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Patch Clamp Recordings (patch + clamp_recording)
Kinds of Patch Clamp Recordings Selected AbstractsSynaptic release of dopamine in the subthalamic nucleusEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 7 2004Stephanie J. Cragg Abstract The direct modulation of subthalamic nucleus (STN) neurons by dopamine (DA) neurons of the substantia nigra (SN) is controversial owing to the thick caliber and low density of DA axons in the STN. The abnormal activity of the STN in Parkinson's disease (PD), which is central to the appearance of symptoms, is therefore thought to result from the loss of DA in the striatum. We carried out three experiments in rats to explore the function of DA in the STN: (i) light and electron microscopic analysis of tyrosine hydroxylase (TH)-, dopamine ,-hydroxylase (D,H)- and DA-immunoreactive structures to determine whether DA axons form synapses; (ii) fast-scan cyclic voltammetry (FCV) to determine whether DA axons release DA; and (iii) patch clamp recording to determine whether DA, at a concentration similar to that detected by FCV, can modulate activity and synaptic transmission/integration. TH- and DA-immunoreactive axons mostly formed symmetric synapses. Because D,H-immunoreactive axons were rare and formed asymmetric synapses, they comprised the minority of TH-immunoreactive synapses. Voltammetry demonstrated that DA release was sufficient for the activation of receptors and abolished by blockade of voltage-dependent Na+ channels or removal of extracellular Ca2+. The lifetime and concentration of extracellular DA was increased by blockade of the DA transporter. Dopamine application depolarized STN neurons, increased their frequency of activity and reduced the impact of ,-aminobutyric acid (GABA)-ergic inputs. These findings suggest that SN DA neurons directly modulate the activity of STN neurons and their loss may contribute to the abnormal activity of STN neurons in PD. [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] Spontaneous Ca2+ Waves in Rabbit Corpus Cavernosum: Modulation by Nitric Oxide and cGMPTHE JOURNAL OF SEXUAL MEDICINE, Issue 4 2009Gerard P. Sergeant PhD ABSTRACT Introduction., Detumescent tone and subsequent relaxation by nitric oxide (NO) are essential processes that determine the erectile state of the penis. Despite this, the mechanisms involved are incompletely understood. It is often assumed that the tone is associated with a sustained high cytosolic Ca2+ level in the corpus cavernosum smooth muscle cells, however, an alternative possibility is that oscillatory Ca2+ signals regulate tone, and erection occurs as a result of inhibition of Ca2+ oscillations by NO. Aims., The aim of this study is to determine if smooth muscle cells displayed spontaneous Ca2+ oscillations and, if so, whether these were regulated by NO. Methods., Male New Zealand white rabbits were euthanized and smooth muscle cells were isolated by enzymatic dispersal for confocal imaging of intracellular Ca2+ (using fluo-4AM) and patch clamp recording of spontaneous membrane currents. Thin tissue slices were also loaded with fluo-4AM for live imaging of Ca2+. Main Outcome Measure., Cytosolic Ca2+ was measured in isolated smooth muscle cells and tissue slices. Results., Isolated rabbit corpus cavernosum smooth muscle cells developed spontaneous Ca2+ waves that spread at a mean velocity of 65 µm/s. Dual voltage clamp/confocal recordings revealed that each of the Ca2+ waves was associated with an inward current typical of the Ca2+ -activated Cl - currents developed by these cells. The waves depended on an intact sarcoplasmic reticulum Ca2+ store, as they were blocked by cyclopiazonic acid (Calbiochem, San Diego, CA, USA) and agents that interfere with ryanodine receptors and IP3 -mediated Ca2+ release. The waves were also inhibited by an NO donor (diethylamine NO; Tocris Bioscience, Bristol, Avon, UK), 3-(5-hydroxymethyl-2-furyl)-1-benzyl indazole (YC-1) (Alexis Biochemicals, Bingham, Notts, UK), 8-bromo-cyclic guanosine mono-phosphate (Tocris), and sildenafil (Viagra, Pfizer, Sandwich, Kent, UK). Regular Ca2+ oscillations were also observed in whole tissue slices where they were clearly seen to precede contraction. This activity was also markedly inhibited by sildenafil, suggesting that it was under NO regulation. Conclusions., These results provide a new basis for understanding detumescent tone in the corpus cavernosum and its inhibition by NO. Sergeant GP, Craven M, Hollywood MA, McHale NG, and Thornbury KD. Spontaneous Ca2+ waves in rabbit corpus cavernosum: Modulation by nitric oxide and cGMP. J Sex Med **;**:**,**. [source] Contribution 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] Effects of nicotine in the dopaminergic system of mice lacking the alpha4 subunit of neuronal nicotinic acetylcholine receptorsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 7 2003L. M. Marubio Abstract The mesostriatal dopaminergic system influences locomotor activity and the reinforcing properties of many drugs of abuse including nicotine. Here we investigate the role of the ,4 nicotinic acetylcholine receptor (nAChR) subunit in mediating the effects of nicotine in the mesolimbic dopamine system in mice lacking the ,4 subunit. We show