Search

Nervous system development is "activity dependent"--activation of neurons controls their development, which controls their activation patterns, which will then influence their further development, and so on. A critical issue is thus the regulation of channel and receptor expression. For nerve cells, the presence of specialized Ca2+-permeable channels in the surface membrane provides a direct link between electrical activity and the intracellular Ca2+ ion concentration ([Ca2+]i), and in many instances [Ca2+]i is thought to link membrane activation and internal biosynthesis. In this context, Ca2+-permeable channels function as "activity sensors," transducing membrane activation by admitting Ca2+ rapidly, locally, and proportionately. In this review, I consider the potential of [Ca2+]i to regulate channel and receptor expression. I emphasize mechanisms by which the Ca2+ concentration of the cytosol and/or the Ca2+ concentrations of membrane-delimited Ca2+ sequestering organelles may influence biosynthetic processes. Here, I use "expression" in the most general sense of referring to the number and location of functional channels and receptors in the plasmalemma; regulation of expression is not limited to transcriptional regulation, but further encompasses translational and posttranslational processes. At the core is the notion of regulation by patterned oscillations in cytosolic [Ca2+], and, in a synchronous or contrapuntal manner, filling and depletion of a series of Ca2+-sequestering organelles--nuclear envelope, endoplasmic reticulum, Golgi, trans-Golgi network, and secretory vesicles--that all also have critical roles in biosynthesis of membrane proteins. These structures provide both an internal Ca2+ regulation and distribution system, and a scaffold for synthesis, targeting, and insertion of channels and receptors.

The pore-forming alpha subunits of voltage-gated potassium channels in neurons and other excitable cells are expressed in association with accessory beta subunits. These subunits both promote insertion of channel complexes into surface membranes and influence their electrophysiological properties. As part of an effort to understand the regulation of voltage-gated potassium channels during development, we cloned the mouse homolog of the rat Kvbeta1 potassium channel subunit. Kvbeta1 subunits are known to associate preferentially with Shaker (Kv1)-related alpha subunits. We then used a digoxigenin-tagged cRNA probe and in situ hybridization techniques to visualize the appearance of Kvbeta1 mRNA transcripts during late embryonic and early neonatal development of the mouse brain. We detected Kvbeta1-specific labeling of cells in hippocampus, cerebral cortex, caudate putamen, colliculus, and cerebellum. In hippocampus, we observed Kvbeta1 mRNA in CA3 pyramidal neurons at the earliest time examined, embryonic day 16 (E16). Between E16 and postnatal day 7 (P7), cell labeling increased uniformly across the pyramidal neurons of Ammon's horn (CA1, CA2, and CA3). Subsequently, between P7 and P22, regional differences characteristic of mature hippocampus appeared-intense labeling of neurons in CA3 and CA1, and less in CA2. In cortex, labeling of cells in the subplate and cortical plate layers was observed at E16. During development, the intensity of this labeling increased, and labeled cells persisted into the adult stage in the deep cortical layer (VIb) formed from subplate neurons. Additional labeling of scattered solitary cells in cortical layers II-VIa emerged between P3 and P7 and was prominent in mature cortex. In caudate putamen, Kvbeta1-labeled cells were observed at P1 and were restricted to the lateral and rostral half of the caudate. During development, labeling expanded caudally and medially and eventually filled the mature caudate putamen. In colliculus, a small population of inferior colliculus cells showed labeling at P7, and additional labeling of scattered cells appeared during development. In superior colliculus, labeling was observed only in the adult deep gray layer. In cerebellum, intense labeling was observed in Purkinje cells at all stages between P1 and adult. Labeling was also seen in granule neurons in the external granule layer at early postnatal stages and in the inner granule layer beginning at P7.

