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.

Action potential waveform voltage clamp shows significance of different Ca2+ channel types in developing ascidian muscle.

1. Early in development, ascidian muscle cells generate spontaneous, long-duration action potentials that are mediated by a high-threshold, inactivating Ca2+ current. This spontaneous activity is required for appropriate physiological development. 2. Mature muscle cells generate brief action potentials only in response to motor neuron input. The mature action potential is mediated by a high-threshold sustained Ca2+ current. 3. Action potentials recorded from these two stages were imposed as voltage-clamp commands on cells of the same and different stages from which they were recorded. This strategy allowed us to study how immature and mature Ca2+ currents are optimized to their particular functions. 4. Total Ca2+ entry during an action potential did not change during development. The developmental increase in Ca2+ current density exactly compensated for decreased spike duration. This compensation was a function purely of Ca2+ current density, not of the transition from immature to mature Ca2+ current types. 5. In immature cells, Ca2+ entry was spread out over the entire waveform of spontaneous activity, including the interspike voltage trajectory. This almost continuous Ca2+ entry may be important in triggering Ca2+-dependent developmental programmes, and is a function of the slightly more negative voltage dependence of the immature Ca2+ current. 6. In contrast, Ca2+ entry in mature cells was confined to the action potential itself, because of the slightly more positive voltage dependence of the mature Ca2+ current. This may be important in permitting rapid contraction-relaxation cycles during larval swimming. 7. The inactivation of the immature Ca2+ current serves to limit the frequency and burst duration of spontaneous activity. The sustained kinetics of the mature Ca2+ current permit high-frequency firing during larval swimming.

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.

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.

Spontaneous activity regulates calcium-dependent K+ current expression in developing ascidian muscle.

1. In embryonic ascidian muscle, outward K+ currents develop in two stages: the initial expression of a slowly activating, voltage-gated K+ current (IKv) near the time of neurulation is followed about 6 h later by a rapidly activating calcium-activated K+ current (IK(Ca)). During this 6 h interval, inward Ca2+ currents (ICa) appear and the inward rectifier (IK(IR)), the sole resting conductance, is transiently downregulated. These events predict a period of spontaneous activity. The following experiments were designed to test this prediction and to examine the relevance of spontaneous activity for muscle cell development. 2. By recording activity in cell-attached patches, we have found that muscle cells generate spontaneous action potentials during this 6 h window of time when IK(IR) is downregulated and outward K+ currents are slow. Action potentials occur at a mean frequency of 13.9 min-1.3. When activity is blocked by the transient application of the Ca2+ channel blocker Cd2+, IK(Ca) fails to develop. This disruption is specific for IK(Ca): IK(IR) and ICa develop normally in activity-blocked cells. Application of Cd2+ either before or after the window of activity has no effect. 4. The reappearance of IK(IR) and the development of IK(Ca) and the mature form of ICa are all prevented by transcription blockers, with a sensitive period corresponding to the period of activity. 5. These data show that, although the expression of three channel types depends on transcription during the period of spontaneous activity, only the development of IK(Ca) depends on activity.

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.

Co-ordinated modulation of Ca2+ and K+ currents during ascidian muscle development.

1. The development of Ca2+ and K+ currents was studied in ascidian muscle cells at twelve embryonic stages from gastrulation to the mature cell, a period of 24 h. A high degree of co-ordination occurs between the development of the inwardly rectifying K+ current (IK(IR)), which sets the resting potential, and Ca2+ and outward K+ currents, which determine action potential waveform. 2. At neurulation IK(IR), which had been present since fertilization, begins to decrease, reaching 12% of its previous density in 6 h. IK(IR) then immediately begins to increase again, reaching its previous density in another 6 h. 3. When IK(IR) begins to decrease, a high-threshold inactivating Ca2+ current and a slowly activating voltage-gated K+ current appear. 4. When IK(IR) returns to its previous density, two new currents appear: a sustained Ca2+ current with the same voltage dependence, but different conotoxin sensitivity than the inactivating Ca2+ current; and a Ca(2+)-dependent K+ current, which activates 8-10 times faster and at potentials 20-30 mV more negative than the voltage-dependent K+ current. 5. The transient downregulation of IK(IR) destabilizes the resting potential and causes spontaneous action potentials to occur. Because IK(IR) is absent when only a slowly activating high-threshold outward K+ current is present, these action potentials are long in duration. 6. The return of IK(IR) and the appearance of the rapidly activating Ca(2+)-dependent K+ current eventually terminate this activity. The action potentials of the mature cell occur only on stimulation, and are 10 times shorter in duration than those in the immature cell.