that there are two distinct populations of receptors in the substantia nigra and striatum by using autoradiographic labelling with 125I ,-conotoxin MII. These receptors are comprised of the ,4, ,2 and ,6 nAChR subunits and non-,4, ,2, and ,6 nAChR subunits. Non-,4 subunit-containing nAChRs are located on dopaminergic neurons, are functional and respond to nicotine as demonstrated by patch clamp recordings. In vivo microdialysis performed in awake, freely moving mice reveal that mutant mice have basal striatal dopamine levels which are twice as high as those observed in wild-type mice. Despite the fact that both wild-type and ,4 null mutant mice show a similar increase in dopamine release in response to intrastriatal KCl perfusion, a nicotine-elicited increase in dopamine levels is not observed in mutant mice. Locomotor activity experiments show that there is no difference between wild-type and mutant mice in basal activity in both habituated and non-habituated environments. Interestingly, mutant mice sustain an increase in cocaine-elicited locomotor activity longer than wild-type mice. In addition, mutant mice recover from depressant locomotor activity in response to nicotine at a faster rate. Our results indicate that ,4-containing nAChRs exert a tonic control on striatal basal dopamine release, which is mediated by a heterogeneous population of nAChRs. [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] 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] Dissecting the pathogenic mechanisms of mutations in the pore region of the human cone photoreceptor cyclic nucleotide-gated channel,HUMAN MUTATION, Issue 7 2010Katja Koeppen Abstract The CNGA3 gene encodes the A3 subunit of the cone photoreceptor cyclic nucleotide-gated (CNG) channel, an essential component of the phototransduction cascade. Certain mutations in CNGA3 cause autosomal recessive achromatopsia, a retinal disorder characterized by severely reduced visual acuity, lack of color discrimination, photophobia, and nystagmus. We identified three novel mutations in the pore-forming region of CNGA3 (L363P, G367V, and E376K) in patients diagnosed with achromatopsia. We assessed the expression and function of channels with these three new and two previously described mutations (S341P and P372S) in a heterologous HEK293 cell expression system using Western blot, subcellular localization on the basis of immunocytochemistry, calcium imaging, and patch clamp recordings. In this first comparative functional analysis of disease-associated mutations in the pore of a CNG channel, we found impaired surface expression of S341P, L363P, and P372S mutants and reduced macroscopic currents for channels with the mutations S341P, G367V, and E376K. Calcium imaging and patch clamp experiments after incubation at 37°C revealed nonfunctional homo- and heteromeric channels in all five mutants, but incubation at 27°C combined with coexpression of the B3 subunit restored residual function of channels with the mutations S341P, G367V, and E376K. Hum Mutat 31:830,839, 2010. © 2010 Wiley-Liss, Inc. [source] Differentiation dependent expression of TRPA1 and TRPM8 channels in IMR-32 human neuroblastoma cellsJOURNAL OF CELLULAR PHYSIOLOGY, Issue 1 2009Lauri M. Louhivuori TRPA1 and TRPM8 are transient receptor potential (TRP) channels involved in sensory perception. TRPA1 is a non-selective calcium permeable channel activated by irritants and proalgesic agents. TRPM8 reacts to chemical cooling agents such as menthol. The human neuroblastoma cell line IMR-32 undergoes a remarkable differentiation in response to treatment with 5-bromo-2-deoxyuridine. The cells acquire a neuronal morphology with increased expression of N-type voltage gated calcium channels and neurotransmitters. Here we show using RT-PCR, that mRNA for TRPA1 and TRPM8 are strongly upregulated in differentiating IMR-32 cells. Using whole cell patch clamp recordings, we demonstrate that activators of these channels, wasabi, allyl-isothiocyanate (AITC) and menthol activate membrane currents in differentiated cells. Calcium imaging experiments demonstrated that AITC mediated elevation of intracellular calcium levels were attenuated by ruthenium red, spermine, and HC-030031 as well as by siRNA directed against the channel. This indicates that the detected mRNA level correlate with the presence of functional channels of both types in the membrane of differentiated cells. Although the differentiated IMR-32 cells responded to cooling many of the cells showing this response did not respond to TRPA1/TRPM8 channel activators (60% and 90% for AITC and menthol respectively). Conversely many of the cells responding to these activators did not respond to cooling (30%). This suggests that these channels have also other functions than cold perception in these cells. Furthermore, our results suggest that IMR-32 cells have sensory characteristics and can be used to study native TRPA1 and TRPM8 channel function as well as developmental expression. J. Cell. Physiol. 