Lineage-specific development of calcium currents during embryogenesis.

The development of electrophysiological properties of isolated, identified ascidian blastomeres was followed from the fertilized egg to the neurula, and the stage at which cells of different lineages first express different functional ion channel populations was determined. Little has been known about such events because of the difficulties of making voltage-clamp recordings from small embryonic cells and of identifying their developmental fates in dissociated preparations. The problem of small cell size was circumvented by using the whole-cell patch clamp, and identification was facilitated by the use of a species of ascidian, Boltenia villosa, in which endogenous pigment marks cells of specific developmental fates. Within approximately 3 hours after gastrulation, muscle-lineage blastomeres in these embryos developed a voltage-dependent calcium current while surrounding blastomeres of other lineages did not. At about the same time, all cells developed delayed outward potassium currents and lost the inwardly rectifying potassium currents present at earlier stages.

A voltage-dependent chloride current linked to the cell cycle in ascidian embryos.

A voltage-dependent chloride current has been found in early ascidian embryos that is a minor conductance in the oocyte and in interphase blastomeres but that increases transiently in amplitude by more than tenfold during each cell division. Repeated cycles in the density of this chloride current could be recorded for up to 6 hours (four cycles) in cleavage-arrested embryos, whether they were activated by sperm or calcium ionophore. These data suggest that there is a direct link between the cell cycle clock and the properties of this channel, a link that results in pronounced cyclical changes in the electrical properties of early blastomeres.

Distinct calcium signals in developing cortical interneurons persist despite disorganization of cortex by Tbr1 KO.

Cortical development involves the structuring of network features by genetically programmed molecular signaling pathways. Additionally, spontaneous ion channel activity refines neuronal connections. We examine Ca(2+) fluctuations in the first postnatal week of normal mouse neocortex and that expressing knockout of the transcription factor T-brain-1 (Tbr1): a signaling molecule in cortical patterning and differentiation of excitatory neurons. In cortex, glutamatergic neurons express Tbr1 just before the onset of population electrical activity that is accompanied by intracellular Ca(2+) increases. It is known that glutamatergic cells are disordered with Tbr1 KO such that normal laying of the cortex, with newer born cells residing in superficial layers, does not occur. However, the fate of cortical interneurons is not well studied, nor is the ability of Tbr1 deficient cortex to express normal physiological activity. Using fluorescent proteins targeted to interneurons, we find that cortical interneurons are also disordered in the Tbr1 knockout. Using Ca(2+) imaging we find that population activity in mutant cortex occurs at normal frequencies with similar sensitivity to GABAA receptor blockade as in nonmutant cortex. Finally, using multichannel fluorescence imaging of Ca(2+) indicator dye and interneurons labeled with red fluorescent protein, we identify an additional Ca(2+) signal in interneurons distinct from population activity and with different pharmacological sensitivities. Our results show the population activity described here is a robust property of the developing network that continues in the absence of an important signaling molecule, Tbr1, and that cortical interneurons generate distinct forms of activity that may serve different developmental functions. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 705-720, 2016.

Block of inward rectification by intracellular H+ in immature oocytes of the starfish Mediaster aequalis.

Intracellular pH was recorded in immature starfish oocytes using pH-sensitive microelectrodes, and inwardly rectifying potassium currents were measured under voltage clamp. When the intracellular pH was lowered using acetate-buffered artificial sea water from the normal value of 7.09 to 5.9, inward rectification was completely blocked. The relationship between inward rectification and internal pH between 7.09 and 5.9 could be fit by a titration curve for the binding of three H ions to a site with a pK of 6.26 to block the channel. The H+ block showed no voltage dependence, and the activation kinetics of the inwardly rectifying currents were not affected by the changes in internal pH.

The role of extracellular potassium in hippocampal epilepsy.

It has been proposed that the notable capacity for epileptogenesis in the hippocampus may be related to potassium accumulation in extracellular spaces. To investigate this hypothesis more directly, we measured changes in extracellular potassium concentration ([K+]o) during focal hippocampal epilepsy using potassium-sensitive microelectrodes. Interictal and ictal electroencephalographic events were accompanied by increases in [K+]o that varied systematically with depth from the ependymal surface and lateral distance from the focus. Maximal [K+]o changes during interictal and ictal discharges occurred in the stratum pyramidale. Initiation of ictal activity did not correlate with a particular "threshold" [K+]o. Comparing these results with similar data from neocortex, we observed that interictal K+ responses in hippocampus lasted longer and had slower rise times, and that peak interictal and ictal [K+]o values were consistently lower. Increases in [K+]o cannot be the sole explanation for regional variations in seizure susceptibility, interictal-ictal transitions, or termination of ictal episodes.

