A Time Course Analysis of the Electrophysiological Properties of Neurons Differentiated from Human Induced Pluripotent Stem Cells (iPSCs)

Many protocols have been designed to differentiate human embryonic stem cells (ESCs) and human induced pluripotent stem cells (iPSCs) into neurons. Despite the relevance of electrophysiological properties for proper neuronal function, little is known about the evolution over time of important neuronal electrophysiological parameters in iPSC-derived neurons. Yet, understanding the development of basic electrophysiological characteristics of iPSC-derived neurons is critical for evaluating their usefulness in basic and translational research. Therefore, we analyzed the basic electrophysiological parameters of forebrain neurons differentiated from human iPSCs, from day 31 to day 55 after the initiation of neuronal differentiation. We assayed the developmental progression of various properties, including resting membrane potential, action potential, sodium and potassium channel currents, somatic calcium transients and synaptic activity. During the maturation of iPSC-derived neurons, the resting membrane potential became more negative, the expression of voltage-gated sodium channels increased, the membrane became capable of generating action potentials following adequate depolarization and, at day 48–55, 50% of the cells were capable of firing action potentials in response to a prolonged depolarizing current step, of which 30% produced multiple action potentials. The percentage of cells exhibiting miniature excitatory post-synaptic currents increased over time with a significant increase in their frequency and amplitude. These changes were associated with an increase of Ca²⁺ transient frequency. Co-culturing iPSC-derived neurons with mouse glial cells enhanced the development of electrophysiological parameters as compared to pure iPSC-derived neuronal cultures. This study demonstrates the importance of properly evaluating the electrophysiological status of the newly generated neurons when using stem cell technology, as electrophysiological properties of iPSC-derived neurons mature over time.     

Tonic and phasic receptor neurons in the vertebrate olfactory epithelium

Olfactory receptor neurons (ORNs) respond to odorants with characteristic patterns of action potentials that are relevant for odor coding. Prolonged odorant exposures revealed three populations of dissociated toad ORNs, which were mimicked by depolarizing currents: tonic (TN, displaying sustained firing, 49% of 102 cells), phasic (PN, exhibiting brief action potential trains, 36%) and intermediate neurons (IN, generating trains longer than PN, 15%). We studied the biophysical properties underlying the differences between TNs and PNs, the most extreme cases among ORNs. TNs and PNs possessed similar membrane capacitances (approximately 4 pF), but they differed in resting potential (-82 versus -64 mV), input resistance (4.2 versus 2.9 G(Omega)) and unspecific current, I(u) (TNs: 0 < I(u) 1 pA/pF). Firing behavior did not correlate with differences in voltage-gated conductances. We developed a mathematical model that accurately simulates tonic and phasic patterns. Whole cell recordings from rat ORNs in fragments (approximately 4 mm(2)) of olfactory epithelium showed that such a tissue normally contains tonic and phasic receptor neurons, suggesting that this feature is common across a wide range of vertebrates. Our findings show that the individual passive electrical properties can govern the firing patterns of ORNs.     

The action potential in mammalian central neurons

The action potential of the squid giant axon is formed by just two voltage-dependent conductances in the cell membrane, yet mammalian central neurons typically express more than a dozen different types of voltage-dependent ion channels. This rich repertoire of channels allows neurons to encode information by generating action potentials with a wide range of shapes, frequencies and patterns. Recent work offers an increasingly detailed understanding of how the expression of particular channel types underlies the remarkably diverse firing behaviour of various types of neurons.     

