Rapid and MR-Independent IK1 activation by aldosterone during ischemia-reperfusion

In ST elevation myocardial infarction (STEMI) context, clinical studies have shown the deleterious effect of high aldosterone levels on ventricular arrhythmia occurrence and cardiac mortality. Previous in vitro reports showed that during ischemia-reperfusion, aldosterone modulates K+ currents involved in the holding of the resting membrane potential (RMP). The aim of this study was to assess the electrophysiological impact of aldosterone on IK1 current during myocardial ischemia-reperfusion. We used an in vitro model of “border zone” using right rabbit ventricle and standard microelectrode technique followed by cell-attached recordings from freshly isolated rabbit ventricular cardiomyocytes. In microelectrode experiments, aldosterone (10 and 100 nmol/L, n=7 respectively) increased the action potential duration (APD) dispersion at 90% between ischemic and normoxic zones (from 95±4ms to 116±6 ms and 127±5 ms respectively, P<0.05) and reperfusion-induced sustained premature ventricular contractions occurrence (from 2/12 to 5/7 preparations, P<0.05). Conversely, potassium canrenoate 100 nmol/L and RU 28318 1 μmol/l alone did not affect AP parameters and premature ventricular contractions occurrence (except Vmax which was decreased by potassium canrenoate during simulated-ischemia). Furthermore, aldosterone induced a RMP hyperpolarization, evoking an implication of a K+ current involved in the holding of the RMP. Cell-attached recordings showed that aldosterone 10 nmol/L quickly activated (within 6.2±0.4 min) a 30 pS K+-selective current, inward rectifier, with pharmacological and biophysical properties consistent with the IK1 current (NPo =1.9±0.4 in control vs NPo=3.0±0.4, n=10, P<0.05). These deleterious effects persisted in presence of RU 28318, a specific MR antagonist, and were successfully prevented by potassium canrenoate, a non specific MR antagonist, in both microelectrode and patch-clamp recordings, thus indicating a MR-independent IK1 activation. In this ischemia-reperfusion context, aldosterone induced rapid and MR-independent deleterious effects including an arrhythmia substrate (increased APD90 dispersion) and triggered activities (increased premature ventricular contractions occurrence on reperfusion) possibly related to direct IK1 activation

The Generation of Direction Selectivity in the Auditory System

Both human speech and animal vocal signals contain frequency-modulated (FM) sounds. Although central auditory neurons that selectively respond to the direction of frequency modulation are known, the synaptic mechanisms underlying the generation of direction selectivity (DS) remain elusive. Here we show the emergence of DS neurons in the inferior colliculus by mapping the three major subcortical auditory nuclei. Cell-attached recordings reveal a highly reliable and precise firing of DS neurons to FM sweeps in a preferred direction. By using in vivo whole-cell current-clamp and voltage-clamp recordings, we found that the synaptic inputs to DS neurons are not direction selective, but temporally reversed excitatory and inhibitory synaptic inputs are evoked in response to opposing directions of FM sweeps. The construction of such temporal asymmetry, resulting DS, and its topography can be attributed to the spectral disparity of the excitatory and the inhibitory synaptic tonal receptive fields

NMDA receptors as voltage sensors

© Springer Science+Business Media LLC 2017. The membrane potential is an essential parameter of a living cell. However, measurements of the membrane potential using conventional techniques are associated with a number of artifacts. Cell-attached recordings of the currents through NMDA receptor channels enable noninvasive measurements of the neuronal membrane potential. This approach overcomes the problem of a leak conductance introduced during intracellular sharp electrode recordings and whole-cell patch-clamp recordings. Here, we describe the procedures of using cell-attached recordings of NMDA receptor channels to measure the true membrane potential

Nanoscale-targeted patch-clamp recordings of functional presynaptic ion channels

Important modulatory roles have been attributed to presynaptic NMDA receptors (NMDARs) located on cerebellar interneuron terminals. Evidence supporting a presynaptic location includes an increase in the frequency of mini events following the application of NMDA and gold particle-labelled NMDA receptor antibody localisation. However, more recent work, using calcium indicators, casts doubt on the idea of presynaptic NMDARs because basket cell varicosities did not show the expected calcium rise following either the local iontophoresis of L-aspartate or the two-photon uncaging of glutamate. (In theory such calcium imaging is sensitive enough to detect the calcium rise from even a single activated receptor.) It has therefore been suggested that the effects of NMDA are mediated via the activation of somatodendritic channels, which subsequently cause a subthreshold depolarization of the axon. Here we report results from a vibrodissociated preparation of cerebellar Purkinje cells, in which the interneuron cell bodies are no longer connected but many of their terminal varicosities remain attached and functional. This preparation can retain both inhibitory and excitatory inputs. We find that the application of NMDA increases the frequency of both types of synaptic event. The characteristics of these events suggest they can originate from interneuron, parallel fiber and even climbing fiber terminals. Interestingly, retrograde signalling seems to activate only the inhibitory terminals. Finally, antibody staining of these cells shows NMDAR-like immunoreactivity co-localised with synaptic markers. Since the Purkinje cells show no evidence of postsynaptic NMDAR-mediated currents, we conclude that functional NMDA receptors are located on presynaptic terminals

Serotonin drives a novel GABAergic synaptic current recorded in rat cerebellar purkinje cells: a lugaro cell to Purkinje cell synapse

