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Whole-cell Patch-clamp Recordings (whole-cell + patch-clamp_recording)
Selected AbstractsIntracellular Calcium Increase in Epileptiform Activity: Modulation by Levetiracetam and LamotrigineEPILEPSIA, Issue 7 2004Antonio Pisani Summary:,Purpose: Alterations in neuronal calcium (Ca2+) homeostasis are believed to play an essential role in the generation and propagation of epileptiform events. Levetiracetam (LEV) and lamotrigine (LTG), novel antiepileptic drugs (AEDs), were tested on epileptiform events and the corresponding elevations in intracellular Ca2+ concentration ([Ca2+]i) recorded from rat neocortical slices. Methods: Electrophysiological recordings were performed from single pyramidal neurons from a slice preparation. Spontaneous epileptiform events consisting of long-lasting, repetitive paroxysmal depolarization shifts (PDSs) and interictal spike activity were induced by reducing the magnesium concentration from the solution and by adding bicuculline and 4-aminopyridine. Simultaneously, microfluorimetric measurements of [Ca2+]i were performed. Optical imaging with Ca2+ indicators revealed a close correlation between Ca2+ transients and epileptiform events. Results: Both LEV and LTG were able to reduce both amplitude and duration of PDSs, as well as the concomitant elevation in [Ca2+]i, in a dose-dependent fashion. Whole-cell patch-clamp recordings from isolated neocortical neurons revealed that LEV significantly reduced N-, and partially P/Q-type high-voltage-activated (HVA) Ca2+ currents, whereas sodium currents were unaffected. Interestingly, the inhibitory effects of LEV were mimicked and occluded by LTG or by a combination of ,-conotoxin GVIA and ,-agatoxin IVA, selective blockers of N- and P/Q-type HVA channels, respectively, suggesting a common site of action for these AEDs. Conclusions: These results demonstrate that large, transient elevations in neuronal [Ca2+]i correlate to epileptiform discharges. The antagonistic effects of LEV and LTG on [Ca2+]i overload might represent the basis for their anticonvulsant efficacy and could preserve neuronal viability. [source] Differential sensitivity of medium- and large-sized striatal neurons to NMDA but not kainate receptor activation in the ratEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 10 2001Carlos Cepeda Abstract Infrared videomicroscopy and differential interference contrast optics were used to identify medium- and large-sized neurons in striatal slices from young rats. Whole-cell patch-clamp recordings were obtained to compare membrane currents evoked by application of N -methyl- d -aspartate (NMDA) and kainate. Inward currents and current densities induced by NMDA were significantly smaller in large- than in medium-sized striatal neurons. The negative slope conductance for NMDA currents was greater in medium- than in large-sized neurons and more depolarization was required to remove the Mg2+ blockade. In contrast, currents induced by kainate were significantly greater in large-sized neurons whilst current densities were approximately equal in both cell types. Spontaneous excitatory postsynaptic currents occurred frequently in medium-sized neurons but were relatively infrequent in large-sized neurons. Excitatory postsynaptic currents evoked by electrical stimulation were smaller in large- than in medium-sized neurons. A final set of experiments assessed a functional consequence of the differential sensitivity of medium- and large-sized neurons to NMDA. Cell swelling was used to examine changes in somatic area in both neuronal types after prolonged application of NMDA or kainate. NMDA produced a time-dependent increase in somatic area in medium-sized neurons whilst it produced only minimal changes in large interneurons. In contrast, application of kainate produced significant swelling in both medium- and large-sized cells. We hypothesize that reduced sensitivity to NMDA may be due to variations in receptor subunit composition and/or the relative density of receptors in the two cell types. These findings help define the conditions that put neurons at risk for excitotoxic damage in neurological disorders. [source] Acute action of rotenone on nigral dopaminergic neurons , involvement of reactive oxygen species and disruption of Ca2+ homeostasisEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 10 2009Peter S. Freestone Abstract Rotenone is a toxin used to generate animal models of Parkinson's disease; however, the mechanisms of toxicity in substantia nigra pars compacta (SNc) neurons have not been well characterized. We have