Ion conductances related to development of repetitive firing in mouse retinal ganglion neurons in situ

ABSTRACT: In the retina, the ability to encode graded depolarizations into spike trains of variable frequency appears to be a specific property of retinal ganglion neurons (RGNs). To deduce the developmental changes in ion conductances underlying the transition from single to repetitive firing, patch-clamp recordings were performed in the isolated mouse retina between embryonic day 15 (E15) and postnatal day 5 (P5). Immature neurons of the E15 retina were selected according to their capacity to generate voltage-activated Na+ currents (I(Na)(v)). Identification of P5 RGNs was based on retrograde labeling, visualization of the axon, or the amplitude of I(Na)(v). At E15, half of the cells were excitable but none of them generated more than one spike. At P5, all cells were excitable and a majority discharged in tonic fashion. Ion conductances subserving maintenance of repetitive discharge were identified at P5 by exposure to low extracellular Ca2+, Cd2+, and charybdotoxin, all of which suppressed repetitive discharge. omega-Conotoxin GVIA and nifedipine had no effect. We compared passive membrane properties and a variety of voltage-activated ion channels at E15 and P5. It was found that the density of high voltage-activated (HVA) Ca2+ currents increased in parallel with the development of repetitive firing, while the density of Ni2+-sensitive low voltage-activated (LVA) Ca2+ currents decreased. Changes in density and activation kinetics of tetrodotoxin-sensitive Na+ currents paralleled changes in firing thresholds and size of action potentials, but seemed to be unrelated to maintenance of repetitive firing. Densities of A-type K+ currents and delayed rectifier currents did not change. The results suggest that HVA Ca2+ channels, and among them a toxin-resistant subtype, are specifically engaged in activation of Ca2+-sensitive K+ conductance and thereby account for frequency coding in postnatal RGNs.     

Electrical Properties of the Pacemaker Neurons in the Heart Ganglion of a Stomatopod, Squilla oratoria

In the Squilla heart ganglion, the pacemaker is located in the rostral group of cells. After spontaneous firing ceased, the electrophysiological properties of these cells were examined with intracellular electrodes. Cells respond to electrical stimuli with all-or-none action potentials. Direct stimulation by strong currents decreases the size of action potentials. Comparison with action potentials caused by axonal stimulation and analysis of time relations indicate that with stronger currents the soma membrane is directly stimulated whereas with weaker currents the impulse first arises in the axon and then invades the soma. Spikes evoked in a neuron spread into all other neurons. Adjacent cells are interconnected by electrotonic connections. Histologically axons are tied with the side-junction. B spikes of adjacent cells are blocked simultaneously by hyperpolarization or by repetitive stimulation. Experiments show that under such circumstances the B spike is not directly elicited from the A spike but is evoked by invasion of an impulse or electrotonic potential from adjacent cells. On rostral stimulation a small prepotential precedes the main spike. It is interpreted as an action potential from dendrites.     

Membrane and firing properties of avian medial vestibular nucleus neuronsin vitro

The intrinsic membrane and firing properties of medial vestibular nucleus (MVN) neurons were investigated in slices of the chick brainstem using intracellular recording and current injection. Avian MVN neurons fired spontaneous action potentials with very regular interspike intervals. The rapid repolarization of all action potentials was followed by an after-hyperpolarization. Intracellular injection of steps of hyperpolarizing current revealed both an inward rectification of the membrane potential during the step and a rebound depolarization following the offset of the step. In some neurons, the rebound depolarization resulted in bursts of action potentials. Steps of depolarizing current applied to spontaneously active neurons evoked increases in firing rate that were higher at the onset of the step than during the steady-state response. The relationship between current and firing rate was linear. The membrane and firing properties of avian MVN neurons were distributed continuously across the population of recorded neurons. These properties appear identical to those of rodent MVN neurons, suggesting that the composition and distribution of ion channels in the MVN neuronal membrane has been highly conserved across vertebrate species.Abbreviations MVN medial vestibular nucleus - VOR vestibulo-ocular reflex - AHP after-hyperpolarization     

The inhibitory effects of dantrolene on action potential-induced calcium transients in cultured rat dorsal root ganglion neurons