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.

A voltage-gated chloride channel in ascidian embryos modulated by both the cell cycle clock and cell volume.

1. Eggs of the ascidian Boltenia villosa have an inwardly rectifying Cl- current whose amplitude varies by more than 10-fold during each cell cycle, the largest amplitude being at exit from M-phase. We examined whether this current was also sensitive to changes in cell volume. 2. Cell swelling, produced by direct inflation through a whole-cell recording pipette, greatly increased the amplitude of the Cl- current at all stages of the cell cycle in activated eggs. Swelling was much less effective in unfertilized eggs. 3. The increase in Cl- current amplitude continued for 10-20 min after an increase in diameter that was complete in 10 s, suggesting the involvement of a second messenger system in the response. 4. Treatment of unfertilized eggs with 6-dimethylaminopurine (DMAP), an inhibitor of cell cycle-dependent protein kinases, increased the amplitude of the Cl- current and its sensitivity to swelling to levels characteristic of fertilized eggs. 5. Osmotically produced swelling also increased Cl- current amplitude in unfertilized eggs. 6. We propose that dephosphorylation renders the Cl- channel functional, and that swelling or activation of the egg increases the sensitivity of the channel to dephosphorylation, perhaps by disrupting its links to the cytoskeleton.

Comparison of ionic currents expressed in immature and mature muscle cells of an ascidian larva.

We have compared the voltage-gated ion channels present in larval ascidian muscle at two developmental stages: muscle precursor cells just after the terminal cell division and mature contractile muscle, 7-11 hr later. All precursor cells express a high-threshold transient Ca current and a slowly activating delayed K current, and about half the cells express a low-threshold transient Ca current. An inwardly rectifying K current, which had been present from fertilization until just before the terminal cell division, is absent. Mature muscle retains two of the tailbud currents: the low-threshold transient Ca current and the slow delayed K current, although at larger densities, and also expresses a high-threshold Ca current that is similar in most respects to the precursor cell current but that lacks inactivation. In addition, mature muscle expresses two rapidly activating outward K currents, one voltage and one Ca dependent, that generate a composite outward K current that is eight times larger and activates eight times faster than the tailbud K current. Mature muscle also reexpresses the inward rectifier. We propose that the transient absence of the inward rectifier and the slow activation of the delayed K current early in development create a window of developmental time when spontaneous electrical activity is likely.

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.

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.

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.

Evidence for a peripheral olfactory memory in imprinted salmon.

The remarkable homing ability of salmon relies on olfactory cues, but its cellular basis is unknown. To test the role of peripheral olfactory receptors in odorant memory retention, we imprinted coho salmon (Oncorhynchus kisutch) to micromolar concentrations of phenyl ethyl alcohol during parr-smolt transformation. The following year, we measured phenyl ethyl alcohol responses in the peripheral receptor cells using patch clamp. Cells from imprinted fish showed increased sensitivity to phenyl ethyl alcohol compared either to cells from naive fish or to sensitivity to another behaviorally important odorant (L-serine). Field experiments verified an increased behavioral preference for phenyl ethyl alcohol by imprinted salmon as adults. Thus, some component of the imprinted olfactory homestream memory appears to be retained peripherally.

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.

Changes in voltage-dependent ion currents during meiosis and first mitosis in eggs of an ascidian.