investigated rotenone (0.05,1 ,m) effects on SNc neurons in acute rat midbrain slices, using whole-cell patch-clamp recording combined with microfluorometry. Rotenone evoked a tolbutamide-sensitive outward current (94 ± 15 pA) associated with increases in intracellular [Ca2+] ([Ca2+]i) (73.8 ± 7.7 nm) and intracellular [Na+] (3.1 ± 0.6 mm) (all with 1 ,m). The outward current was not affected by a high ATP level (10 mm) in the patch pipette but was decreased by Trolox. The [Ca2+]i rise was abolished by removing extracellular Ca2+, and attenuated by Trolox and a transient receptor potential M2 (TRPM2) channel blocker, N -(p -amylcinnamoyl) anthranilic acid. Other effects included mitochondrial depolarization (rhodamine-123) and increased mitochondrial reactive oxygen species (ROS) production (MitoSox), which was also abolished by Trolox. A low concentration of rotenone (5 nm) that, by itself, did not evoke a [Ca2+]i rise resulted in a large (46.6 ± 25.3 nm) Ca2+ response when baseline [Ca2+]i was increased by a ,priming' protocol that activated voltage-gated Ca2+ channels. There was also a positive correlation between ,naturally' occurring variations in baseline [Ca2+]i and the rotenone-induced [Ca2+]i rise. This correlation was not seen in non-dopaminergic neurons of the substantia nigra pars reticulata (SNr). Our results show that mitochondrial ROS production is a key element in the effect of rotenone on ATP-gated K+ channels and TRPM2-like channels in SNc neurons, and demonstrate, in these neurons (but not in the SNr), a large potentiation of rotenone-induced [Ca2+]i rise by a small increase in baseline [Ca2+]i. [source] Differential responses to NMDA receptor activation in rat hippocampal interneurons and pyramidal cells may underlie enhanced pyramidal cell vulnerabilityEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 12 2005E. Avignone Abstract Hippocampal interneurons are generally more resistant than pyramidal cells to excitotoxic insults. Because NMDA receptors play a crucial role in neurodegeneration, we have compared the response to exogenous NMDA in CA1 pyramidal cells and interneurons of the stratum oriens using combined whole-cell patch-clamp recording and ratiometric Ca2+ imaging. In voltage-clamp, current-clamp or in nominally Mg2+ -free medium, NMDA (10 µm; 3,5 min exposure in the presence of tetrodotoxin) induced a markedly larger inward current and Ca2+ rise in pyramidal cells than in interneurons. Pyramidal cells also showed a more pronounced voltage dependence in their response to NMDA. We hypothesized that this enhanced response to NMDA receptor activation in pyramidal cells could underlie their increased vulnerability to excitotoxicity. Using loss of dye as an indicator of degenerative membrane disruption, interneurons tolerated continuous exposure to a high concentration of NMDA (30 µm) for longer periods than pyramidal cells. This acute neurodegeneration in pyramidal cells was independent of intracellular Ca2+, because high intracellular BAPTA (20 mm) did not prolong survival time. Thus, a plausible explanation for the enhanced sensitivity of pyramidal neurons to excitotoxic insults associated with cerebral ischemia is their greater response to NMDA receptor activation, which may reflect differences in NMDA receptor expression and/or subunit composition. [source] Differential modulation of AMPA receptors by cyclothiazide in two types of striatal neuronsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 8 2000Vladimir S. Vorobjev Abstract The modulation of ,-amino-3-hydroxy-5-methyl-4-isoxazol-propionate (AMPA) receptor-mediated currents by cyclothiazide was investigated in acutely isolated cells from rat striatum with whole-cell patch-clamp recording. Single-cell reverse transcriptase-polymerase chain reaction (RT-PCR) was used to identify medium spiny and giant aspiny neurons and to determine their AMPA receptor subunit composition mostly in separate experiments. After pretreatment with cyclothiazide, kainate-induced AMPA responses were more strongly potentiated in medium spiny than in giant aspiny neurons; cyclothiazide induced a ninefold leftward shift in the kainate concentration,response curve for medium spiny neurons (not giant aspiny neurons). The EC50s for the cyclothiazide potentiation did not differ substantially between medium spiny neurons and giant aspiny neurons. The recovery of kainate-activated currents from modulation by cyclothiazide was slower for medium spiny neurons than for giant aspiny neurons. Medium spiny neurons expressed GluR-A, GluR-B and GluR-C, but not GluR-D subunits in both flip and flop