We investigated the actions of dantrolene Ca(2+)-induced on Ca(2+)-release (CICR) evoked by action potentials in cultured rat sensory neurons. The effect of dantrolene on action potential after-depolarization and voltage-activated calcium currents was studied in cultured neonatal rat dorsal root ganglion cells (DRG) using the whole-cell patch-clamp technique. Depolarizing current injection evoked action potentials and depolarizing after-potentials, which are activated as a result of CICR following a single action potential in some cells. The type of after-potentials was determined by inducing action potentials from the resting membrane potential. Extracellular application of dantrolene (10 microM) abolished after-depolarizations without affecting action potential properties. Furthermore, dantrolene significantly reduced repetitive action potentials after depolarizing current injection into these neurons, but had no significant effect on the steady-state current voltage relationship of calcium currents in these neurons. We conclude that dantrolene inhibits the induction of action potential after depolarizations by inhibiting CICR in cultured rat sensory neurons.     

Functional role of receptor neurons in encoding olfactory information

In the present review we have considered the properties of the olfactory receptor neurons and discuss the strategy these cells use to perform their signaling task. Special emphasis is laid on the mechanisms for setting the membrane potential at rest and the mechanisms that the cell can use to respond with action potentials to significant stimuli only. We demonstrate that the firing properties of the receptor neurons depend upon the initial level of the membrane potential. We present the idea that the olfactory glomerulus can function as a unit in olfactory processing. In this perspective the olfactory receptor neuron is a subunit of the olfactory glomerulus. © 1996 John Wiley & Sons, Inc.     

CRH-induced electrical activity and calcium signalling in pituitary corticotrophs

Pituitary corticotroph cells generate repetitive action potentials and associated Ca2+ transients in response to the agonist corticotropin releasing hormone (CRH). There is indirect evidence suggesting that the agonist, by way of complex intracellular mechanisms, modulates the voltage sensitivity of the L-type Ca2+ channels embedded in the plasma membrane. We have previously constructed a Hodgkin-Huxley-type model of this process, which indicated that an increase in the L-type Ca2+ current is sufficient to generate repetitive action potentials (LeBeau et al. (1997). Biophys. J.73, 1263-1275). CRH is also believed to inhibit an inwardly rectifying K+ current. In this paper, we have found that a CRH-induced inhibition of the inwardly rectifying K+ current increases the model action potential firing frequency, [Ca2+]i transients and membrane excitability. This dual modulatory action of CRH on inward rectifier and voltage-gated Ca2+ channels better describes the observed CRH-induced effects. This structural alteration to the model along with parameter changes bring the model firing frequency in line with experimental data. We also show that the model exhibits experimentally observed bursting behaviour, where the depolarization spike is followed by small oscillations in the membrane potential.     

Electrophysiological and Morphological Properties of Neurons Acutely Isolated from the Rostral Gustatory Zone of the Rat Nucleus of the Solitary Tract

The biophysical and morphological characteristics of acutelyisolated neurons from the rostral nucleus of the solitary tract(rNST) were investigated under current clamp conditions andcompared with the results obtained from neurons recorded inbrain slices. The passive membrane properties of the isolatedneurons were similar to rNST neurons in brain slices and theneurons maintained their morphological characteristics althoughtheir dendritic tree was truncated. The isolated neurons alsoretained their characteristic repetitive firing properties.In addition we also noted developmental changes in the intrinsicmembrane properties of the isolated neurons, such as a shorteningin action potential duration, decrease in membrane time constantand input resistance, that occurred when these parameters werecompared in neurons isolated from young (5–10 days) andolder animals. These enzymatically dispersed neurons thereforeretained both the membrane properties and morphology observedin the intact brainstem and in vitro brain slice preparation.The use of this isolated neuron preparation provides a basisfor further study of rNST neurobiology. Chem. Senses 21: 729–737,1996     

Electrical Inexcitability of the Frog Neuromuscular Synapse

Frog muscle endplates were explored with an extracellular microelectrode. An intracellular microelectrode nearby simultaneously monitored invasion of the endplate by a spike directly evoked by a third microelectrode placed away from the endplate in the same fiber. External positivities were seen only at sites generating miniature endplate potentials. The external positivity reached a maximum prior to the internally recorded potential and was followed by a small late negativity. Small movements away from active synaptic sites resulted in positive-negative-positive potential sequences characteristic of activity and propagation. Since the external potential is a function of membrane current, the absence of negativity associated with the rising phase of the spike indicates the absence of inward current at synaptic sites. Thus, the synaptic membrane appears not to be excited by a depolarization of the magnitude of an action potential. In an Appendix it is shown that the late negativity and earlier maximum of the external potential can be accounted for by capacitative current through passive membrane.     

