Home About us Contact
|
Home About us Contact | ||
|
|
||
Whole-cell Recordings (whole-cell + recording)
Selected AbstractsSuppression of excitatory cholinergic synaptic transmission by Drosophila dopamine D1-like receptorsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 9 2007Ning Yuan Abstract The physiological function of dopamine is mediated through its G-protein-coupled receptor family. In Drosophila, four dopamine receptors have been molecularly characterized so far. However, due largely to the absence of a suitable preparation, the role of Drosophila dopamine receptors in modulating central synaptic transmission has not been examined. The present study investigated mechanisms by which dopamine modulates excitatory cholinergic synaptic transmission in Drosophila using primary neuronal cultures. Whole-cell recordings demonstrated that cholinergic excitatory postsynaptic currents (EPSCs) were down-regulated by focally applied dopamine (10,500 µm). The vertebrate D1 specific agonists SKF38393 and 6-chloro-APB (10 µm) mimicked dopamine-mediated suppression of cholinergic synaptic transmission with higher potency. In contrast, the D2 agonists quinpirole and bromocriptine did not alter cholinergic EPSCs, demonstrating that dopamine-mediated suppression of cholinergic synaptic transmission is specifically through activation of Drosophila D1-like receptors. Biophysical analysis of miniature EPSCs indicated that cholinergic suppression by activation of D1-like receptors is presynaptic in origin. Dopamine modulation of cholinergic transmission is not mediated through the cAMP/protein kinase A signaling pathway as cholinergic suppression by dopamine occurred in the presence of the protein kinase A inhibitor H-89. In addition, an adenylate cyclase activator, forskolin, led to an increase, not a decrease, of cholinergic EPSC frequency. Finally, we showed that activation of D1-like receptors decreased the frequency of action potentials in cultured Drosophila neurons by inhibiting excitatory cholinergic transmission. All our data demonstrated that activation of D1-like receptors in Drosophila neurons negatively modulates excitatory cholinergic synaptic transmission and thus inhibits neuronal excitability. [source] Environmental manipulations early in development alter seizure activity, Ih and HCN1 protein expression later in lifeEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 12 2006Ulrich Schridde Abstract Although absence epilepsy has a genetic origin, evidence from an animal model (Wistar Albino Glaxo/Rijswijk; WAG/Rij) suggests that seizures are sensitive to environmental manipulations. Here, we show that manipulations of the early rearing environment (neonatal handling, maternal deprivation) of WAG/Rij rats leads to a pronounced decrease in seizure activity later in life. Recent observations link seizure activity in WAG/Rij rats to the hyperpolarization-activated cation current (Ih) in the somatosensory cortex, the site of seizure generation. Therefore, we investigated whether the alterations in seizure activity between rats reared differently might be correlated with changes in Ih and its channel subunits hyperpolarization-activated cation channel HCN1, 2 and 4. Whole-cell recordings from layer 5 pyramidal neurons, in situ hybridization and Western blot of the somatosensory cortex revealed an increase in Ih and HCN1 in neonatal handled and maternal deprived, compared to control rats. The increase was specific to HCN1 protein expression and did not involve HCN2/4 protein expression, or mRNA expression of any of the subunits (HCN1, 2, 4). Our findings provide the first evidence that relatively mild changes in the neonatal environment have a long-term impact of absence seizures, Ih and HCN1, and suggest that an increase of Ih and HCN1 is associated with absence seizure reduction. Our findings shed new light on the role of Ih and HCN in brain functioning and development and demonstrate that genetically determined absence seizures are quite sensitive for early interventions. [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] Potentiation of glycine responses by dideoxyforskolin and tamoxifen in rat spinal neuronsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 4 2003Dominique Chesnoy-Marchais Abstract Dideoxyforskolin, a forskolin analogue unable to stimulate adenylate cyclase, and tamoxifen, an antioestrogen widely used against breast cancer, are both known to block some Cl, channels. Their effects on Cl, responses to glycine or GABA have been tested here by using whole-cell recording from cultured spinal neurons. Dideoxyforskolin (4 or 16 µm) and tamoxifen (0.2,5 µm) both potentiate responses to low glycine concentrations. They also induce blocking effects, predominant at high glycine concentrations. At 5 µm, tamoxifen increased responses to 15 µm glycine by a factor >4.5, reaching 20 in some neurons. Potentiation by extracellular dideoxyforskolin or tamoxifen persisted after intracellular application of the modulator and was not due to Zn2+ contamination. Potentiation by tamoxifen also persisted in a Ca2+ -free extracellular solution, after intracellular Ca2+ buffering and protein kinase C blockade. Thus, the critical sites of action are