splice variants. All giant aspiny neurons expressed GluR-A and GluR-D, exclusively in the flop form, half of them also expressed GluR-B and GluR-C. This is in keeping with slow and fast desensitization kinetics in medium spiny neurons and giant aspiny neurons, respectively, and differences in cyclothiazide modulation. The rate of cyclothiazide dissociation from the AMPA receptor, activated by glutamate, was ,,90 times slower in medium spiny neurons than in giant aspiny neurons. In giant aspiny neurons (not medium spiny neurons) this rate was strongly dependent on the presence of an agonist; 1 m m glutamate increased it 30-fold. Thus, two major cell groups in the striatum display distinct AMPA receptor compositions carrying specific properties of glutamate responses. Excitatory transmission will thus be differentially affected by cyclothiazide-type compounds. [source] Electrophysiological Identification of the Functional Presynaptic Nerve Terminals on an Isolated Single Vasopressin Neurone of the Rat Supraoptic NucleusJOURNAL OF NEUROENDOCRINOLOGY, Issue 5 2010T. Ohbuchi Release of arginine vasopressin (AVP) and oxytocin from magnocellular neurosecretory cells (MNCs) of the supraoptic nucleus (SON) is under the control of glutamate-dependent excitation and GABA-dependent inhibition. The possible role of the synaptic terminals attached to SON neurones has been investigated using whole-cell patch-clamp recording in in vitro rat brain slice preparations. Recent evidence has provided new insights into the repercussions of glial environment modifications on the physiology of MNCs at the synaptic level in the SON. In the present study, excitatory glutamatergic and inhibitory GABAergic synaptic inputs were recorded from an isolated single SON neurone cultured for 12 h, using the whole-cell patch clamp technique. Neurones expressed an AVP-enhanced green fluorescent protein (eGFP) fusion gene in MNCs. In addition, native synaptic terminals attached to a dissociated AVP-eGFP neurone were visualised with synaptic vesicle markers. These results suggest that the function of presynaptic nerve terminals may be evaluated directly in a single AVP-eGFP neurone. These preparations would be helpful in future studies aiming to electrophysiologically distinguish between the functions of synaptic terminals and glial modifications in the SON neurones. [source] A novel Nav1.7 mutation producing carbamazepine-responsive erythromelalgia,ANNALS OF NEUROLOGY, Issue 6 2009Tanya Z. Fischer MD Objective Human and animal studies have shown that Nav1.7 sodium channels, which are preferentially expressed within nociceptors and sympathetic neurons, play a major role in inflammatory and neuropathic pain. Inherited erythromelalgia (IEM) has been linked to gain-of-function mutations of Nav1.7. We now report a novel mutation (V400M) in a three-generation Canadian family in which pain is relieved by carbamazepine (CBZ). Methods We extracted genomic DNA from blood samples of eight members of the family, and the sequence of SCN9A coding exons was compared with the reference Nav1.7 complementary DNA. Wild-type Nav1.7 and V400M cell lines were then analyzed using whole-cell patch-clamp recording for changes in activation, deactivation, steady-state inactivation, and ramp currents. Results Whole-cell patch-clamp studies of V400M demonstrate changes in activation, deactivation, steady-state inactivation, and ramp currents that can produce dorsal root ganglia neuron hyperexcitability that underlies pain in these patients. We show that CBZ, at concentrations in the human therapeutic range, normalizes the voltage dependence of activation and inactivation of this inherited erythromelalgia mutation in Nav1.7 but does not affect these parameters in wild-type Nav1.7. Interpretation Our results demonstrate a normalizing effect of CBZ on mutant Nav1.7 channels in this kindred with CBZ-responsive inherited erythromelalgia. The selective effect of CBZ on the mutant Nav1.7 channel appears to explain the ameliorative response to treatment in this kindred. Our results suggest that functional expression and pharmacological studies may provide mechanistic insights into hereditary painful disorders. Ann Neurol 2009;65:733,741 [source] FORMALIN-INDUCED INCREASE IN P2X3 RECEPTOR EXPRESSION IN DORSAL ROOT GANGLIA: IMPLICATIONS FOR NOCICEPTIONCLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, Issue 8 2009Ai-Hua Pan SUMMARY 1ATP-gated P2X receptors in nociceptive sensory neurons participate in the transmission of pain signals from the periphery to the spinal cord. The effect of formalin on the expression of P2X3 receptors in dorsal root ganglia (DRG) was characterized using molecular and immunological approaches and the patch-clamp technique. 