Voltage dependence of subthreshold resonance frequency in layer II of medial entorhinal cortex

The resonance properties of individual neurons in entorhinal cortex (EC) may contribute to their functional properties in awake, behaving rats. Models propose that entorhinal grid cells could arise from shifts in the intrinsic frequency of neurons caused by changes in membrane potential owing to depolarizing input from neurons coding velocity. To test for potential changes in intrinsic frequency, we measured the resonance properties of neurons at different membrane potentials in neurons in medial and lateral EC. In medial entorhinal neurons, the resonant frequency of individual neurons decreased in a linear manner as the membrane potential was depolarized between -70 and -55 mV. At more hyperpolarized membrane potentials, cells asymptotically approached a maximum resonance frequency. Consistent with the previous studies, near resting potential, the cells of the medial EC possessed a decreasing gradient of resonance frequency along the dorsal to ventral axis, and cells of the lateral EC lacked resonant properties, regardless of membrane potential or position along the medial to lateral axis within lateral EC. Application of 10 μM ZD7288, the H-channel blocker, abolished all resonant properties in MEC cells, and resulted in physiological properties very similar to lateral EC cells. These results on resonant properties show a clear change in frequency response with depolarization that could contribute to the generation of grid cell firing properties in the medial EC.     

Voltage-gated calcium channels

Voltage-gated calcium (Ca(2+)) channels are key transducers of membrane potential changes into intracellular Ca(2+) transients that initiate many physiological events. There are ten members of the voltage-gated Ca(2+) channel family in mammals, and they serve distinct roles in cellular signal transduction. The Ca(V)1 subfamily initiates contraction, secretion, regulation of gene expression, integration of synaptic input in neurons, and synaptic transmission at ribbon synapses in specialized sensory cells. The Ca(V)2 subfamily is primarily responsible for initiation of synaptic transmission at fast synapses. The Ca(V)3 subfamily is important for repetitive firing of action potentials in rhythmically firing cells such as cardiac myocytes and thalamic neurons. This article presents the molecular relationships and physiological functions of these Ca(2+) channel proteins and provides information on their molecular, genetic, physiological, and pharmacological properties.     

Electrophysiologic study of cultured neurons dissociated from spinal cords and dorsal root ganglia of fetal mice

Spontaneously occurring action potentials and postsynaptic potentials were recorded intracellularly from mouse spinal cord (SC) neurons and dorsal root ganglion (DRG) neurons in mixed SC and DRG cell cultures. In some SC cells, excitatory postsynaptic potentials were evoked by electrical stimulation of a nearby SC or DRG cell. SC and DRG neurons had distinguishing morphologic and electrophysiologic properties. SC neurons usually were elliptical or stellate and had several branched processes whereas DRG cells were most commonly round and had on the average only one process, but occasionally 3 or 4. Calculations from cell measurements revealed that SC neurons had less soma surface area and more process surface area than DRG cells, with a similar total surface area for each class. Lower resting membrane potentials were recorded from SC neurons, but when the capability for action potential generation was tested at comparable steady membrane potentials, most SC and less than half of DRG neurons fired repetitively to electrical stimulation. During the depolarizing and repolarizing phases of SC cell action potentials the rates of change of membrane potential were lower than for DRG cells, which had rapidly rising action potentials and a markedly negative afterpotential. An initially delayed repolarization phase was characteristic of the DRG cell action potential. Cell cultures were prepared by trypsin dissociation of spinal cords with attached spinal ganglia from fetuses of 10, 11, 12, 13, 14, and 17 days gestational age. Cell cultures grown on plastic or collagen were studied electrophysiologically at times from 16 to 94 days.     