The ionic mechanism of intracellular pH regulation in crayfish neurones.

1. Intracellular pH (pHi) regulation in crayfish neurones was studied using pH-, Na+-, and Cl- sensitive micro-electrodes. Neuronal pH regulation has previously been studied only in molluscs. 2. The average resting pHi of crayfish neurones was 7.12 +/- 0.09, which is 1 pH unit more alkaline than that predicted were H+ ions distributed in equilibrium with the membrane potential. 3. When the cytoplasm was acidified (by NH4Cl loading, CO2 application, or HCl injection), pHi recovered towards its resting value. 4. Removal of Na+ from the external solution inhibited pHi recovery from an acid load by more than 90%. pHi recovery resumed immediately when external Na+ was reintroduced. 5. The resting intracellular Na+ concentration ([Na+]i) of crayfish neurones was 15-25 mM. During pHi recovery from an acid load, [Na+]i increased by 10-50 mM. 6. Reducing the external HCO3(-) concentration from 5 mM to 0 mM slowed pHi recovery by an average of about 45%. This slowing was appreciable even in cells in which Na+ removal almost totally blocked pHi recovery. 7. The resting intracellular Cl- concentration ([Cl-]i) was 30-40 mM, indicating that these cells actively accumulate Cl-. During pHi recovery from an acid load, [Cl-]i decreased by 3-5 mM. 8. In the presence of the anion exchange inhibitor SITS (4-acetamide-4'-isothiocyanostilbene-2,2'-disulphonic acid), pHi recovery was slowed to the rate which was normally seen in HCO3(-)-free Ringer solution. SITS abolished the dependence of pHi recovery on the external HCO3(-) concentration. 9. It is concluded that pHi regulation in crayfish neurones involves two separate mechanisms: a Na+-dependent, HCO3(-)-independent acid extrusion process, and a Cl---HCO3(-) exchange which is probably also Na+-dependent.

Critical periods of early development created by the coordinate modulation of ion channel properties.

The development of ion channel properties in excitable cells begins in the very early embryo and continues throughout differentiation. The pattern of ion channel development in a given cell type is not a simple linear progression to the mature state, but rather is a complex sequence of modulatory events that create windows of time during which excitability is qualitatively different from that in the mature cell. These windows are likely candidates for critical periods when electrical activity influences later development.

The development of voltage-gated ion channels and its relation to activity-dependent development events.

Spontaneous activity is an essential feature in the development of the nervous system. The patterns of activity and the waveform and ionic dependence of the action potentials that occur during such activity are fine-tuned to carry out certain developmental functions, and are therefore generally not compatible with the mature physiological function of the cell. For this reason, the patterns of ion channel development that create spontaneous activity early in the development of a given cell type are complex and not easily predicted from the mature properties of that same cell. Ion channels are often found that are specific to early stages of development, and that either are not retained in the mature cell or whose properties are greatly changed during later differentiation. The exact significance of such patterns of channel development is just now becoming clear, as we understand more about the mechanisms linking spontaneous activity to later developmental events.

The development of calcium and potassium currents during oogenesis in the starfish, Leptasterias hexactis.

The development of membrane electrical properties of oocytes of the starfish Leptasterias hexactis during oogenesis was studied using voltage- and current-clamp techniques. Two voltage-dependent K currents--the fast transient and inwardly rectifying--are present early in oogenesis, before the rapid growth phase, and are maintained throughout oogenesis at the same current density and kinetics. The inward current, which is composed of a Ca current and a slower Ca-dependent inward sodium current, is also present early in oogenesis, but at very low current density. Late in oogenesis, after the oocyte has grown to full size, the inward current increases in amplitude by about fivefold, and undergoes major changes in kinetics. These changes are closely associated with the migration of the germinal vesicle to the cell periphery. The relationship of these events to electrophysiological changes during subsequent maturation and fertilization of the oocytes is discussed.

Control of spontaneous activity during development.

Electrical activity participates in the development of the nervous system and comes in two general forms. Use-dependent or experience-driven activity occurs relatively late in development, and is important in events of terminal nervous system differentiation, such as stabilization of synaptic connections. Earlier in development, activity is spontaneous, occurring independently of normal sensory input and motor output. Spontaneous activity participates in many of the initial events of axon outgrowth, pruning of synaptic connections, and maturation of neuronal signaling properties. Despite its importance, the genesis of spontaneous activity is poorly understood. What is clear is that spontaneous activity must be regulated by the patterns with which voltage- and ligand-gated ion channels develop in individual neurons. This review explores how that regulation most likely occurs.