not intracellular. The EC50 for glycine was lowered 6.6-fold by 5 µm tamoxifen. The kinetics and voltage-dependence of the effects of tamoxifen on glycine responses support the idea that this hydrophobic drug may act from a site located within the membrane. Tamoxifen (5 µm) also increased responses to 2 µm GABA by a factor of 3.5, but barely affected peak responses to 20 µm GABA. The demonstration that tamoxifen affects some of the main inhibitory receptors should be useful for better evaluating its neurological effects. Furthermore, the results identify a new class of molecules that potentiate glycine receptor function. [source] Modulation of glycine responses by dihydropyridines and verapamil in rat spinal neuronsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 12 2001Dominique Chesnoy-Marchais Abstract Although glycine receptors (GlyRs) are responsible for the main spinal inhibitory responses in adult vertebrates, in the embryo they have been reported to mediate depolarizing responses, which can sometimes activate dihydropyridine-sensitive l -type calcium channels. However, these channels are not the only targets of dihydropyridines (DHPs), and we questioned whether GlyRs might be directly modulated by DHPs. By whole-cell recording of cultured spinal neurons, we investigated modulation of glycine responses by the calcium channel antagonists, nifedipine, nitrendipine, nicardipine and (R)-Bay K 8644, and by the calcium channel, agonist (S)-Bay K 8644. At concentrations between 1 and 10 µm, all these DHPs could block glycine responses, even in the absence of extracellular Ca2+. The block was stronger at higher glycine concentrations, and increased with time during each glycine application. Nicardipine blocked GABAA responses from the same neurons in a similar manner. In addition to their blocking effects, nitrendipine and nicardipine potentiated the peak responses to low glycine concentrations. Both effects of extracellular nitrendipine on glycine responses persisted when the drug was present in the intracellular solution. Thus, these modulations are related neither to calcium channel modulation nor to possible intracellular effects of DHPs. Another type of calcium antagonist, verapamil (10,50 µm), also blocked glycine responses. Our results suggest that some of the effects of calcium antagonists, including the neuroprotective and anticonvulsant effects of DHPs, might result partly from their interactions with ligand-gated chloride channels. [source] 1-Methyl-4-phenylpridinium (MPP+)-induced functional run-down of GABAA receptor-mediated currents in acutely dissociated dopaminergic neuronsJOURNAL OF NEUROCHEMISTRY, Issue 1 2002Jie Wu Abstract We have evaluated GABAA receptor function during treatment of 1-methyl-4-phenylpridinium (MPP+) using patch-clamp perforated whole-cell recording techniques in acutely dissociated dopaminergic (DAergic) neurons from rat substantia nigra compacta (SNc). ,-Aminobutyric acid (GABA), glutamate or glycine induced inward currents (IGABA, IGlu, IGly) at a holding potential (VH) of ,45 mV. The IGABA was reversibly blocked by the GABAA receptor antagonist, bicuculline, suggesting that IGABA is mediated through the activation of GABAA receptors. During extracellular perfusion of MPP+ (1,10 ,m), IGABA, but neither IGlu nor IGly, declined (termed run-down) with repetitive agonist applications, indicating that the MPP+ -induced IGABA run-down occurred earlier than IGly or IGlu under our experimental conditions. The MPP+ -induced IGABA run-down can be prevented by a DA transporter inhibitor, mazindol, and can be mimicked by a metabolic inhibitor, rotenone. Using conventional whole-cell recording with different concentrations of ATP in the pipette solution, IGABA run-down can be induced by decreasing intracellular ATP concentrations, or prevented by supplying intracellular ATP, indicating that IGABA run-down is dependent on intracellular ATP concentrations. A GABAA receptor positive modulator, pentobarbital (PB), potentiated the declined IGABA and eliminated IGABA run-down. Corresponding to these patch-clamp data, tyrosine hydroxylase (TH) immunohistochemical staining showed that TH-positive cell loss was protected by PB during MPP+ perfusion. It is concluded that extracellular perfusion of MPP+ induces a functional run-down of GABAA receptors, which may cause an imbalance of excitation and inhibition of DAergic neurons. [source] Acute exposure to low-level CW and GSM-modulated 900 MHz radiofrequency does not affect Ba2+ currents through voltage-gated calcium channels in rat cortical neuronsBIOELECTROMAGNETICS, Issue 8 2007Daniela Platano Abstract We have studied the non-thermal effects of radiofrequency (RF) electromagnetic fields (EMFs) on Ba2+ currents () through voltage-gated calcium channels (VGCC), recorded in primary cultures of rat cortical neurons using the patch-clamp technique. To assess whether low-level acute RF field exposure could modify the amplitude and/or the voltage-dependence of , Petri dishes containing cultured neurons were exposed for 1,3 periods of 90 s to 900 MHz RF-EMF continuous wave (CW) or amplitude-modulated according to global system mobile communication standard (GSM) during whole-cell recording. The