William J. Moody

Blocking effects of barium and hydrogen ions on the potassium current during anomalous rectification in the starfish egg.

1. The blocking effects of Ba+ and H+ on the inward K current during anomalous rectification of the giant egg membrane of the starfish, Mediaster aequalis, were studied using voltage clamp techniques. 2. External Ba2+ at a low concentration (10‐‐100 micron) suppresses the inward K current; the extent of suppression, expressed as the ratio of currents with and without Ba2+, can be described by a conventional bimolecular adsorption isotherm, K/(K + [Ba2+]o), K being an apparent dissociation constant. 3. The dissociation constant, K, decreases as the membrane potential V becomes more negative and can be expressed by K(V) = K(0) exp (zmuFV/RT), where K(0) is the K at V = 0, z is the charge of the blocking ion, and mu is a parameter for the membrane potential dependence of Ba2+ blockage. The value of mu ranges between 0.64 and 0.68. 4. Upon a sudden change in membrane potential the change in the blocking effect of Ba2+ follows first order kinetics; the forward rate constant is membrane‐potential‐dependent whereas the backward constant is potential‐independent. 5. The blocking effect of Ba2+ appears to be independent of the activation of K channels during anomalous rectification. 6. The blocking effect of Ba2+ depends on V alone, in contrast to the activation of the K channel during anomalous rectification which depends on V‐‐VK. 7. In these respects, the effect of Ba2+ is equivalent to the introduction of inactivation into the anomalous rectification. 8. SI2+ and Ca2+ show small but observable blocking effects only at much higher concentrations (about 10‐‐20 mM). 9. The inward K current is suppressed when the external pH is reduced below 6.0. The blocking effect of H+ shows no significant potential dependence. The concentration dependence suggests that three H+ ions simultaneously titrate the acidic groups of each channel (pK = 5.3‐‐5.4). 10. The implications of these results are discussed in terms of molecular models of the potassium channel of anomalous rectification and possible mechanisms of K channel inactivation.

Spontaneous, synchronous electrical activity in neonatal mouse cortical neurones

Spontaneous [Ca2+]i transients were measured in the mouse neocortex from embryonic day 16 (E16) to postnatal day 6 (P6). On the day of birth (P0), cortical neurones generated widespread, highly synchronous [Ca2+]i transients over large areas. On average, 52% of neurones participated in these transients, and in 20% of slices, an average of 80% participated. These transients were blocked by TTX and nifedipine, indicating that they resulted from Ca2+ influx during electrical activity, and occurred at a mean frequency of 0.91 min−1. The occurrence of this activity was highly centred at P0: at E16 and P2 an average of only 15% and 24% of neurones, respectively, participated in synchronous transients, and they occurred at much lower frequencies at both E16 and P2 than at P0. The overall frequency of [Ca2+]i transients in individual cells did not change between E16 and P2, just the degree of their synchronicity. The onset of this spontaneous, synchronous activity correlated with a large increase in Na+ current density that occurred just before P0, and its cessation with a large decrease in resting resistance that occurred just after P2. This widespread, synchronous activity may serve a variety of functions in the neonatal nervous system.

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.

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.

The self‐regulating nature of spontaneous synchronized activity in developing mouse cortical neurones

Waves of spontaneous electrical activity that are highly synchronized across large populations of neurones occur throughout the developing mammalian central nervous system. The stages at which this activity occurs are tightly regulated to allow activity‐dependent developmental programmes to be initiated correctly. What determines the onset and cessation of spontaneous synchronous activity (SSA) in a particular region of the nervous system, however, remains unclear. We have tested the hypothesis that activity itself triggers developmental changes in intrinsic and circuit properties that determine the stages at which SSA occurs. To do this we exposed cultured slices of mouse neocortex to tetrodotoxin (TTX) to block SSA, which normally occurs between embryonic day 17 (E17) and postnatal day 3 (P3). In control cultured slices, SSA rarely occurs after P3. In TTX‐treated slices, however, SSA was generated from P3 (the day of TTX removal) until at least P10. This indicates that in the absence of spontaneous activity, the mechanisms that normally determine the timing of SSA are not initiated, and that a compensatory response occurs that shifts the time of SSA occurrence to later developmental stages.