Na+ channel mis-expression accelerates K+ channel development in embryonic Xenopus laevis skeletal muscle.

1. The normal developmental pattern of voltage-gated ion channel expression in embryonic skeletal muscle cells of the frog Xenopus laevis was disrupted by introduction of cloned rat brain Na+ channels. 2. Following injection of channel mRNA into fertilized eggs, large Na+ currents were observed in muscle cells at the earliest developmental stage at which they could be uniquely identified. Muscle cells normally have no voltage-gated currents at this stage. 3. Muscle cells expressing exogenous Na+ channels showed increased expression of at least two classes of endogenous K+ currents. 4. This increase in K+ current expression was inhibited by the Na+ channel blocker tetrodotoxin, suggesting that increased electrical activity caused by Na+ channel mis-expression triggers a compensatory increase in K+ channel expression. 5. Block of endogenous Na+ channels in later control myocytes retards K+ current development, indicating that a similar compensatory mechanism to that triggered by Na+ channel mis-expression operates to balance Na+ and K+ current densities during normal muscle development.

Membrane currents of internally perfused neurones of the snail, Lymnaea stagnalis, at low intracellular pH.

The effects of low intracellular pH (pHi) on the membrane currents of snail neurone somata were studied using the internal perfusion and ion-sensitive micro-electrode techniques. Recordings with pH-sensitive micro-electrodes made while the pH of the perfusion solution was changed between 7.3 and 6.3 indicated that only with high buffer concentrations (100 mM) could pHi be changed effectively. H+ was slower to exchange into the cytoplasm than an unbuffered ion such as K+. When pHi was decreased to 5.9, large outward H+ currents could be recorded at voltages positive to -30 mV. The time course and amplitude of these currents were such that they did not affect the measurement of the peak amplitude of the fast transient K+ current (A-current), but severely contaminated both Ca2+ and delayed K+ current measurements. Low pHi blocked the A-current. The titration curve was consistent with the binding of two H ions to a site with a pK of 6.05 to block the channel. Low pHi appeared to block the slow inactivation of the delayed outward current without greatly changing its peak amplitude. However, when correction was made for the increase of H+ current at low pHi, the effect of internal H+ was found to be a block of the delayed K+ current with no consistent effect on inactivation. The Ca2+ current was also decreased at low pHi, but we were unable to determine whether this was a direct effect of pHi or secondary to a rise in internal free [Ca2+]. If no correction was made for H+ currents, the block of the Ca2+ current appeared greater and more reversible than it actually was. We conclude that under certain conditions, such as low pHi, the H+ current is a significant fraction of the total outward current in snail neurones, and may also be in a variety of other cells. The H+ currents must be accounted for under such conditions in order to study accurately the properties of K+ and Ca2+ currents.

Effects of intracellular pH and calcium activity on ion currents in internally perfused neurons of the snail Lymnaea stagnalis.

The suction pipet method of intracellular dialysis and voltage clamp of cells has proven extremely useful in analysing the electrical properties of cells too small for the application of conventional microelectrode techniques and in larger cells for studying the effects of alterations in the internal ionic composition. Using neurons of the snail Lymnaea stagnalis, we have analysed several problems involved in the latter application of this technique and present several solutions to them. One major problem centers around the degree of control over the ionic composition of the cytoplasm achieved by altering the pipet solution. Using ion-sensitive microelectrodes during internal dialysis, we found that the efficiency of exchange between pipet and cytoplasm was much poorer for highly buffered ions such as H+ and Ca2+, than for K+, for example. Special precautions are described that can help this situation. The second problem involves the study of the effects of low internal pH on ion-channel properties. We summarize evidence for a specific voltage-dependent hydrogen ion channel, current through which becomes prominent at low internal pH. We analyse how the presence of this heretofore unrecognized current can seriously confuse the results of experiments designed to study the effects of low internal pH on other voltage-dependent currents.

Electrical activity and calcium influx regulate ion channel development in embryonic Xenopus skeletal muscle.