Different patterns of voltage-dependent ion currents are present in mature eggs and in early embryos of the ascidian Boltenia villosa, as if each ion current is regulated in a different manner between fertilization and the early cleavages of embryogenesis. The ion currents appear and/or disappear with precise timing suggesting that they play important roles at specific times during early development. We investigated changes in three voltage-dependent ion currents (an inwardly rectifying chloride current, a calcium current, and a sodium current) and membrane surface area over time between the resumption of meiosis (with fertilization or activation) and the first mitotic cleavage. Using time-lapse video recordings made during whole-cell patch-clamp experiments, we were able to correlate electrophysiological changes with morphological changes and cell cycle related events. Between fertilization and first cleavage, INa was lost exponentially, the density of ICa remained relatively constant, and the amplitudes of both ICl and membrane surface area fluctuated in time with the cell cycle. ICl and surface area increased whenever the cell began dividing--with the polar body extrusions and the formation of the first cleavage furrow. This suggested that the values of ICl and surface area were largest during interphase and smallest during M-phase of each cell cycle. This hypothesis was supported by an experiment in which entry into M-phase was blocked in fertilized eggs by inhibiting protein synthesis. This prevented the decreases of ICl and surface area but allowed the increases to occur normally. Patterns of change in ion currents are current specific and, as is the case with ICl, are tightly correlated with developmental events.

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.

Dependence of Ca2+ and K+ current development on RNA and protein synthesis in muscle-lineage cells of the ascidian Boltenia villosa.

The early development of excitability of muscle-lineage cells of the ascidian Boltenia villosa is characterized by the appearance, just after gastrulation, of a Ca2+ current and a delayed outward K+ current, while an inwardly rectifying K+ current, present since fertilization, disappears. The muscle-lineage cells are the first cells in which we detect tissue-specific electrical properties after gastrulation. Here, we show that the development of electrical properties in these cells involves RNA and protein synthesis. If transcription or translation is blocked, the Ca2+ and outward K+ currents fail to appear, whereas the inward K+ current disappears normally. For the Ca2+ current, the sensitive period for transcription extends until just before gastrulation, while the sensitive period for translation extends until after gastrulation. The oocyte has a Ca2+ current present at about 5-10% the density of that in the muscle-lineage cells; this current disappears by gastrulation. A comparison of the oocyte and muscle Ca2+ currents indicates that they are similar in voltage dependence and inactivation mechanism. A small difference in permeability sequence can be attributed to different surface charge properties at the two stages of development.

Macroscopic and single-channel studies of two Ca2+ channel types in oocytes of the ascidian Ciona intestinalis.

Whole-cell and single-channel patch-clamp experiments were performed on unfertilized oocytes of the ascidian Ciona intestinalis to investigate the properties of two voltage-dependent Ca2+ currents found in this cell. The peak of the low threshold current (channel I) occurred at -20 mV, the peak of the high-threshold current (channel II) at +20 mV. The two currents could be distinguished by voltage dependence, kinetics of inactivation and ion selectivity. During large depolarizing voltage pulses, a transient outward current was recorded which appeared to be due to potassium efflux through channel II. When the external concentrations of Ca2+ and Mg2+ were reduced sufficiently, large inward Na currents flowed through both channels I and II. Using divalent-free solutions in cell-attached patch recordings, single-channel currents representing Na influx through channels I and II were recorded. The two types of unitary events could be distinguished on the basis of open time (channel I longer) and conductance (channel I smaller). Blocking events during channel I openings were recorded when micromolar concentrations of Ca2+ or Mg2+ were added to the patch pipette solutions. Slopes of the blocking rate constant vs. concentration gave binding constants of 6.4 X 10(6) M-1 sec-1 for Mg2+ and 4.5 X 10(8) M-1 sec-1 for Ca2+. The Ca2+ block was somewhat relieved at negative potentials, whereas the Mg2+ block was not, suggesting that Ca2+, but not Mg2+, can exit from the binding site toward the cell interior.