specific absorption rates (SARs) were 2 W/kg for CW and 2 W/kg (time average value) for GSM-modulated signals, respectively. The results obtained indicate that single or multiple acute exposures to either CW or GSM-modulated 900 MHz RF-EMFs do not significantly alter the current amplitude or the current,voltage relationship of , through VGCC. Bioelectromagnetics 28:599,607, 2007. © 2007 Wiley-Liss, Inc. [source] Single mechano-gated channels activated by mechanical deformation of acutely isolated cardiac fibroblasts from ratsACTA PHYSIOLOGICA, Issue 3 2010A. Kamkin Abstract Aim:, Mechanosensitive conductances were reported in cardiac fibroblasts, but the properties of single channels mediating their mechanosensitivity remain uncharacterized. The aim of this work was to investigate single mechano-gated channels (MGCs) activated by mechanical deformations of cardiac fibroblasts. Methods:, Currents through single MGCs and mechanosensitive whole-cell currents were recorded from isolated rat atrial fibroblasts using the cell-attached and whole-cell patch-clamp configurations respectively. Defined mechanical stress was applied via the patch pipette used for the whole-cell recordings. Results:, Under resting conditions occasional short openings of two types of single MGCs with conductances of 43 and 87 pS were observed. Both types of channels displayed a linear current,voltage relationship with the reversal potential around 0 mV. Small (1 ,m) mechanical deformations affected neither single nor whole-cell mechano-gated currents. Cell compressions (2, 3 and 4 ,m) augmented the whole-cell currents and increased the frequency and duration of single channel openings. Cell stretches (2, 3 and 4 ,m) inactivated the whole-cell currents and abolished the activity of single MGCs. Gd3+ (8 ,m) blocked the whole-cell currents within 5 min. No single channel activity was observed in the cell-attached mode when Gd3+ was added to the intrapipette solution. Cytochalasin D and colchicine (100 ,m each) completely blocked both the whole-cell and single channel currents. Conclusions:, These findings show that rat atrial fibroblasts express two types of MGCs whose activity is governed by cell deformation. We conclude that fibroblasts can sense the direction of applied stress and contribute to mechano-electrical coupling in the heart. [source] Genetic manipulation, whole-cell recordings and functional imaging of the sensorimotor cortex of behaving miceACTA PHYSIOLOGICA, Issue 1 2009C. C. H. Petersen Abstract Sensory processing, sensorimotor integration and motor control are amongst the most basic functions of the brain and yet our understanding of how the underlying neuronal networks operate and contribute to behaviour is very limited. The relative simplicity of the mouse whisker sensorimotor system is helpful for detailed quantitative analyses of motor control and perception during active sensory processing. Recent technical advances now allow the measurement of membrane potential in awake-behaving mice, using whole-cell recordings and voltage-sensitive dye imaging. With these recording techniques, it is possible to directly correlate neuronal activity with behaviour. However, in order to obtain causal evidence for the specific contributions of different neuronal networks to behaviour, it is critical to manipulate the system in a highly controlled manner. Advances in molecular neurobiology, gene delivery and mouse genetics provide techniques capable of layer, column and cell-type specific control of gene expression in the mouse neocortex. Over the next years, we anticipate considerable advances in our understanding of brain function through measuring and manipulating neuronal activity with unprecedented precision to probe the molecular and synaptic mechanisms underlying simple forms of active sensory perception and associative learning. [source] Roles of glutamate and GABA receptors in setting the developmental timing of spontaneous synchronized activity in the developing mouse cortexDEVELOPMENTAL NEUROBIOLOGY, Issue 12 2007Annette K. McCabe Abstract Spontaneous, synchronized electrical activity (SSA) plays important roles in nervous system development, but it is not clear what causes it to start and stop at the appropriate times. In previous work, we showed that when SSA in neonatal mouse cortex is blocked by TTX in cultured slices during its normal time of occurrence (E17,P3), it fails to stop at P3 as it does in control cultured slices, but instead persists through at least P10. This indicates that SSA is self-extinguishing. Here we use whole-cell recordings and [Ca2+]i imaging to compare control and TTX-treated slices to isolate the factors that normally extinguish SSA on schedule. In TTX-treated slices, SSA bursts average 4 s in duration, and have two components. The first, lasting about 1 s, is mediated by AMPA receptors; the second, which extends the burst to 4 s and is responsible for most of the action potential generation during the burst, is mediated by NMDA receptors. In later stage (P5,P9) control slices, after SSA has declined to about 4% of its peak frequency, bursts lack this long NMDA component. Blocking this NMDA component in P5,P9 TTX-treated slices reduces