The development of electrical excitability involves complex coordinated changes in ion channel activity. Part of this coordination appears to be due to the fact that the expression of some channels is dependent on electrical activity mediated by other channel types. For example, we have previously shown that normal potassium current development in embryonic skeletal muscle cells of the frog Xenopus laevis is dependent on sodium channel activity. To examine the interrelationships between the development of different ionic currents, we have made a detailed study of electrical development in cultured Xenopus myocytes using whole-cell patch-clamp recording. The initial expression of potassium, sodium, and calcium currents is followed by a brief period during which the densities of potassium currents decrease, while at the same time sodium and calcium current densities continue to increase, which may increase electrical excitability during this time. The normal developmental increase in both potassium and sodium currents is inhibited by the sodium channel blocker tetrodotoxin, suggesting that electrical activity normally stimulates the expression of both these currents. These effects of electrical activity appear to be mediated via activation of voltage-gated calcium channels. We suggest that the developmental acquisition of sodium and calcium channels by these cells, possibly coupled with a transient decrease in potassium current density, lead to an increase in electrical excitability and calcium entry, and that this calcium entry provides a critical developmental cue controlling the subsequent development of mature electrical properties.

An electrophysiological characterization of ciliated olfactory receptor cells of the coho salmon Oncorhynchus kisutch.

Electrical properties of ciliated olfactory receptor cells isolated from coho salmon (Oncorhynchus kisutch) were studied using the whole-cell mode of the patch-clamp recording technique. 1. Voltage-dependent currents could be separated into two inward and three outward conductances, including a Na+ current, Ca2+ current and three K+ currents. 2. The components of the outward current varied with the life stage of the salmon from which cells had been isolated. In cells isolated from juvenile fish (parr), a Ca(2+)-dependent K+ current dominated the outward current, whereas in cells isolated from older fish (i.e. fish that had undergone smoltification), a transient K+ current became prominent. 3. Differences in response characteristics of outward currents to internal dialysis with cyclic GMP (but not cyclic AMP) were also correlated to the life stage of salmon. Under conditions in which the Ca(2+)-activated current was blocked, relaxation of the outward current was slowed by dialysis with cyclic GMP only in cells isolated from smolts and sea-run fish, but not in those isolated from mature spawners. 4. From these results, we suggest that hormone modulation of olfactory receptor cell development or differentiation may play a role in establishing these differences.

Developmental sequence of expression of voltage-dependent currents in embryonic Xenopus laevis myocytes.

Although the development of several of the voltage-dependent currents in embryonic amphibian myocytes has been described, the overall muscle electrical development, particularly the relative times of expression of different voltage-dependent currents, has not been addressed in a single study under one set of conditions. We have found that, in mesoderm isolated and cultured from neurula stage embryos, myocytes are identifiable before they express voltage-gated currents. These ionic currents are absent from all Xenopus mesodermal cells during the late gastrula/early neurula stages of embryonic development. At about the time of first somite segregation an inward rectifier K+ current is expressed in some myocytes, followed within 2 hr by a delayed rectifier K+ current. The density of both currents increases fourfold over the next 24 hr in culture. A Na+ current is not expressed in large numbers of myocytes until late in this culture period, at about the time that a slow Ca2+ current appears. Under our culture conditions the myocytes have a very low chloride conductance. A fast inactivating component to the outward K+ current is expressed in all myocytes by 24 hr in culture. In some experiments we dissociated embryos at later times and made recordings when all previously isolated myocytes expressed currents. In the late dissociations, most myocytes did not express currents, but developed them after a short period in culture. Because we have evidence that in vivo development is more closely approximated by the early dissociations, these results suggest that dissociation causes some degree of dedifferentiation.

Time course of ion channel development in Xenopus muscle induced in vitro by activin.

During the process of mesoderm specification in Xenopus embryos, cells of the equatorial region are induced to form mesoderm in response to signals from the underlying endodermal cells. One mesodermal cell type resulting from this in vivo induction is skeletal muscle, which has a very specific and tightly regulated course of electrical and morphological development. Previously, electrical development could be analyzed only after neurulation, once myocytes could be morphologically identified. In vitro, activin triggers a cascade of events leading to the development of specific mesodermal tissues, including skeletal muscle; however, the precise role of activin in vivo is less clear. Much is now known about the mechanism and control of activin action, but very little is known about the subsequent time course of differentiation of activin-induced muscle. Such muscle is routinely identified by the presence of a small number of specific markers which, although they accurately confirm the presence of muscle, give little indication of the time course or quantitative aspects of muscle development. One of the most important functional aspects of muscle development is the acquisition of the complex electrical properties which allow it to function normally. Here we assess the ability of activin to drive in vitro the normal highly regulated sequence of electrical development in skeletal muscle. We find that in most, but not all, respects the normal time course of development of voltage-gated ion currents is well reproduced in activin-induced muscle. This characterization strengthens the case for activin as an agent capable of inducing the detailed developmental program of muscle and now allows for analysis of the regulation of electrical development prior to neurulation.