2Adult Sprague-Dawley rats were injected with 100 µL of 5% formalin in the planar surface of the hindpaw and were killed 30 min and 1, 3, 6, 12, 24 and 48 h later for in vitro analyses. The expression and distribution of P2X3 receptors in the lumbar spinal cord and in L5/L6 DRG were examined; 24 and 48 h after formalin injection, currents in neurons were examined using whole-cell patch-clamp recording. 3Western blots showed that anti-P2X3 antibody recognized a major monomer of approximately 64 kDa in DRG. Immunoreactivity for P2X3 receptors was detected predominantly in the cytoplasm and plasma membrane of small (< 25 µm) and middle-sized (25,50 µm) DRG neurons. Expression of the P2X3 transcript in the DRG was unchanged 30 min and 1 h after formalin injection, but increased after 12 h. There was no distinct change in P2X3 immunostaining of the spinal cord lamina at 30 min or 1 h after injection, but after 24 h P2X3 labelling increased. At 24 h after the formalin injection, currents in isolated small and middle-sized DRG neurons were increased by 1 µmol/L ,,,-methylene-ATP. These currents were completely inhibited by 1 µmol/L A-317491, a potent and selective P2X3 receptor antagonist. 4These data suggest that formalin injection leads to early upregulation of P2X3 expression in the spinal cord and DRG and that this may be one of the mechanisms giving rise to nociception. [source] Developmental shift in bidirectional functions of taurine-sensitive chloride channels during cortical circuit formation in postnatal mouse brainDEVELOPMENTAL NEUROBIOLOGY, Issue 2 2004Mika Yoshida Abstract Taurine (2-aminoethanesulfonic acid) is the most abundant free amino acid in the developing mammalian cerebral cortex, however, few studies have reported its neurobiological functions during development. In this study, by means of whole-cell patch-clamp recordings, we examined the effects of taurine on chloride channel receptors in neocortical neurons from early to late postnatal stages, which cover a critical period in cortical circuit formation. We show here that taurine activates chloride channels in cortical neurons throughout the postnatal stages examined (from postnatal day 2 to day 36). The physiological effects of taurine changed from excitatory to inhibitory due to variations in the intracellular Cl, concentration during development. An antagonist blocking analysis also demonstrated a developmental shift in the receptor target of taurine, from glycine receptors to GABAA receptors. Taken together, these results may reflect genetically programmed, bidirectional functions of taurine. At the early developmental stage, taurine acting on glycine receptors would serve to promote cortical circuit formation. As cortical circuit has to be regulated in the later stages, taurine would serve as a safeguard against hyperexcitable circuit. © 2004 Wiley Periodicals, Inc. J Neurobiol 60: 166,175, 2004 [source] Inhibition of superior colliculus neurons by a GABAergic input from the pretectal nuclear complex in the ratEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 12 2004Gesche Born Abstract The mammalian pretectal nuclear complex (PNC) is a visual and visuomotor control structure which is strongly connected to other subcortical visual structures. This indicates that the PNC also controls subcortical visual information flow during the execution of various oculomotor programs. A prominent, presumably GABAergic, projection from the PNC targets the superficial grey layer of the superior colliculus (SC), which itself is a central structure for visual information processing necessary for the generation of saccadic eye movements. In order to characterize the pretecto-tectal projection in vitro, we performed whole-cell patch-clamp recordings from SC and PNC neurons in slices obtained from 3,6-week-old pigmented rats. Focal glutamate injections into the PNC and electrical PNC stimulation were used to induce postsynaptic responses in SC neurons. Electrical stimulation of the SC allowed electrophysiological identification of PNC neurons that provide the inhibitory pretecto-tectal input. Only inhibitory postsynaptic currents could be elicited in SC neurons both by pharmacological and by electrical activation of the ipsilateral PNC. Concomitantly, a small number of PNC neurons could be antidromically activated from the ipsilateral SC. Most SC cells postsynaptic to the prectectal input showed the dendritic morphology of wide-field and narrow-field cells and