SSA frequency, but not to the low values found in control slices, implying that additional factors help extinguish SSA. GABAA inhibitors restore SSA in control slices, indicating that the emergence of GABAA -mediated inhibition is another major factor that helps terminate SSA. © 2007 Wiley Periodicals, Inc. Develop Neurobiol, 2007 [source] Interneuron subtype specific activation of mGluR1/5 during epileptiform activity in hippocampusEPILEPSIA, Issue 8 2010Nathalie T Sanon Summary Purpose:, Specific inhibitory interneurons in area CA1 of the hippocampus, notably those located in stratum oriens,alveus (O/A-INs), are selectively vulnerable in patients and animal models of temporal lobe epilepsy (TLE). The excitotoxic mechanisms underlying the selective vulnerability of interneurons have not been identified but could involve group I metabotropic glutamate receptor subtypes (mGluR1/5), which have generally proconvulsive actions and activate prominent cationic currents and calcium responses specifically in O/A-INs. Methods:, In this study, we examine the role of mGluR1/5 in interneurons during epileptiform activity using whole-cell recordings from CA1 O/A-INs and selective antagonists of mGluR1, (LY367385) and mGluR5 (MPEP) in a disinhibited rat hippocampal slice model of epileptiform activity. Results:, Our data indicate more prominent epileptiform burst discharges and paroxysmal depolarizations (PDs) in O/A-INs than in interneurons located at the border of strata radiatum and lacunosum/moleculare (R/LM-INs). In addition, mGluR1 and mGluR5 significantly contributed to epileptiform responses in O/A-INs but not in R/LM-INs. Epileptiform burst discharges in O/A-INs were partly dependent on mGluR5. PDs and associated postsynaptic currents were dependent on both mGluR1, and mGluR5. These receptors contributed differently to postsynaptic currents underlying PDs, with mGluR5 contributing to the fast and slow components and mGluR1, to the slow component. Discussion:, These findings support interneuron subtype-specific activation and differential contributions of mGluR1, and mGluR5 to epileptiform activity in O/A-INs, which could be important for their selective vulnerability in TLE. [source] Vasopressin modulates lateral septal network activity via two distinct electrophysiological mechanismsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 9 2007G. Allaman-Exertier Abstract The lateral septal area is rich in vasopressin V1A receptors and is densely innervated by vasopressinergic axons, originating mainly from the bed nucleus of the stria terminalis and the amygdala. Genetic and behavioral studies provide evidence that activation of vasopressin receptors in this area plays a determinant role in promoting social recognition. What could be the neuronal mechanism underlying this effect? Using rat brain slices and whole-cell recordings, we found that lateral septal neurons are under the influence of a basal GABAergic inhibitory input. Vasopressin, acting via V1A but not V1B receptors, greatly enhanced this input in nearly all neurons. The peptide had no effect on miniature inhibitory postsynaptic currents, indicating that it acted on receptors located in the somatodendritic membrane, rather than on axon terminals, of GABAergic interneurons. Cell-attached recordings showed that vasopressin can cause a direct excitation of a subpopulation of lateral septal neurons by acting via V1A but not V1B receptors. The presence in the lateral septum of V1A but not of V1B receptors was confirmed by competition binding studies using light microscopic autoradiography. In conclusion, vasopressin appears to act in the lateral septum in a dual mode: (i) by causing a direct excitation of a subpopulation of neurons, and (ii) by causing an indirect inhibition of virtually all lateral septal neurons. This modulation by vasopressin of the lateral septal circuitry may be part of the neuronal mechanism by which the peptide, acting via V1A receptors, promotes social recognition. [source] Postsynaptic M1 and M3 receptors are responsible for the muscarinic enhancement of retrograde endocannabinoid signalling in the hippocampusEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 1 2003Takako Ohno-Shosaku Abstract The cholinergic system is crucial for higher brain functions including learning and memory. These functions are mediated primarily by muscarinic acetylcholine receptors (mAChRs) that consist of five subtypes (M1,M5). A recent study suggested a novel role of acetylcholine as a potent enhancer of endocannabinoid signalling that acts retrogradely from postsynaptic to presynaptic neurons. In the present study, we further investigated the mechanisms of this cholinergic effect on endocannabinoid signalling. We made paired whole-cell recordings from cultured hippocampal neurons, and monitored inhibitory postsynaptic currents (IPSCs). The postsynaptic depolarization induced a transient suppression of IPSCs (DSI), a phenomenon known to involve retrograde signalling by endocannabinoids. The cholinergic agonist carbachol (CCh) markedly enhanced DSI at 0.01,0.3 µm without changing the presynaptic cannabinoid sensitivity. The facilitating effect of CCh on DSI was mimicked by the muscarinic agonist