Alon Korngreen

Voltage-gated K+ channels in layer 5 neocortical pyramidal neurones from young rats: subtypes and gradients

1 We investigated the types and distribution of voltage‐gated K+ channels in the soma and apical dendrite of layer 5 (L5) neocortical pyramidal neurones, of young rats (postnatal days 13–15), in acute brain slices. 2 A slow inactivating outward K+ current and a fast inactivating outward K+ current were detected in nucleated patches. The slow K+ current was completely blocked by tetraethylammonium (TEA) with an IC50 of 5 ± 1 mm (mean ±s.e.m.) and was partially blocked by 4‐aminopyridine (4‐AP). The fast K+ current was blocked by 4‐AP with an IC50 of 4.2 ± 0.5 mM, but was not blocked by TEA. 3 The activation kinetics of the slow K+ current were described by a second order Hodgkin‐Huxley model. The slow K+ current displayed bi‐exponential inactivation. A fourth order Hodgkin‐Huxley model for activation and first order for inactivation described the kinetics of the fast K+ current. 4 In somatic cell‐attached recordings, three classes of single K+ channels could be differentiated based on their unitary conductance and inactivation kinetics, a fast inactivating channel having a conductance of 13 ± 1 pS, a slow inactivating channel having a conductance of 9.5 ± 0.5 pS, and a very slowly inactivating channel having a conductance of 16 ± 1 pS. 5 The inactivation time constants of the slow and of the very slow K+ channel corresponded to the two inactivation time constants of the slow K+ current observed in nucleated patches. This suggested that two distinct K+ channels mediated the slow K+ current in nucleated patches. 6 The three subtypes of K+ channels that were observed in somatic recordings were present along the apical dendrite. The amplitude of ensemble K+ currents in cell‐attached patches decreased along the apical dendrite as the distance from the soma increased, with a slope of −0.9 ± 0.3 pA per 100 μm. 7 The results suggest that the decrease of the voltage‐gated K+ channel density from the soma along the apical dendrite of L5 pyramidal neurones helps to define a distal, low threshold region for the initiation of dendritic regenerative potentials.

PA6-induced human embryonic stem cell-derived neurospheres: a new source of human peripheral sensory neurons and neural crest cells

Human embryonic stem cells (hESC) have been directed to differentiate into CNS cells with clinical importance. However, for study of development and regeneration of the human PNS, and peripheral neuropathies, it would be useful to have a source of human PNS derivatives. We have demonstrated that peripheral sensory neuron-like cells (PSN) can also be derived from hESC via neural crest-like (NC) intermediates, and from neural progenitors induced from hESC using noggin. Here we report the generation of higher purity PSN from passagable neurospheres (NSP) induced by murine PA6 stromal cells. hESC were cultured with PA6, and colonies that developed a specific morphology were cut from the plates. Culture of these colonies under non-adhesive conditions yielded NSPs. Several NC marker genes were expressed in the NSP, and these were also detected in 3-5week gestation human embryos containing migrating NC. These NSPs passaged for 2-8weeks and re-plated on PA6 gave rise to many Brn3a+/peripherin+ cells, characteristic of early sensory-like neurons. Re-culturing PA6-induced NSP cells with PA6 resulted in about 25% of the human cells in the co-cultures differentiating to PSN after 1week, compared to only about 10% PSN obtained after 3 weeks when noggin-induced NSP were used. Two month adherent cultures of PA6-induced NSP cells contained neurons expressing several PSN neuropeptides, and voltage-dependent currents and action potentials were obtained from a molecularly identified PSN. hESC-derived PA6-induced NSP cells are therefore an excellent potential source of human PSN for study of differentiation and modeling of PNS disease.

Pore properties and pharmacological features of the P2X receptor channel in airway ciliated cells

Airway ciliated cells express an ATP‐gated P2X receptor channel of unknown subunit composition (P2Xcilia) which is modulated by Na+ and by long exposures to ATP. P2Xcilia was investigated by recording currents from freshly dissociated rabbit airway ciliated cells with the patch‐clamp technique in the whole‐cell configuration. During the initial continuous exposure to extracellular ATP, P2Xcilia currents gradually increase in magnitude (priming), yet the permeability to N‐methyl‐d‐glucamine (NMDG) does not change, indicating that priming does not arise from a progressive change in pore diameter. Na+, which readily permeates P2Xcilia receptor channels, was found to inhibit the channel extracellular to the electric field. The rank order of permeability to various monovalent cations is: Li+, Na+, K+, Rb+, Cs+, NMDG+ and TEA+, with a relative permeability of 1.35, 1.0, 0.99, 0.91, 0.79, 0.19 and 0.10, respectively. The rank order for the alkali cations follows an Eisenman series XI for a high‐strength field site. Ca2+ has been estimated to be 7‐fold more permeant than Na+. The rise in [Ca2+]i in ciliated cells, induced by the activation of P2Xcilia, is largely inhibited by either Brilliant Blue G or KN‐62, indicating that P2X7 may be a part of P2Xcilia. P2Xcilia is augmented by Zn2+ and by ivermectin, and P2X4 receptor protein is detected by immunolabelling at the basal half of the cilia, strongly suggesting that P2X4 is a component of P2Xcilia receptor channels. Taken together, these results suggest that P2Xcilia is either assembled from P2X4 and P2X7 subunits, or formed from modified P2X4 subunits.