1. The regulation of A-current, one of several transient voltage-gated potassium currents, was studied using whole cell gigaohm seal voltage-clamp techniques on hippocampal pyramidal neurons that were either acutely dissociated from postnatal mouse brain or isolated from embryonic mouse brain and grown in dissociated culture. These neurons also express gamma-aminobutyric acid-A (GABAA) receptors, the activation of which can, under some circumstances, depolarize immature neurons and the dendrites of more mature neurons. 2. Application of GABA (50 microM) reduced the amplitude of A-current when potassium current amplitude was measured during a period of slow and incomplete desensitization of IGABA. A-current was reduced to 67 +/- 9% of control (mean +/- SD, n - 14) in acutely dissociated neurons, and to 64 +/- 11% of control (n = 15) in cultured neurons. Similar A-current reductions were seen in large outside-out membrane patches pulled from somata of cultured neurons, an observation suggesting that imperfect control of membrane voltage was not responsible for A-current inhibition. 3. A-current inhibition exhibited the sensitivity expected of a GABAA-sensitive process. It was mimicked by muscimol and blocked by bicuculline, picrotoxin, and reduction of [Cl-] in the external solution. Baclophen and phaclophen, effective as agonist and antagonist on GABAB receptors, did not affect A-currents or their inhibition. Reduction in extracellular osmolarity (to increase cell swelling as might occur with Cl- entry), or removal of external HCO3- (which might flow inward through GABAA channels and cause local external acidification), did not affect A-current or its inhibition. The mechanisms of inhibition is not clear at present. 4. We suggest that reduced A-current may favor GABA-induced depolarization and consequent activation of voltage-gated calcium channels.

Glial cells have long been recognized as participating in development of neural tissue by providing a scaffolding for migration and by synthesis and secretion of a variety of growth factors and extracellular matrix components. An additional role for glia and other non-neuronal cells in the electrical differentiation of neurons has been suggested by several recent articles, in particular a group that has focused on the potassium currents that influence action potential repolarization and repetitive activity. In this review I present examples of developmental regulation of potassium currents by interactions with non-neuronal cells and discuss some implications of this regulation.

We have investigated the initial appearance of voltage-gated Ca channels and kainate- and NMDA-type glutamate receptors in cultured embryonic mouse hippocampal neurons. The Ca-dependent fluorescence change of the dye fura-2 was used as a sensitive assay for the presence of functional channels and receptors. Expression of functional NMDA receptors was observed on some hippocampal neurons as early as E14. By the equivalent of E15-16, 40-50% of cells responded to Ko-depolarization (50 mM), indicating the presence of functional voltage-gated Ca channels, approximately 20% of cells responded to kainate (50 microM), and just under 20% responded to NMDA (50 microM; in the presence of glycine and strychnine). By the equivalent of the end of the embryonic period 70-80% of cells responded to all 3 stimuli. As approximately 20% of cells in these cultures are glia, these data indicate that by the time of birth close to 100% of neurons express functioning kainate and NMDA receptors, and voltage-gated Ca channels. Increases in [Ca2+]i in embryonic neurons after application of NMDA were sensitive to APV and to external Mg, as are responses in mature neurons. The IC50 for block by external Mg of the [Ca2+]i increase induced by NMDA was 130 microM, and there was a slight positive correlation between the amplitude of the response to NMDA and sensitivity to external Mg.

A model of the membrane currents in embryonic amphibian neurons has been developed in order to investigate the ionic mechanisms underlying developmental changes in excitability. Differentiating amphibian neurons both in situ and in vitro show a gradual change in the ionic sensitivity of their action potential from Ca- to Na-dependent, with an intermediate period in which the action potential shows a mixed ionic sensitivity. The model developed incorporates quantitative descriptions of Na, Ca and voltage-dependent K currents recorded (using whole-cell gigaohm-seal recording techniques) from presumptive neurons isolated from neural plate stage embryos of the axolotl Amblystoma during their in vitro differentiation. The kinetic descriptions of the currents were developed using the form of those of Hodgkin and Huxley. Na and K currents were modeled as voltage-dependent conductances showing m3h and n4 kinetics, respectively. Ca current was modeled (using the constant field relation) as a voltage-dependent permeability with s2 activation kinetics. Ca currents in whole-cell recordings were non-inactivating, and no inactivation mechanism was specified in the model. When modeled current amplitudes and kinetics were scaled as appropriate for a cell at an intermediate stage of differentiation (when the action potential recorded with microelectrodes shows a mixed Na- and Ca-dependence), the action potential predicted by the model showed a similar waveform and ionic dependence. The utility of this model lies in its ability to indicate the activity of each ionic current during an action potential at any developmental stage, and to unambiguously test the effects on neuronal excitability of alterations in the magnitude and/or kinetics of any of the ionic currents present in the membrane.(ABSTRACT TRUNCATED AT 250 WORDS)