oxotremorine-M, whereas it was eliminated by the muscarinic antagonist atropine. It was also blocked by a non-hydrolizable analogue of GDP (GDP-,-S) that was applied intracellularly to postsynaptic neurons. The muscarinic enhancement of DSI persisted to a substantial degree in the neurons prepared from M1 -knockout and M3 -knockout mice, but was virtually eliminated in the neurons from M1/M3 -compound-knockout mice. CCh still enhanced DSI significantly under the blockade of postsynatpic K+ conductance, and did not significantly influence the depolarization-induced Ca2+ transients. These results indicate that the activation of postsynaptic M1 and M3 receptors facilitates the depolarization-induced release of endocannabinoids. [source] Innervation of interneurons immunoreactive for VIP by intrinsically bursting pyramidal cells and fast-spiking interneurons in infragranular layers of juvenile rat neocortexEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 1 2002Jochen F. Staiger Abstract Cortical columns contain specific neuronal populations with characteristic sets of connections. This wiring forms the structural basis of dynamic information processing. However, at the single-cell level little is known about specific connectivity patterns. We performed experiments in infragranular layers (V and VI) of rat somatosensory cortex, to clarify further the input patterns of inhibitory interneurons immunoreactive (ir) for vasoactive intestinal polypeptide (VIP). Neurons in acute slices were electrophysiologically characterized using whole-cell recordings and filled with biocytin. This allowed us to determine their firing pattern as regular-spiking, intrinsically bursting and fast-spiking, respectively. Biocytin was revealed histochemically and VIP immunohistochemically. Sections were examined for contacts between the axons of the filled neurons and the VIP-ir targets. Twenty pyramidal cells and five nonpyramidal (inter)neurons were recovered and sufficiently stained for further analysis. Regular-spiking pyramidal cells displayed no axonal boutons in contact with VIP-ir targets. In contrast, intrinsically bursting layer V pyramidal cells showed four putative single contacts with a proximal dendrite of VIP neurons. Fast-spiking interneurons formed contacts with two to six VIP neurons, preferentially at their somata. Single as well as multiple contacts on individual target cells were found. Electron microscopic examinations showed that light-microscopically determined contacts represent sites of synaptic interactions. Our results suggest that, within infragranular local cortical circuits, (i) fast-spiking interneurons are more likely to influence VIP cells than are pyramidal cells and (ii) pyramidal cell input probably needs to be highly convergent to fire VIP target cells. [source] Riluzole inhibits the persistent sodium current in mammalian CNS neuronsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 10 2000Andrea Urbani Abstract The effects of 0.1,100 ,m riluzole, a neuroprotective agent with anticonvulsant properties, were studied on neurons from rat brain cortex. Patch-clamp whole-cell recordings in voltage-clamp mode were performed on thin slices to examine the effects of the drug on a noninactivating (persistent) Na+ current (INa,p). INa,p was selected because it enhances neuronal excitability near firing threshold, which makes it a potential target for anticonvulsant drugs. When added to the external solution, riluzole dose-dependently inhibited INa,p up to a complete blocking of the current (EC50 2 ,m), showing a significant effect at therapeutic drug concentrations. A comparative dose-effect study was carried out in the same cells for the other main known action of riluzole, the inhibitory effect on the fast transient sodium current. This effect was confirmed in our experiments, but we found that it was achieved at levels much higher than putative therapeutic concentrations. Only the effect on INa,p, and not that on fast sodium current, can account for the reduction in neuronal excitability observed in cortical neurons following riluzole treatment at therapeutic concentrations, and this might represent a novel mechanism accounting for the anticonvulsant and neuroprotective properties of riluzole. [source] A new class of neurotoxin from wasp venom slows inactivation of sodium currentEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 6 2000Yoshinori Sahara Abstract The effects of ,-pompilidotoxin (,-PMTX), a new neurotoxin isolated from the venom of a solitary wasp, were studied on the neuromuscular synapses in lobster walking leg and the rat trigeminal ganglion (TG) neurons. Paired intracellular recordings from the presynaptic axon terminals and the innervating lobster leg muscles revealed that ,-PMTX induced long bursts of action potentials in the presynaptic axon, which resulted in facilitated excitatory and inhibitory synaptic transmission. The action of ,-PMTX was distinct from that of other known facilitatory presynaptic toxins, including sea anemone toxins and ,-scorpion toxins, which modify the fast inactivation of Na+ current. We further characterized the action of ,-PMTX on Na+ channels by whole-cell recordings from rat trigeminal neurons. We