We recorded a novel fast GABAergic synaptic current in cerebellar Purkinje cells in rat brain slices using patch-clamp techniques. Because of a relatively low sensitivity to bicuculline, these currents can be recorded under conditions in which basket and stellate cell inputs are blocked. The observations that the novel synaptic currents occur spontaneously only in the presence of serotonin, and the specific limited positions in the slice from which they can be electrically evoked, suggest that the presynaptic cell is the Lugaro cell. Cell-attached recordings confirm that the Lugaro cell is the only interneuron in the cerebellar cortex with firing behavior consistent with the spontaneous activity recorded in Purkinje cells. The input shows a strong presynaptic modulation mediated by GABAA receptors, resulting in a dynamic range from almost 0 to >90% release probability. Modeling GABAA receptor responses to different GABA transients suggests that the relatively low sensitivity of the synaptic currents to bicuculline, compared with the higher affinity GABAA receptor antagonist SR-95531 (2-(3-carboxypropyl)-3-amino-6-(4-methoxyphenyl) pyridazinium), is attributable to an unusually long GABA dwell time and/or high GABA concentration in the synaptic cleft. The significance of this novel input is discussed in relation to other GABAergic synapses impinging on Purkinje cells. We suggest that the release of GABA onto Purkinje cells from Lugaro cells would primarily occur during motor activity under conditions in which the activity of basket and stellate cells might be inhibited

Serotonin drives a novel GABAergic synaptic current recorded in rat cerebellar purkinje cells: a lugaro cell to purkinje cell synapse

We recorded a novel fast GABAergic synaptic current in cerebellar Purkinje cells in rat brain slices using patch-clamp techniques. Because of a relatively low sensitivity to bicuculline, these currents can be recorded under conditions in which basket and stellate cell inputs are blocked. The observations that the novel synaptic currents occur spontaneously only in the presence of serotonin, and the specific limited positions in the slice from which they can be electrically evoked, suggest that the presynaptic cell is the Lugaro cell. Cell-attached recordings confirm that the Lugaro cell is the only interneuron in the cerebellar cortex with firing behavior consistent with the spontaneous activity recorded in Purkinje cells. The input shows a strong presynaptic modulation mediated by GABAA receptors, resulting in a dynamic range from almost 0 to >90% release probability. Modeling GABAA receptor responses to different GABA transients suggests that the relatively low sensitivity of the synaptic currents to bicuculline, compared with the higher affinity GABAA receptor antagonist SR-95531 (2-(3-carboxypropyl)-3-amino-6-(4-methoxyphenyl) pyridazinium), is attributable to an unusually long GABA dwell time and/or high GABA concentration in the synaptic cleft. The significance of this novel input is discussed in relation to other GABAergic synapses impinging on Purkinje cells. We suggest that the release of GABA onto Purkinje cells from Lugaro cells would primarily occur during motor activity under conditions in which the activity of basket and stellate cells might be inhibited

A reinforcing circuit action of extrasynaptic GABAA receptor modulators on cerebellar granule cell inhibition.

GABAA receptors (GABARs) are the targets of a wide variety of modulatory drugs which enhance chloride flux through GABAR ion channels. Certain GABAR modulators appear to acutely enhance the function of δ subunit-containing GABAR subtypes responsible for tonic forms of inhibition. Here we identify a reinforcing circuit mechanism by which these drugs, in addition to directly enhancing GABAR function, also increase GABA release. Electrophysiological recordings in cerebellar slices from rats homozygous for the ethanol-hypersensitive (α6100Q) allele show that modulators and agonists selective for δ-containing GABARs such as THDOC, ethanol and THIP (gaboxadol) increased the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) in granule cells. Ethanol fails to augment granule cell sIPSC frequency in the presence of glutamate receptor antagonists, indicating that circuit mechanisms involving granule cell output contribute to ethanol-enhancement of synaptic inhibition. Additionally, GABAR antagonists decrease ethanol-induced enhancement of Golgi cell firing. Consistent with a role for glutamatergic inputs, THIP-induced increases in Golgi cell firing are abolished by glutamate receptor antagonists. Moreover, THIP enhances the frequency of spontaneous excitatory postsynaptic currents in Golgi cells. Analyses of knockout mice indicate that δ subunit-containing GABARs are required for enhancing GABA release in the presence of ethanol and THIP. The limited expression of the GABAR δ subunit protein within the cerebellar cortex suggests that an indirect, circuit mechanism is responsible for stimulating Golgi cell GABA release by drugs selective for extrasynaptic isoforms of GABARs. Such circuit effects reinforce direct actions of these positive modulators on tonic GABAergic inhibition and are likely to contribute to the potent effect of these compounds as nervous system depressants

Depolarizing Effect of Neocortical Chandelier Neurons

Chandelier (or axo-axonic) cells are one of the most distinctive types of GABAergic interneurons in the cortex. Although they have traditionally been considered inhibitory neurons, data from rat and human neocortical preparations suggest that chandelier cells have a depolarizing effect on pyramidal neurons at resting membrane potential, and could even activate synaptic chains of neurons. At the same time, recent results from rat hippocampal chandeliers indicate a predominantly inhibitory effect on their postsynaptic targets. To better understand the function of chandelier neurons, we generated Nkx2.1Cre MADM mice, a strain of genetically engineered animals that, by expressing GFP in a subset of neocortical interneurons, enable the identification and targeting of chandelier cells in living brain slices. Using these mice, we characterized the basic electrophysiological properties of a homogeneous population of chandelier neurons from upper layers of somatosensory cortical slices. These chandelier cells have characteristic axon cartridges and stereotypical electrophysiological features, distinguishable from basket cells. To investigate the effect of chandelier cells on target neurons, we performed paired recordings from chandeliers and postsynaptic pyramidal cells. In both perforated patch and cell-attached configurations, chandelier PSPs have in every case a reversal potential that is depolarized from rest. Our results support the idea that chandelier cells depolarize pyramidal neurons and could potentially have an excitatory effect on the network at rest