A nonselective cation channel activated by membrane deformation in oocytes of the ascidianBoltenia villosa

SummaryCell-attached patch clamp recordings from unfertilized oocytes of the ascidianBoltenia villosa reveal an ion channel which is activated by mechanical deformation of the membrane. These channels are seen when suction is applied to the patch pipette, but not in the absence of suction or during voltage steps. The estimated density of these stretch-activated channels is about 1.5/μm2, a figure equal to or greater than the density of known voltage-dependent channels in the oocyte. Ion substitution experiments done with combined whole-cell and attached patch recording, so absolute potentials are known, indicate that the channel passes Na+, Ca2+ and K+, but not Cl−. The channel has at least two open and two closed states, with the rate constant that leaves the longer-lived closed state being the primary site of stretch sensitivity. External Ca2+ concentration affects channel kinetics: at low calcium levels, long openings predominate, whereas at high calcium virtually all openings are to the short-lived open state. In multiple channel patches, the response to a step change in suction is highly phasic, with channel open probability decreasing over several hundred milliseconds to a nonzero steady-state level after an initial rapid increase. This channel may play a role in the physiological response of cells of the early embryo to the membrane strains associated with morphogenetic events.

Early Expression of Sodium Channel Transcripts and Sodium Current by Cajal-Retzius Cells in the Preplate of the Embryonic Mouse Neocortex

In mouse, the first neurons are generated at embryonic day (E) 12 and form the preplate (PP), which contains a mix of future marginal zone cells, including Cajal-Retzius cells, and subplate cells. To detect developmental changes in channel populations in these earliest-generated neurons of the cerebral cortex, we studied the electrophysiological properties of proliferative cells of the ventricular zone and postmitotic neurons of the PP at E12 and E13, using whole-cell patch-clamp recordings. We found an inward sodium current in 55% of PP cells. To determine whether sodium currents occur in a specific cell type, we stained recorded cells with an antibody for calretinin, a calcium-binding protein found specifically in Cajal-Retzius cells. All calretinin-positive cells had sodium currents, although so did some calretinin-negative cells. To correlate the Na current expression to Na channel gene expression with the Cajal-Retzius cell phenotype, we performed single-cell reverse transcription-PCR on patch-clamp recorded cells to detect expression of the Cajal-Retzius cell marker reelin and the Na channel isoforms SCN 1, 2, and 3. These results showed that virtually all Cajal-Retzius cells (97%), as judged by reelin expression, express the SCN transcript identified as the SCN3 isoform. Of these, 41% presented a functional Na current. There is, however, a substantial SCN-positive population in the PP (27% of SCN-positive cells) that does not express reelin. These results raise the possibility that populations of pioneer neurons of the PP, including Cajal-Retzius cells, gain neuronal physiological properties early in development via expression of the Nav1.3 (SCN3) Na channel isoform.

Changes in sodium, calcium and potassium currents during early embryonic development of the ascidian Boltenia villosa.

1. The whole‐cell variation of the patch clamp was used to study ion channel properties in the unfertilized oocyte, and in surgically isolated blastomeres from 2‐, 4‐, and 8‐cell embryos of the ascidian, Boltenia villosa. 2. The unfertilized oocyte has three major voltage‐dependent currents: a transient, inward Na+ current; a transient, inward Ca2+ current; and an inwardly rectifying K+ current. 3. The total surface area of the embryo, either measured by capacitance or calculated from cell diameters, increased about 2.5‐fold between fertilization and the 8‐cell stage. 4. The Na+ current almost completely disappeared from the embryo by the time of first cleavage and was undetectable in any of the blastomeres at the 8‐cell stage. This loss was too large to be explained by the dilution of channels in the oocyte due to newly added membrane. 5. Both the Ca2+ current and the inwardly rectifying K+ current were maintained at constant or slightly increased density through the first three cleavage cycles. This suggests that these channels are added along with new membrane during these stages. 6. No differences in mean current densities of blastomeres of different developmental fates were detected through the 8‐cell stage. 7. Continuous recordings in single egg cells between fertilization and first cleavage, using two‐microelectrode voltage clamp, revealed the increase in capacitance, Ca2+ current amplitude, and K+ current amplitude, and the loss of Na+ current predicted from the blastomere studies.

5 The Development of Voltage-Gated Ion Channels and Its Relation to Activity-Dependent Developmental 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.