Amphetamine elicited potential changes in vertebrate and invertebrate central neurons

The effects of amphetamine on potential changes in both vertebrate and invertebrate central neurons and factors affecting the potential changes were tested. The animals studied included mice, newborn rat and African snail. Seizure was elicited after lethal doses of d-amphetamine (75 mg/kg, i.p.) administration in mice. Repetitive firing of the action potentials were elicited after d-amphetamine (1-30 microM) administration in thin thalamic brain slices of newborn rat. Bursting firing of action potentials in the giant African central RP4 neuron were also elicited after d-amphetamine or l-amphetamine (0.27 mM) administration. The amphetamine elicited bursting firing of action potentials was not blocked even after high concentrations of d-tubocurarine, atropine, haloperidol, hexamethonium administration. Therefore, the amphetamine elicited potential changes may not be directly related to the activation of the receptors of the neuron. The bursting firing of action potentials elicited by amphetamine occurred 20-30 min after amphetamine administration extracellularly, even after high concentrations of d-amphetamine administration (0.27, 1 mM). However, the bursting firing of potentials occurred immediately if amphetamine was administrated intracellularly at lower concentration. Extracellular application of ruthenium red, the calcium antagonist, abolished the amphetamine elicited bursting firing of action potentials. If intracellular injection of EGTA, a calcium ion chelator, or injection with high concentrations of magnesium, the bursting firing of potentials were immediately abolished. These results suggested that the active site of amphetamine may be inside of the neuron and the calcium ion in the neuron played an important role on the bursting of potentials. In two-electrode voltage clamped RP4 neuron, amphetamine, at 0.27 mM, decreased the total inward and steady outward currents of the RP4 neuron. d-Amphetamine also decreased the calcium, Ia and the steady-state outward currents of the RP4 neuron. Besides, amphetamine elicited a negative slope resistance (NSR) if membrane potential was in the range of -50 to -10 mV. The NSR was decreased in cobalt substituted calcium free and sodium free solution. The effects of secondary messengers on the amphetamine elicited potential changes were tested. The bursting firing of action potentials elicited by amphetamine in central snail neurons decreased following extracellular application of H8 (N-(2-methyl-amino) ethyl-3-isoquinoline sulphonamide dihydrochloride), a specific protein kinase A inhibitor and anisomycin, a protein synthesis inhibitor. However, the bursting firing of action potentials were not affected after extracellular application of H7 (1,(5-isoquinolinesulphonyl)-2-methylpiperasine dihydrochloride), a specific protein kinase C (PKC) inhibitor, or intracellular application of GDPbetaS, a G protein inhibitor. The oscillation of membrane potential of the bursting activity was blocked after intracellular injection of 3'-deoxyadenosine, an adenylyl-cyclase inhibitor. These results suggested that the bursting firing of action potentials elicited by d-amphetamine in snail neuron may be associated with the cyclic AMP second messenger system; on the other hand, it may not be associated with the G protein and protein kinase C activity. It is concluded that amphetamine elicited potential changes in both vertebrate and invertebrate central neurons. The changes are closely related to the ionic currents and second messengers of the neurons.     

Synaptic mechanisms underlying sparse coding of active touch

Sensory information is actively gathered by animals, but the synaptic mechanisms driving neuronal circuit function during active sensory processing are poorly understood. Here, we investigated the synaptically driven membrane potential dynamics during active whisker sensation using whole-cell recordings from layer 2/3 pyramidal neurons in the primary somatosensory barrel cortex of behaving mice. Although whisker contact with an object evoked rapid depolarization in all neurons, these touch responses only drove action potentials in ～10% of the cells. Such sparse coding was ensured by cell-specific reversal potentials of the touch-evoked response that were hyperpolarized relative to action potential threshold for most neurons. Intercontact interval profoundly influenced touch-evoked postsynaptic potentials, interestingly without affecting the peak membrane potential of the touch response. Dual whole-cell recordings indicated highly correlated membrane potential dynamics during active touch. Sparse action potential firing within synchronized cortical layer 2/3 microcircuits therefore appears to robustly signal each active touch response.     