are therefore regarded as projection neurons. All inhibitory currents evoked by PNC activation could be completely blocked by bath application of the selective GABAA receptor antagonist bicuculline. Together these results indicate that SC projection neurons receive a direct inhibitory input from the ipsilateral PNC and that this input is mediated by GABAA receptors. [source] No evidence for calcium electrogenic exchanger in frog semicircular canal hair cellsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 9 2002M. Martini Abstract We investigated the possibility that, in hair cells mechanically isolated from frog semicircular canals, Ca2+ extrusion occurs via a Na+ : Ca2+ (cardiac type) or a Na+ : Ca2+,K+ (retinal type) exchanger. Cells concurrently imaged during whole-cell patch-clamp recordings using the Ca2+ sensitive fluorescent dye Oregon Green 488 BAPTA-1 (100 µm) showed no voltage dependence of Ca2+ clearance dynamics following a Ca2+ load through voltage-gated Ca2+ channels. Reverse exchange was probed in hair cells dialyzed with a Ca2+ - and K+ -free solution, containing a Na+ concentration that saturates the exchanger, after zeroing the contribution to the whole-cell current from Ca2+ and K+ conductances. In these conditions, no reverse exchange current was detected upon switching from a Ca2+ -free external solution to a solution containing concentrations of Ca2+ alone, or Ca2+ + K+ that saturated the exchanger. By contrast, the same experimental protocol elicited peak exchange currents exceeding 100 pA in gecko rod photoreceptors, used as positive controls. In both cell types, we also probed the forward mode of the exchanger by rapidly increasing the intracellular Ca2+ concentration using flash photolysis of two novel caged Ca2+ complexes, calcium 2,2,-{[1-(2-nitrophenyl)ethane-1,2-diyl]bis(oxy)}bis(acetate) and calcium 2,2,-{[1-(4,5-dimethoxy-2-nitrophenyl)ethane-1,2-diyl]bis(oxy)} bis(acetate), in the presence of internal K+ and external Na+. No currents were evoked by UV-triggered Ca2+ jumps in hair cells, whereas exchanger conformational currents up to 400 pA, followed by saturating forward exchange currents up to 40 pA, were recorded in rod photoreceptors subjected to the same experimental conditions. We conclude that no functional electrogenic exchanger is present in this hair cell population, which leaves the abundant plasma membrane Ca2+ -ATPases as the primary contributors to Ca2+ extrusion. [source] An essential role for the H218/AGR16/Edg-5/LPB2 sphingosine 1-phosphate receptor in neuronal excitabilityEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 2 2001A. John MacLennan Abstract A wealth of indirect data suggest that the H218/AGR16/Edg-5/LPB2 sphingosine 1-phosphate (S1P) receptor plays important roles in development. In vitro, it activates several forms of development-related signal transduction and regulates cellular proliferation, differentiation and survival. It is expressed during embryogenesis, and mutation of an H218 -like gene in zebrafish leads to profound defects in embryonic development. Nevertheless, the in vivo functions served by H218 signalling have not been directly investigated. We report here that mice in which the H218 gene has been disrupted are unexpectedly born with no apparent anatomical or physiological defects. In addition, no abnormalities were observed in general neurological development, peripheral axon growth or brain structure. However, between 3 and 7 weeks of age, H218,/, mice have seizures which are spontaneous, sporadic and occasionally lethal. Electroencephalographic abnormalities were identified both during and between the seizures. At a cellular level, whole-cell patch-clamp recordings revealed that the loss of H218 leads to a large increase in the excitability of neocortical pyramidal neurons. Therefore, H218 plays an essential, unanticipated and functionally important role in the proper development and/or mediation of neuronal excitability. [source] The retrograde inhibition of IPSCs in rat cerebellar Purkinje cells is highly sensitive to intracellular Ca2+EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 3 2000Maike Glitsch Abstract The Ca2+ -dependent retrograde inhibition of inhibitory postsynaptic currents (depolarization-induced-suppression of inhibition; DSI) was investigated using fura-2 Ca2+ measurements and whole-cell patch-clamp recordings in rat cerebellar Purkinje cells. DSI was studied in cells loaded with different concentrations of the Ca2+ chelators BAPTA and EGTA. A concentration of 40 m m BAPTA was required to significantly interfere with DSI, whereas 10 m m BAPTA was almost ineffective. 