found that ,-PMTX slowed the Na+ channels inactivation process without changing the peak current,voltage relationship or the activation time course of tetrodotoxin (TTX)-sensitive Na+ currents, and that ,-PMTX had voltage-dependent effects on the rate of recovery from Na+ current inactivation and deactivating tail currents. The results suggest that ,-PMTX slows or blocks conformational changes required for fast inactivation of the Na+ channels on the extracellular surface. The simple structure of ,-PMTX, consisting of 13 amino acids, would be advantageous for understanding the functional architecture of Na+ channel protein. [source] Age-dependent enhancement of inhibitory synaptic transmission in CA1 pyramidal neurons via GluR5 kainate receptorsHIPPOCAMPUS, Issue 8 2009Changqing Xu Abstract Changes in hippocampal synaptic networks during aging may contribute to age-dependent compromise of cognitive functions such as learning and memory. Previous studies have demonstrated that GABAergic synaptic transmission exhibits age-dependent changes. To better understand such age-dependent changes of GABAergic synaptic inhibition, we performed whole-cell recordings from pyramidal cells in the CA1 area of acute hippocampal slices on aged (24,26 months old) and young (2,4 months old) Brown-Norway rats. We found that the frequency and amplitude of spontaneous inhibitory postsynaptic current (IPSCs) were significantly increased in aged rats, but the frequency and amplitude of mIPSCs were decreased. Furthermore, the regulation of GABAergic synaptic transmission by GluR5 containing kainate receptors was enhanced in aged rats, which was revealed by using LY382884 (a GluR5 kainate receptor antagonist) and ATPA (a GluR5 kainate receptor agonist). Moreover, we demonstrated that vesicular glutamate transporters are involved in the kainate receptor dependent regulation of sIPSCs. Taken together, these results suggest that GABAergic synaptic transmission is potentiated in aged rats, and GluR5 containing kainate receptors regulate the inhibitory synaptic transmission through endogenous glutamate. These alterations of GABAergic input with aging could contribute to age-dependent cognitive decline. © 2009 Wiley-Liss, Inc. [source] Few cultured rat primary sensory neurons express a tolbutamide-sensitive K+ currentJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 2 2002Violeta Ristoiu Abstract The response of dorsal root ganglion (DRG) neurons to metabolic inhibition is known to involve calcium-activated K+ channels; in most neuronal types ATP-sensitive K+ channels (KATP) also contribute, but this is not yet established in the DRG. We have investigated the presence of a KATP current using whole-cell recordings from rat DRG neurons, classifying the neurons functionally by their "current signature" (Petruska et al, J Neurophysiol 84: 2365,2379, 2000). We clearly identified a KATP current in only 1 out of 62 neurons, probably a nociceptor. The current was activated by cyanide (2 mM NaCN) and was sensitive to 100 ,M tolbutamide; the relation between reversal potential and external K+ concentration indicated it was a K+ current. In a further two neurons, cyanide activated a K+ current that was only partially blocked by tolbutamide, which may also be an atypical KATP current. We conclude that KATP channels are expressed in normal DRG, but in very few neurons and only in nociceptors. [source] Transient Expression of NMDA Receptor Subunit NR2B in the Developing Rat HeartJOURNAL OF NEUROCHEMISTRY, Issue 6 2000Silke Seeber Abstract: NMDA receptors represent a subtype of the ionotropicglutamate receptor family, comprising three classes of subunits (NR1, NR2A-D,NR3), which exhibit distinct patterns of regional and developmental expressionin the CNS. Recently, some NMDA receptor subunits have also been described inadult extraneuronal tissues and keratinocytes. However, their developmentalexpression patterns are currently unknown. With use of RT-PCR and western blotanalysis, the expression of NMDA receptor subunit NR2B was investigated in thedeveloping rat heart. NR2B mRNA and protein were detected in heart tissue ofrats from embryonic day 14 until postnatal day 21 but disappeared 10 weeksafter birth. In contrast, no NMDA receptor subunit NR1,,-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor subunitGluR2, or anchoring postsynaptic density protein-95 could be detected in ratheart at any developmental stage. Confocal microscopy of cultured cardiacmyocytes (CMs) from neonatal rats revealed distinct NR2B staining mainly ofintracellular structures. However, no functional NMDA receptor could bedetected on CMs by whole-cell recordings. In conclusion, high concentrationsof NR2B protein can be detected in early rat heart development, but itsfunction still remains elusive. [source] Properties of glycine receptors underlying synaptic currents in presynaptic axon terminals of rod bipolar cells in the rat retinaTHE JOURNAL OF PHYSIOLOGY, Issue 15 2009Svein Harald Mørkve The excitability of presynaptic terminals can be controlled by synaptic input that directly targets the terminals. Retinal rod bipolar axon terminals receive presynaptic input from different types of amacrine cells, some of which