Extracellular sodium regulates airway ciliary motility by inhibiting a P2X receptor

The mucociliary system is responsible for clearing inhaled particles and pathogens from the airways. This important task is performed by the beating of cilia and the consequent movement of mucus from the lungs to the upper airways,. Because ciliary motility is enhanced by elevated intracellular calcium concentrations, inhibition of calcium influx could lead to disease by jeopardizing mucociliary clearance. Several hormones and neurotransmitters stimulate ciliary motility, one of the most potent of which is extracellular ATP (ATPo), which acts by releasing calcium ions from internal stores and by activating calcium influx. Here we show that, in airway ciliated cells, extracellular sodium ions (Nao+) specifically and competitively inhibit an ATPo-gated channel that is permeable to calcium ions, and thereby attenuate ATPo-induced ciliary motility. Our finding points to a physiological role for Nao+ in ciliary function, and indicates that mucociliary clearance might be improved in respiratory disorders such as chronic bronchitis and cystic fibrosis by decreasing the sodium concentration of the airway surface fluid in which the cilia are bathed.

Space-Clamp Problems When Voltage Clamping Neurons Expressing Voltage-Gated Conductances

The voltage-clamp technique is applicable only to spherical cells. In nonspherical cells, such as neurons, the membrane potential is not clamped distal to the voltage-clamp electrode. This means that the current recorded by the voltage-clamp electrode is the sum of the local current and of axial currents from locations experiencing different membrane potentials. Furthermore, voltage-gated currents recorded from a nonspherical cell are, by definition, severely distorted due to the lack of space clamp. Justifications for voltage clamping in nonspherical cells are, first, that the lack of space clamp is not severe in neurons with short dendrites. Second, passive cable theory may be invoked to justify application of voltage clamp to branching neurons, suggesting that the potential decay is sufficiently shallow to allow spatial clamping of the neuron. Here, using numerical simulations, we show that the distortions of voltage-gated K(+) and Ca(2+) currents are substantial even in neurons with short dendrites. The simulations also demonstrate that passive cable theory cannot be used to justify voltage clamping of neurons due to significant shunting to the reversal potential of the voltage-gated conductance during channel activation. Some of the predictions made by the simulations were verified using somatic and dendritic voltage-clamp experiments in rat somatosensory cortex. Our results demonstrate that voltage-gated K(+) and Ca(2+) currents recorded from branching neurons are almost always severely distorted.

Mechanisms of Magnetic Stimulation of Central Nervous System Neurons

Transcranial magnetic stimulation (TMS) is a stimulation method in which a magnetic coil generates a magnetic field in an area of interest in the brain. This magnetic field induces an electric field that modulates neuronal activity. The spatial distribution of the induced electric field is determined by the geometry and location of the coil relative to the brain. Although TMS has been used for several decades, the biophysical basis underlying the stimulation of neurons in the central nervous system (CNS) is still unknown. To address this problem we developed a numerical scheme enabling us to combine realistic magnetic stimulation (MS) with compartmental modeling of neurons with arbitrary morphology. The induced electric field for each location in space was combined with standard compartmental modeling software to calculate the membrane current generated by the electromagnetic field for each segment of the neuron. In agreement with previous studies, the simulations suggested that peripheral axons were excited by the spatial gradients of the induced electric field. In both peripheral and central neurons, MS amplitude required for action potential generation was inversely proportional to the square of the diameter of the stimulated compartment. Due to the importance of the fibers diameter, magnetic stimulation of CNS neurons depolarized the soma followed by initiation of an action potential in the initial segment of the axon. Passive dendrites affect this process primarily as current sinks, not sources. The simulations predict that neurons with low current threshold are more susceptible to magnetic stimulation. Moreover, they suggest that MS does not directly trigger dendritic regenerative mechanisms. These insights into the mechanism of MS may be relevant for the design of multi-intensity TMS protocols, may facilitate the construction of magnetic stimulators, and may aid the interpretation of results of TMS of the CNS.