40 m m EGTA reduced DSI, but was less effective than 40 m m BAPTA. Ratiometric Ca2+ measurements indicated that the extent of DSI depended critically on the changes in intracellular calcium ([Ca2+]i). The relationship between DSI and peak ,[Ca2+]i could be approximated by a hyperbolic function, with apparent half-saturation concentrations of 200 and 40 n m for dendritic and somatic [Ca2+]i, respectively. It is suggested that DSI is due to somatodendritic exocytosis of a retrograde messenger, and that this exocytosis is highly sensitive to [Ca2+]i. [source] Adenosine inhibits paraventricular pre-sympathetic neurons through ATP-dependent potassium channelsJOURNAL OF NEUROCHEMISTRY, Issue 2 2010De-Pei Li J. Neurochem. (2010) 113, 530,542. Abstract Adenosine produces cardiovascular depressor effects in various brain regions. However, the cellular mechanisms underlying these effects remain unclear. The pre-sympathetic neurons in the hypothalamic paraventricular nucleus (PVN) play an important role in regulating arterial blood pressure and sympathetic outflow through projections to the spinal cord and brainstem. In this study, we performed whole-cell patch-clamp recordings on retrogradely labeled PVN neurons projecting to the intermediolateral cell column of the spinal cord in rats. Adenosine (10,100 ,M) decreased the firing activity in a concentration-dependent manner, with a marked hyperpolarization in 12 of 26 neurons tested. Blockade of A1 receptors with the adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine or intracellular dialysis of guanosine 5,- O -(2-thodiphosphate) eliminated the inhibitory effect of adenosine on labeled PVN neurons. Immunocytochemical labeling revealed that A1 receptors were expressed on spinally projecting PVN neurons. Also, blocking ATP-dependent K+ (KATP) channels with 100 ,M glibenclamide or 200 ,M tolbutamide, but not the G protein-coupled inwardly rectifying K+ channels blocker tertiapin-Q, abolished the inhibitory effect of adenosine on the firing activity of PVN neurons. Furthermore, glibenclamide or tolbutamide significantly decreased the adenosine-induced outward currents in labeled neurons. The reversal potential of adenosine-induced currents was close to the K+ equilibrium potential. In addition, adenosine decreased the frequency of both spontaneous and miniature glutamatergic excitatory post-synaptic currents and GABAergic inhibitory post-synaptic currents in labeled neurons, and these effects were also blocked by 8-cyclopentyl-1,3-dipropylxanthine. Collectively, our findings suggest that adenosine inhibits the excitability of PVN pre-sympathetic neurons through A1 receptor-mediated opening of KATP channels. [source] Efficient generation of mature cerebellar Purkinje cells from mouse embryonic stem cellsJOURNAL OF NEUROSCIENCE RESEARCH, Issue 2 2010Osamu Tao Abstract Mouse embryonic stem cells (ESCs) can generate cerebellar neurons, including Purkinje cells (PCs) and their precursor cells, in a floating culture system called serum-free culture of embryoid body-like aggregates (SFEB) treated with BMP4, Fgf8b, and Wnt3a. Here we successfully established a coculture system that induced the maturation of PCs in ESC-derived Purkinje cell (EDPC) precursors in SFEB, using as a feeder layer a cerebellum dissociation culture prepared from mice at postnatal day (P) 6,8. PC maturation was incomplete or abnormal when the adherent culture did not include feeder cells or when the feeder layer was from neonatal cerebellum. In contrast, EDPCs exhibited the morphology of mature PCs and synaptogenesis with other cerebellar neurons when grown for 4 weeks in coculture system with the postnatal cerebellar feeder. Furthermore, the electrophysiological properties of these EDPCs were compatible with those of native mature PCs in vitro, such as Na+ or Ca2+ spikes elicited by current injections and excitatory or inhibitory postsynaptic currents, which were assessed by whole-cell patch-clamp recordings. Thus, EDPC precursors in SFEB can mature into PCs whose properties are comparable with those of native PCs in vitro. © 2009 Wiley-Liss, Inc. [source] Analysis of neural potential of human umbilical cord blood,derived multipotent mesenchymal stem cells in response to a range of neurogenic stimuliJOURNAL OF NEUROSCIENCE RESEARCH, Issue 9 2008Isabel Zwart Abstract We investigated the neurogenic potential of full-term human umbilical cord blood (hUCB),derived multipotent mesenchymal stem cells (MSCs) in response to neural induction media or coculture with rat neural cells. Phenotypic and functional changes