are glycinergic. Here, we have performed patch-clamp recordings from rod bipolar axon terminals in rat retinal slices. We used whole-cell recordings to study glycinergic inhibitory postsynaptic currents (IPSCs) under conditions of adequate local voltage clamp and outside-out patch recordings to study biophysical and pharmacological properties of the glycine receptors with ultrafast application. Glycinergic IPSCs, recorded in both intact cells and isolated terminals, were strychnine sensitive and displayed fast kinetics with a double-exponential decay. Ultrafast application of brief (,1 ms) pulses of glycine (3 mm) to patches evoked responses with fast, double-exponential deactivation kinetics, no evidence of desensitization in double-pulse experiments, relatively low apparent affinity (EC50,100 ,m), and high maximum open probability (,0.9). Longer pulses evoked slow, double-exponential desensitization and double-pulse experiments indicated slow, double-exponential recovery from desensitization. Non-stationary noise analysis of IPSCs and patch responses yielded single-channel conductances of ,41 pS and ,64 pS, respectively. Directly observed single-channel gating occurred at ,40,50 pS and ,80,90 pS in both types of responses, suggesting a mixture of heteromeric and homomeric receptors. Synaptic release of glycine leads to transient receptor activation, with about eight receptors available to bind transmitter after release of a single vesicle. With a low intracellular chloride concentration, this leads to either hyperpolarizing or shunting inhibition that will counteract passive and regenerative depolarization and depolarization-evoked transmitter release. [source] Switching between transient and sustained signalling at the rod bipolar-AII amacrine cell synapse of the mouse retinaTHE JOURNAL OF PHYSIOLOGY, Issue 11 2009Josefin Snellman At conventional synapses, invasion of an action potential into the presynaptic terminal produces a rapid Ca2+ influx and ultimately the release of synaptic vesicles. However, retinal rod bipolar cells (RBCs) generally do not produce action potentials, and the rate of depolarization of the axon terminal is instead governed by the rate of rise of the light response, which can be quite slow. Using paired whole-cell recordings, we measured the behaviour of the RBC-AII amacrine cell synapse while simulating light-induced depolarizations either by slowly ramping the RBC voltage or by depolarizing the RBC with the mGluR6 receptor antagonist (R,S)-,-cyclopropyl-4-phosphonophenylglycine (CPPG). Both voltage ramps and CPPG evoked slow activation of presynaptic Ca2+ currents and severely attenuated the early, transient component of the AII EPSC compared with voltage steps. We also found that the duration of the transient component was limited in time, and this limitation could not be explained by vesicle depletion, inhibitory feedback, or proton inhibition. Limiting the duration of the fast transient insures the availability of readily releasable vesicles to support a second, sustained component of release. The mGluR6 pathway modulator cGMP sped the rate of RBC depolarization in response to puffs of CPPG and consequently potentiated the transient component of the EPSC at the expense of the sustained component. We conclude that the rod bipolar cell is capable of both transient and sustained signalling, and modulation of the mGluR6 pathway by cGMP allows the RBC to switch between these two time courses of transmitter release. [source] Coincidence detection of convergent perforant path and mossy fibre inputs by CA3 interneuronsTHE JOURNAL OF PHYSIOLOGY, Issue 11 2008Eduardo Calixto We performed whole-cell recordings from CA3 s. radiatum (R) and s. lacunosum-moleculare (L-M) interneurons in hippocampal slices to examine the temporal aspects of summation of converging perforant path (PP) and mossy fibre (MF) inputs. PP EPSPs were evoked from the s. lacunosum-moleculare in area CA1. MF EPSPs were evoked from the medial extent of the suprapyramidal blade of the dentate gyrus. Summation was strongly supralinear when examining PP EPSP with MF EPSP in a heterosynaptic pair at the 10 ms ISI, and linear to sublinear at longer ISIs. This pattern of nonlinearities suggests that R and L-M interneurons act as coincidence detectors for input from PP and MF. Summation at all ISIs was linear in voltage clamp mode demonstrating that nonlinearities were generated by postsynaptic voltage-dependent conductances. Supralinearity was not detected when the first EPSP in the pair was replaced by a simulated EPSP injected into the soma, suggesting that the conductances underlying the EPSP boosting were located in distal dendrites. Supralinearity was selectively eliminated with either Ni2+ (30 ,m), mibefradil (10 ,m) or nimodipine (15 ,m), but was unaffected by QX-314. This pharmacological profile indicates that supralinearity is due to recruitment of dendritic T-type Ca2+channels by the first subthreshold EPSP in the pair. Results with the hyperpolarization-activated (Ih) channel blocker ZD 7288 (50 ,m) revealed that Ih restricted the time course of supralinearity for coincidently summed EPSPs, and promoted linear to sublinear summation for asynchronous EPSPs. We