A Numerical Approach to Ion Channel Modelling Using Whole-Cell Voltage-Clamp Recordings and a Genetic Algorithm

The activity of trans-membrane proteins such as ion channels is the essence of neuronal transmission. The currently most accurate method for determining ion channel kinetic mechanisms is single-channel recording and analysis. Yet, the limitations and complexities in interpreting single-channel recordings discourage many physiologists from using them. Here we show that a genetic search algorithm in combination with a gradient descent algorithm can be used to fit whole-cell voltage-clamp data to kinetic models with a high degree of accuracy. Previously, ion channel stimulation traces were analyzed one at a time, the results of these analyses being combined to produce a picture of channel kinetics. Here the entire set of traces from all stimulation protocols are analysed simultaneously. The algorithm was initially tested on simulated current traces produced by several Hodgkin-Huxley–like and Markov chain models of voltage-gated potassium and sodium channels. Currents were also produced by simulating levels of noise expected from actual patch recordings. Finally, the algorithm was used for finding the kinetic parameters of several voltage-gated sodium and potassium channels models by matching its results to data recorded from layer 5 pyramidal neurons of the rat cortex in the nucleated outside-out patch configuration. The minimization scheme gives electrophysiologists a tool for reproducing and simulating voltage-gated ion channel kinetics at the cellular level.

Electrophysiological characteristics of globus pallidus neurons.

Extracellular recordings in primates have identified two types of neurons in the external segment of the globus pallidus (GPe): high frequency pausers (HFP) and low frequency bursters (LFB). The aim of the current study was to test whether the properties of HFP and LFB neurons recorded extracellularly in the primate GPe are linked to cellular mechanisms underlying the generation of action potential (AP) firing. Thus, we recorded from primate and rat globus pallidus neurons. Extracellular recordings in primates revealed that in addition to differences in firing patterns the APs of neurons in these two groups have different widths (APex). To quantitatively investigate this difference and to explore the heterogeneity of pallidal neurons we carried out cell-attached and whole-cell recordings from acute slices of the rat globus pallidus (GP, the rodent homolog of the primate GPe), examining both spontaneous and evoked activity. Several parameters related to the extracellular activity were extracted in order to subdivide the population of recorded GP neurons into groups. Statistical analysis showed that the GP neurons in the rodents may be differentiated along six cellular parameters into three subgroups. Combining two of these groups allowed a better separation of the population along nine parameters. Four of these parameters (Fmax, APamp, APhw, and AHPs amplitude) form a subset, suggesting that one group of neurons may generate APs at significantly higher frequencies than the other group. This may suggest that the differences between the HFP and LFB neurons in the primate are related to fundamental underlying differences in their cellular properties.

Correction of conductance measurements in non−space−clamped structures: 1. Voltage−gated K+ channels

To understand functions of a single neuron, such as propagation and generation of synaptic or action potentials, a detailed description of the kinetics and distribution of the underlying ionic conductances is essential. In voltage-clamp experiments, incomplete space clamp distorts the recorded currents, rendering accurate analysis impossible. Here, we present a simple numerical algorithm that corrects such distortions. The method performs a stepwise approximation of the conductance density at the site of a local voltage clamp. This is achieved by estimating membrane conductances in a simulation that yields simulated clamp currents, which are then fitted to the distorted recordings from the non-space-clamped structure, relying on accurately reconstructed cell morphology and experimentally determined passive properties. The method enabled accurate retrieval of the local densities, kinetics, and density gradients of somatic and dendritic channels. Neither the addition of noise nor variation of passive parameters significantly reduced the performance of the correction algorithm. The correction method was applied to two-electrode voltage-clamp recordings of K(+) currents from the apical dendrite of layer 5 neocortical pyramidal neurons. The generality and robustness of the algorithm make it a useful tool for voltage-clamp analysis of voltage-gated currents in structures of any morphology that is amenable to the voltage-clamp technique.

Dendritic voltage-gated K + conductance gradient in pyramidal neurones of neocortical layer 5B from rats

Voltage‐gated potassium channels effectively regulate dendritic excitability in neurones. It has been suggested that in the distal apical dendrite of layer 5B (L5B) neocortical pyramidal neurones, K+ conductances participate in active dendritic synaptic integration and control regenerative dendritic potentials. The ionic mechanism for triggering these regenerative potentials has yet to be elucidated. Here we used two‐electrode voltage clamp (TEVC) to quantitatively record K+ conductance densities of a sustained K+ conductance in the soma and apical dendrite of L5B neurones of adult rats. We report that the somatic and proximal dendritic sustained voltage‐gated K+ conductance density is more than 10‐fold larger than previous estimates. The results obtained using TEVC were corroborated using current‐clamp experiments in combination with compartmental modelling. Possible error sources, including inaccurate measurement of the passive membrane parameters and unknown axonal and basal dendritic conductance distributions, were shown not to distort the density estimation considerably. The sustained voltage‐gated K+ conductance density was found to decrease steeply along the apical dendrite. The steep negative K+ conductance density gradient along the apical dendrite may help to define a distal, low‐threshold region for amplification of distal synaptic input in L5B pyramidal neurones.