were assessed by immunocytochemistry, RT-PCR, and whole-cell patch-clamp recordings. Naive MSCs expressed both mesodermal and ectodermal markers prior to neural induction. Exposure to retinoic acid, basic fibroblast growth factor, or cyclic adenosine monophosphate (cAMP) did not stimulate neural morphology, whereas exposure to dibutyryl cAMP and 3-isobutyl-1-methylxanthine stimulated a neuron-like morphology but also appeared to be cytotoxic. All protocols stimulated increases in expression of the neural precursor marker nestin, but expression of mature neuronal or glial markers MAP2 and GFAP was not observed. Nestin expression increases were serum level dependent. Electrophysiological properties of MSCs were studied with whole-cell patch-clamp recordings. The MSCs possessed no ionic currents typical of neurons before or after neural induction protocols. Coculture of hUCB-derived MSCs and rat neural cells induced some MSCs to adopt an astrocyte-like morphology and express GFAP protein and mRNA. Our data suggest hUCB-derived MSCs do not transdifferentiate into mature functioning neurons in response to the above neurogenic protocols; however, coculture with rat neural cells led to a minority adopting an astrocyte-like phenotype. © 2008 Wiley-Liss, Inc. [source] Ethanol Acutely Modulates mGluR1-Dependent Long-Term Depression in CerebellumALCOHOLISM, Issue 7 2010Li-Da Su Background:, Acute and chronic ethanol exposure produces profound impairments in motor functioning. Individuals with lower sensitivity to the acute motor impairing effects of ethanol have an increased risk of developing alcohol dependence and abuse, and infants with subtle delays in motor coordination development may have an increased risk for subsequently developing alcoholism. Thus, understanding the mechanism by which ethanol disrupts motor functioning is very important. Methods:, Parasagittal slices of the cerebellar vermis (250 ,M thick) were prepared from P17 to 20 Sprague,Dawley rats. Whole-cell recordings of Purkinje cells were obtained with an Axopatch 200B amplifier. Parallel fiber-Purkinje cell synaptic currents were sampled at 1 kHz and digitized at 10 kHz, and synaptic long-term depression (LTD) was observed in either external or internal application of ethanol for comparison. Results:, We determined whether ethanol acutely affects parallel fiber LTD using whole-cell patch-clamp recordings from Purkinje cells. Application of ethanol both externally (50 mM) and internally (17 and 10 mM) significantly suppressed mGluR-mediate slow currents. Short-term external ethanol exposure (50 but not 17 mM) during tetanus blocked mGluR-dependent parallel fiber LTD. Furthermore, internal 17 and 10 mM ethanol completely inhibited this LTD. Conclusions:, The results of the current study demonstrate that ethanol acutely suppresses parallel fiber LTD and may influence the mGluR-mediated slow current intracellularly. This study, plus previous evidence by Carta and colleagues (2006) and Belmeguenai and colleagues (2008), suggests significant actions of ethanol on mGluR-mediated currents and its dependent plasticity in brain. [source] Efficient Ca2+ buffering in fast-spiking basket cells of rat hippocampusTHE JOURNAL OF PHYSIOLOGY, Issue 8 2008Yexica Aponte Fast-spiking parvalbumin-expressing basket cells (BCs) represent a major type of inhibitory interneuron in the hippocampus. These cells inhibit principal cells in a temporally precise manner and are involved in the generation of network oscillations. Although BCs show a unique expression profile of Ca2+ -permeable receptors, Ca2+ -binding proteins and Ca2+ -dependent signalling molecules, physiological Ca2+ signalling in these interneurons has not been investigated. To study action potential (AP)-induced dendritic Ca2+ influx and buffering, we combined whole-cell patch-clamp recordings with ratiometric Ca2+ imaging from the proximal apical dendrites of rigorously identified BCs in acute slices, using the high-affinity Ca2+ indicator fura-2 or the low-affinity dye fura-FF. Single APs evoked dendritic Ca2+ transients with small amplitude. Bursts of APs evoked Ca2+ transients with amplitudes that increased linearly with AP number. Analysis of Ca2+ transients under steady-state conditions with different fura-2 concentrations and during loading with 200 ,m fura-2 indicated that the endogenous Ca2+ -binding ratio was ,200 (,S= 202 ± 26 for the loading experiments). The peak amplitude of the Ca2+ transients measured directly with 100 ,m fura-FF was 39 nm AP,1. At ,23°C, the decay time constant of the Ca2+ transients was 390 ms, corresponding to an