCa currents by enhancing the activity of cGMP-dependent protein kinase, and in part this is due to increasing cAMP-dependent protein kinase. Physiologically, this activation would result in the closure of voltage-dependent calcium channels and the relaxation of cerebral arteries. British Journal of Pharmacology (2005) 146, 862,871. doi:10.1038/sj.bjp.0706387 [source] | |||||||||||||