Electrical properties of neurons recorded from the rat supraoptic nucleus in vitro

The electrical properties of neurons in the supraoptic nucleus (so.n.) have been studied in the hypothalamic slice preparation by intracellular and extracellular recording techniques, with Lucifer Yellow CH dye injection to mark the recording site as being the so.n. Intracellular recordings from so.n. neurons revealed them to have an average membrane potential of -67 +/- 0.8 mV (mean +/- s.e.m.), membrane resistance of 145 +/- 9 M omega with linear current-voltage relations from 40 mV in the hyperpolarizing direction to the level of spike threshold in the depolarizing direction. Average cell time constant was 14 +/- 2.2 ms. So.n. action potentials ranged in amplitude from 55 to 95 mV, with a mean of 76 +/- 2 mV, and a spike width of 2.6 +/- 0.5 ms at 30% of maximal spike height. Both single spikes and trains of spikes were followed by a strong, long-lasting hyperpolarization with a decay fitted by a single exponential having a time constant of 8.6 +/- 1.8 ms. Action potentials could be blocked by 10(-6) M tetrodotoxin. Spontaneously active so.n. neurons were characterized by synaptic input in the form of excitatory and inhibitory postsynaptic potentials, the latter being apparently blocked when 4 M KCl electrodes were used. Both forms of synaptic activity were blocked by application of divalent cations such as Mg2+, Mn2+ or Co2+. 74% of so.n. neurons fired spontaneously at rates exceeding 0.1 spikes per second, with a mean for all cells of 2.9 +/- 0.2 s-1. Of these cells, 21% fired slowly and continuously at 0.1 - 1.0 s-1, 45% fired continuously at greater than 1 Hz, and the remaining 34% fired phasically in bursts of activity followed by silence or low frequency firing. Spontaneously firing phasic cells showed a mean burst length of 16.7 +/- 4.5 s and a silent period of 28.2 +/- 4.2 s. Intracellular recordings revealed the presence of slow variations in membrane potential which modified the neuron's proximity to spike threshold, and controlled phasic firing. Variations in synaptic input were not observed to influence firing in phasic cells.     

A model of the electrophysiological properties of nucleus reticularis thalami neurons.

A model of the electrophysiological properties of rodent nucleus reticularis thalami (NRT) neurons of the dorsal lateral thalamus was developed using Hodgkin-Huxley style equations. The model incorporated voltage-dependent rate constants and kinetics obtained from recent voltage-clamp experiments in vitro. The intrinsic electroresponsivity of the model cell was found to be similar to several empirical observations. Three distinct modes of oscillatory activity were identified: 1) a pattern of slow rhythmic burst firing (0.5-7 Hz) usually associated with membrane potentials negative to approximately -70 mV which resulted from the interplay of ITs and IK(Ca); 2) at membrane potentials from approximately -69 to -62 mV, rhythmic burst firing in the spindle frequency range (7-12 Hz) developed and was immediately followed by a tonic tail of single spike firing after several bursts. The initial bursting rhythm resulted from the interaction of ITs and IK(Ca), with a slow after-depolarization due to ICAN which mediated the later tonic firing; 3) with further depolarization of the membrane potential positive to approximately -61 mV, sustained tonic firing appeared in the 10-200-Hz frequency range depending on the amplitude of the injected current. The frequency of this firing was also dependent on the maximum conductance of the leak current, IK(leak), and an interaction between the fast currents involved in generating action potentials, INa(fast) and IK(DR), and the persistent Na+ current, INa(P). Transitions between different firing modes were identified and studied parametrically.