conclude that coincidence detection results from the counterbalanced activation of T-type Ca2+ channels and inactivation of Ih. [source] Action potential initiation and propagation in hippocampal mossy fibre axonsTHE JOURNAL OF PHYSIOLOGY, Issue 7 2008Christoph Schmidt-Hieber Dentate gyrus granule cells transmit action potentials (APs) along their unmyelinated mossy fibre axons to the CA3 region. Although the initiation and propagation of APs are fundamental steps during neural computation, little is known about the site of AP initiation and the speed of propagation in mossy fibre axons. To address these questions, we performed simultaneous somatic and axonal whole-cell recordings from granule cells in acute hippocampal slices of adult mice at ,23°C. Injection of short current pulses or synaptic stimulation evoked axonal and somatic APs with similar amplitudes. By contrast, the time course was significantly different, as axonal APs had a higher maximal rate of rise (464 ± 30 V s,1 in the axon versus 297 ± 12 V s,1 in the soma, mean ±s.e.m.). Furthermore, analysis of latencies between the axonal and somatic signals showed that APs were initiated in the proximal axon at ,20,30 ,m distance from the soma, and propagated orthodromically with a velocity of 0.24 m s,1. Qualitatively similar results were obtained at a recording temperature of ,34°C. Modelling of AP propagation in detailed cable models of granule cells suggested that a ,4 times higher Na+ channel density (,1000 pS ,m,2) in the axon might account for both the higher rate of rise of axonal APs and the robust AP initiation in the proximal mossy fibre axon. This may be of critical importance to separate dendritic integration of thousands of synaptic inputs from the generation and transmission of a common AP output. [source] Intrinsic properties and mechanisms of spontaneous firing in mouse cerebellar unipolar brush cellsTHE JOURNAL OF PHYSIOLOGY, Issue 2 2007Marco J. Russo Neuronal firing patterns are determined by the cell's intrinsic electrical and morphological properties and are regulated by synaptic interactions. While the properties of cerebellar neurons have generally been studied in much detail, little is known about the unipolar brush cells (UBCs), a type of glutamatergic interneuron that is enriched in the granular layer of the mammalian vestibulocerebellum and participates in the representation of head orientation in space. Here we show that UBCs can be distinguished from adjacent granule cells on the basis of differences in membrane capacitance, input resistance and response to hyperpolarizing current injection. We also show that UBCs are intrinsically firing neurons. Using action potential clamp experiments and whole-cell recordings we demonstrate that two currents contribute to this property: a persistent TTX-sensitive sodium current and a ruthenium red-sensitive, TRP-like cationic current, both of which are active during interspike intervals and have reversal potentials positive to threshold. Interestingly, although UBCs are also endowed with a large Ih current, this current is not involved in their intrinsic firing, perhaps because it activates at voltages that are more hyperpolarized than those associated with autonomous activity. [source] Spontaneous IPSCs and glycine receptors with slow kinetics in wide-field amacrine cells in the mature rat retinaTHE JOURNAL OF PHYSIOLOGY, Issue 1 2007Margaret Lin Veruki The functional properties of glycine receptors were analysed in different types of wide-field amacrine cells, narrowly stratifying cells considered to play a role in larger-scale integration across the retina. The patch-clamp technique was used to record spontaneous IPSCs (spIPSCs) and glycine-evoked patch responses from mature rat retinal slices (4,7 weeks postnatal). Glycinergic spIPSCs were blocked reversibly by strychnine (300 nm). Compared to previously described spIPSCs in AII amacrine cells, the spIPSCs in wide-field amacrine cells displayed a very slow decay time course (,fast, 15 ms; ,slow, 57 ms). The kinetic properties of spIPSCs in whole-cell recordings were paralleled by even slower deactivation kinetics of responses evoked by brief pulses of glycine (3 mm) to outside-out patches from wide-field amacrine cells (,fast, 45 ms; ,slow, 350 ms). Non-stationary noise analysis of patch responses and spIPSCs yielded similar average single-channel conductances (,31 and ,34 pS, respectively). Similar, as well as both lower- and higher-conductance levels could be identified from directly observed single-channel gating during the decay phase of spIPSCs and patch responses. These results suggest that the slow glycinergic spIPSCs in wide-field amacrine cells involve ,2, heteromeric receptors. Taken together with previous work, the kinetic properties of glycine receptors in different types of amacrine cells display a considerable range that is probably a direct consequence of differential expression of receptor subunits. Unique kinetic properties are likely to differentially shape the glycinergic input to different types of amacrine cells and thereby contribute to distinct integrative properties among these cells. [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] | |||||||||||||