Modifications of current properties by expression of a foreign potassium channel gene in Xenopus embryonic cells.

The development of excitable cells is characterized by highly organized patterns of expression of ion channels. During the terminal differentiation of Xenopus muscle somites, potassium currents are expressed first just after Stage 15 (early-mid neurula), following a long period during which no voltage-dependent currents can be detected in any cell in the dorsal embryo. We have investigated whether early expression of a foreign delayed rectifier potassium channel may affect this endogenous pattern of electrical development. We injected the purified cRNA of the mammalian brain Shaker-like potassium channel, Kv1.1, into fertilized Xenopus eggs. The resulting currents were analyzed in blastomeres during a 12-hr period prior to Stage 15 and in differentiating muscle cells after Stage 15. In injected embryos, a high fraction of blastomeres expressed a delayed rectifier-type current. The Kv1.1 current could be distinguished from the endogenous muscle delayed potassium current (IK,X) by its very different voltage dependence. Separation of currents based on this difference indicated that, in injected embryos, IK,X appeared much earlier in development than in control embryos. Furthermore, even in cells which expressed solely Kv1.1-type current, the sensitivity of the current to dendrotoxin declined dramatically during development, approaching that of IK,X. These data suggest an interaction between Kv1.1 and endogenous channel subunits, and/or modification of the Kv1.1 protein by the embryonic cells in ways not seen in Xenopus oocytes or mammalian cell lines.

Hormone-induced loss of surface membrane during maturation of starfish oocytes: differential effects on potassium and calcium channels.

Prior to fertilization, starfish oocytes undergo meiotic maturation, triggered by the hormone 1-methyladenine (1-MA). Maturation involves a variety of complex biochemical, morphological, and electrical changes, many of which are similar to those caused by progesterone in vertebrates. Using voltage-clamp and ultrastructural techniques to study maturation in starfish, we have discovered a novel process by which 1-MA alters the electrical properties of the oocyte. The surface area of the oocyte decreases by more than 50% during the first hour of maturation, due to the elimination of microvilli, but the calcium and potassium currents present are affected differently by the loss of membrane. The amplitudes of both the transient K current ("A-current") and the inwardly rectifying K current decrease, following the time course of the decrease in surface area, while the Ca current amplitude remains virtually unaffected, and may even increase in some oocytes. The kinetics of the currents do not change. This selective removal of K channels results in a larger and more rapidly rising action potential in the mature egg, which may aid in the fast block to polyspermy. The differential accessibility of various ion channels to mechanisms of membrane removal and insertion may play an important role in the development of excitable cells.

Fertilization alters the spatial distribution and the density of voltage-dependent sodium current in the egg of the ascidian Boltenia villosa.

The spatial distribution of voltage-dependent ionic currents was characterized in Boltenia villosa eggs before and after fertilization using two-microelectrode voltage clamp of paired animal-vegetal halves of eggs (merogones) made surgically. Major voltage-dependent conductances in the Boltenia egg are a transient inward Na current, a transient inward Ca current, and an inwardly rectifying K current. These currents were randomly distributed along the animal-vegetal axis in the unfertilized egg. When paired merogones (surgically prepared egg fragments) were made at the vegetal cap stage, 15-30 min after fertilization, Ca and K currents remained randomly distributed along the animal-vegetal axis. In contrast, the relative Na current density was found to be twofold lower in the vegetal vs the animal merogones made at the vegetal cap stage. By making pairs of merogones from unfertilized eggs and subsequently fertilizing one merogone of a pair, we showed that this change in current density ratio was due to a loss of absolute Na current density in the vegetal hemisphere shortly after fertilization. These results also show that this loss was intrinsic to the vegetal hemisphere, rather than being determined solely by the point of sperm entry. A second decrease in Na current was observed during the hour before first cleavage, 60-120 min after fertilization (M.L. Block and W.J. Moody, 1987, J. Physiol. 393, 619-634), both in fertilized eggs and in animal merogones fertilized after isolation. This second loss of Na current was not observed in vegetal merogones fertilized after isolation or in either animal or vegetal merogones made from fertilized eggs at the vegetal cap stage. Possible mechanisms for te rapid (complete by 40 min after fertilization) and the late (occurring from ca. 60 to 120 minutes after fertilization) Na current losses are discussed.