extrusion rate of ,600 s,1. At 34°C, the decay time constant was 203 ms and the corresponding extrusion rate was ,1100 s,1. At both temperatures, continuous theta-burst activity with three to five APs per theta cycle, as occurs in vivo during exploration, led to a moderate increase in the global Ca2+ concentration that was proportional to AP number, whereas more intense stimulation was required to reach micromolar Ca2+ concentrations and to shift Ca2+ signalling into a non-linear regime. In conclusion, dentate gyrus BCs show a high endogenous Ca2+ -binding ratio, a small AP-induced dendritic Ca2+ influx, and a relatively slow Ca2+ extrusion. These specific buffering properties of BCs will sharpen the time course of local Ca2+ signals, while prolonging the decay of global Ca2+ signals. [source] The voltage-dependent Cl, channel ClC-5 and plasma membrane Cl, conductances of mouse renal collecting duct cells (mIMCD-3)THE JOURNAL OF PHYSIOLOGY, Issue 3 2001J. A. Sayer 1We have tested the hypothesis that the voltage-dependent Cl, channel, ClC-5 functions as a plasma membrane Cl, conductance in renal inner medullary collecting duct cells. 2Full-length mouse kidney ClC-5 (mClC-5) was cloned and transiently expressed in CHO-K1 cells. Fast whole-cell patch-clamp recordings confirmed that mClC-5 expression produces a voltage-dependent, strongly outwardly rectifying Cl, conductance that was unaffected by external DIDS. 3Slow whole-cell recordings, using nystatin-perforated patches from transfected CHO-K1 cells, also produced voltage-dependent Cl, currents consistent with ClC-5 expression. However, under this recording configuration an endogenous DIDS-sensitive Ca2+ -activated Cl, conductance was also evident, which appeared to be activated by green fluorescent protein (GFP) transfection. 4A mClC-5-GFP fusion protein was transiently expressed in CHO-K1 cells; confocal laser scanning microscopy (CLSM) showed localization at the plasma membrane, consistent with patch-clamp experiments. 5Endogenous expression of mClC-5 was demonstrated in mouse renal collecting duct cells (mIMCD-3) by RT-PCR and by immunocytochemistry. 6Using slow whole-cell current recordings, mIMCD-3 cells displayed three biophysically distinct Cl, -selective currents, which were all inhibited by DIDS. However, no cells exhibited whole-cell currents that had mClC-5 characteristics. 7Transient transfection of mIMCD-3 cells with antisense mClC-5 had no effect on the endogenous Cl, conductances. Transient transfection with sense mClC-5 failed to induce the Cl, conductance seen in CHO-K1 cells but stimulated levels of the endogenous Ca2+ -activated Cl, conductance 24 h post-transfection. 8Confocal laser scanning microscopy of mIMCD-3 cells transfected with mClC-5-GFP showed that the protein was absent from the plasma membrane and was instead localized to acidic endosomal compartments. 9These data discount a major role for ClC-5 as a plasma membrane Cl, conductance in mIMCD-3 cells but suggest a role in endosomal function. [source] Electrophysiological classification of P2X7 receptors in rat cultured neocortical astrogliaBRITISH JOURNAL OF PHARMACOLOGY, Issue 8 2010W Nörenberg Background and purpose:, P2X7 receptors are ATP-gated cation channels mediating important functions in microglial cells, such as the release of cytokines and phagocytosis. Electrophysiological evidence that these receptors also occur in CNS astroglia is rare and rather incomplete. Experimental approach:, We used whole-cell patch-clamp recordings to search for P2X7 receptors in astroglial,neuronal co-cultures prepared from the cerebral cortex of rats. Key results:, All the astroglial cells investigated responded to ATP with membrane currents, reversing around 0 mV. These currents could be also detected in isolated outside-out patch vesicles. The results of the experiments with the P2X [,,,-methylene ATP and 2,-3,-O-(4-benzoyl) ATP] and P2Y receptor agonists [adenosine 5,-O-(2-thiodiphosphate), uridine 5,-diphosphate, uridine 5,-triphosphate (UTP) and UDP-glucose] suggested the involvement of P2X receptors in this response. The potentiation of ATP responses in a low divalent cation or alkaline bath, but not by ivermectin, made it likely that a P2X7 receptor is operational. Blockade of the ATP effect by the P2X7 antagonists Brilliant Blue G, calmidazolium and oxidized ATP corroborated this assumption. Conclusions and implications:, Rat cultured cortical astroglia possesses functional P2X7 receptors. It is suggested that astrocytic P2X7 receptors respond to high local ATP concentrations during neuronal injury. [source] | |||||||||||||