221: 67,74, 2009. © 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] Lactobacillus reuteri ingestion and IKCa channel blockade have similar effects on rat colon motility and myenteric neuronesNEUROGASTROENTEROLOGY & MOTILITY, Issue 1 2010B. Wang Abstract, Background, We have previously shown that ingestion of Lactobacillus reuteri may modulate colonic enteric neuron activity but with unknown effects on colon motility. The aim of the present report was to elucidate the neuronal mechanisms of action of the probiotic by comparing the effects on motility of L. reuteri ingestion with blockade of a specific ionic current in enteric neurons. Methods, We have used intraluminal pressure recordings from ex vivo rat colon segments and whole cell patch clamp recordings from neurons of rat longitudinal muscle myenteric plexus preparations to investigate the effects of L. reuteri and TRAM-34 on colon motility and neurophysiology. The effects of daily feeding of 109L. reuteri bacteria or acute application of TRAM-34 on threshold fluid filling pressure or pulse pressure was measured. Key Results,Lactobacillus reuteri increased intraluminal fluid filling pressure thresholds for evoking pressure pulses by 51% from 0.47 ± 0.17 hPa; the probiotic also decreased the pulse pressure amplitudes, but not frequency, by 18% from 3.91 ± 0.52 hPa. The intermediate conductance calcium-dependent potassium (IKCa) channel blocker TRAM-34 (3 ,mol L,1) increased filling threshold pressure by 43% from 0.52 ± 0.22 hPa and reduced pulse pressure amplitude by 40% from 2.63 ± 1.11 hPa; contraction frequency was unaltered. TRAM-34 (3 ,mol L,1) reduced membrane polarization, leak conductance and the slow afterhyperpolarization current in 16/16 myenteric rat colon AH cells but 19/19 S cells were unaffected. Conclusions & Inferences, The present results are consistent with L. reuteri enhancing tonic inhibition of colon contractile activity by acting via the IKCa channel current in AH cells. [source] Electroacupuncture attenuates visceral hyperalgesia and inhibits the enhanced excitability of colon specific sensory neurons in a rat model of irritable bowel syndromeNEUROGASTROENTEROLOGY & MOTILITY, Issue 12 2009G.-y. Xu Abstract, The causes of irritable bowel syndrome remain elusive and there are few effective treatments for pain in this syndrome. Electroacupunture (EA) is used extensively for treatment of various painful conditions including chronic visceral hyperalgesia (CVH). However, mechanism of its analgesic effect remains unknown. This study was designed to investigate effect of EA on colon specific dorsal root ganglion (DRG) neurons in rats with CVH. CVH was induced by intracolonic injection of acetic acid (AA) in 10-day-old rats. Electromyography and patch clamp recordings were performed at age of 8,10 weeks. Colon DRG neurons were labelled by injection of DiI into the colon wall. EA was given at ST36 in both hindlimbs. As adults, neonatal AA-injected rats displayed an increased sensitivity to colorectal distension (CRD) and an enhanced excitability of colon DRG neurons. EA treatment for 40 min significantly attenuated the nociceptive responses to CRD in these rats; this attenuation was reversed by pretreatment with naloxone. EA treatment for 40 min per day for 5 days produced a prolonged analgesic effect and normalized the enhanced excitability of colon DRG neurons. Furthermore, in vitro application of [D-Ala2, N -MePhe4, Gly5 -Ol] enkephalin (DAMGO) suppressed the enhanced excitability of colon neurons from rats with CVH. These findings suggest that EA produced-visceral analgesia, which might be mediated in a large part by endogenous opioids pathways, is associated with reversal of the enhanced excitability of colon DRG neurons in rats with CVH. [source] 5-Hydroxytryptamine selectively activates the vagal nodose C-fibre subtype in the guinea-pig oesophagusNEUROGASTROENTEROLOGY & MOTILITY, Issue 9 2008S. Yu Abstract, The afferent neurons innervating the oesophagus originate from two embryonic sources: neurons located in vagal nodose ganglia originate from embryonic placodes and neurons located in vagal jugular and spinal dorsal root ganglia (DRG) originate from the neural crest. Here, we address the hypothesis that 5-hydroxytryptamine (5-HT) differentially stimulates afferent nerve subtypes in the oesophagus. Extracellular recordings of single unit activity originating from nerve terminals were made in the isolated innervated guinea-pig oesophagus. Whole cell patch clamp recordings (35 °C) were made from the primary afferent neurons retrogradely labelled from the oesophagus. 5-Hydroxytryptamine (10 ,mol L,1) activated vagal nodose C-fibres (70%) in the oesophagus but failed to activate overtly vagal jugular nerve fibres and oesophagus-specific spinal DRG neurons. The response to 5-HT in nodose C-fibre nerve terminals was mimicked by the selective 5-HT3 receptor agonist 2-methyl-5-HT (10 ,mol L,1) and nearly abolished by the 5-HT3 receptor antagonists ondansetron (10 ,mol L,1) and Y-25130 (10 ,mol L,1). In patch clamp studies, 2-methyl-5-HT (10 ,mol L,1) activated a proportion of isolated oesophagus-specific nodose capsaicin-sensitive neurons (putative cell bodies of nodose C-fibres). We conclude that the responsiveness to 5-HT discriminates placode-derived (vagal nodose) C-fibres from the neural crest-derived (vagal jugular and spinal DRG) afferent nerves in the oesophagus. The response to 5-HT in nodose C-fibres is mediated by the 5-HT3 receptor in their neuronal membrane. [source] | |||||||||||||