Stuart E. Dryer

Na+-activated K+ channels: a new family of large-conductance ion channels

Sodium-activated K+ channels (IK(Na)) are a class of large-conductance ion channels expressed in several populations of vertebrate neurons, mammalian cardiac myocytes and Xenopus oocytes. These channels are activated by the binding of Na+ to sites located on the cytoplasmic face of the channel. The physiological functions of IK(Na) channels have been difficult to ascertain, in part because their activation typically requires Na+ concentrations considerably higher than those that are normally present in the cytosol. However, there is now evidence suggesting that IK(Na) can play a role in the regulation of neuronal excitability, the modulation of the action-potential waveform, and the responses of excitable cells to hypoxia and ischemia.

Innervation and target tissue interactions differentially regulate acetylcholine receptor subunit mRNA levels in developing neurons in situ

Neurons engage in two distinct types of cell-cell interactions: they receive innervation and establish synapses on target tissues. Regulatory events that influence synapse formation and function on developing neurons are largely undefined. We show here that nicotinic acetylcholine receptor (AChR) subunit transcript levels are differentially regulated by innervation and target tissue interactions in developing chick ciliary ganglion neurons in situ. Using ganglia that have developed in the absence of pre- or postganglionic tissues and quantitative RT-PCR, we demonstrate that alpha 3 and beta 4 transcript levels are increased by innervation and target tissue interactions. In contrast, alpha 5 transcript levels are increased by innervation, but target tissues have little effect. Whole-cell ACh-induced currents, used to estimate the number of functional AChRs, change in correlation with alpha 3 and beta 4, but not alpha 5, transcript levels. A model is proposed in which the changes in AChR subunit expression regulate levels of synaptic activity, which is a critical determinant of synapse stabilization and elimination, and neuronal cell death.

Changes in the electrical properties of chick ciliary ganglion neurones during embryonic development.

1. Whole‐cell recording techniques were used to examine the expression of ionic currents in chick ciliary ganglion neurones dissociated acutely at various stages of embryonic development. Currents were also examined in dissociated cells that had been maintained in vitro for several days. 2. Voltage‐activated, tetrodotoxin (TTX)‐sensitive Na+ currents (INa) could be detected in all cells tested between stage 25 and stage 40 (embryonic days 4.5‐14). INa increased in both amplitude and density throughout development, but no obvious changes in kinetics or sensitivity to TTX were observed. 3. High‐threshold Ca2+ currents (ICa) were also detectable between stage 25 and stage 40. ICa increased in both amplitude and density throughout this time. No obvious changes in kinetics or voltage dependence were observed. 4. Delayed rectifier K+ currents (IDR) and A‐currents (IA) could be detected in Ca(2+)‐free salines, and distinguished on the basis of differences in kinetics, voltage dependence, and sensitivity to tetraethylammonium (TEA). IA was either absent, or present at very low densities at stages 26‐30, but showed a sharp increase in density thereafter. In contrast, IDR was detectable as early as stage 25, and did not display a significant increase in density during development. 5. Ca(2+)‐activated K+ currents (IK(Ca)) were either undetectable or present at very low density between stage 26 and stage 30 (embryonic days 5‐9) but showed a large increase in amplitude and density thereafter. 6. Ionic currents were examined in age‐matched cells dissociated acutely on embryonic day 13, or isolated on embryonic day 9 and maintained in vitro for an additional 4 days. Most of the cells maintained in culture for 4 days did not express detectable IK(Ca), and had significantly reduced IA compared to acutely isolated controls. The cultured cells expressed normal densities of IDR, ICa and INa. 7. All ionic currents increased in amplitude during normal embryonic development, and all but IDR increased in density. The largest change in density generally occurred between stages 30 and 40, during which time ciliary ganglion neurones form synapses with target tissues. 8. Isolation of ciliary neurones from the in ovo environment prevented the normal development of IA and IK(Ca), suggesting that the expression of these channels is controlled by one or more extrinsic environmental factors. In contrast, the normal expression of INa, ICa and IDR is not dependent upon extrinsic factors.

Na(+)‐activated K+ channels and voltage‐evoked ionic currents in brain stem and parasympathetic neurones of the chick.

1. Patch‐clamp and computer‐modelling techniques were used to study the activation of Na(+)‐activated K+ channels (IK(Na] in dissociated neurones from the embryonic chick ciliary ganglion and the embryonic chick brain stem. 2. Numerical solutions of diffusion equations suggested that Na+ accumulation as a result of Na+ influx through voltage‐sensitive Na+ channels (INa) is insufficient to allow for alteration in the gating of IK(Na) channels. 3. Whole‐cell recordings using two independent micropipettes were made from chick ciliary‐ganglion neurones. These showed that transient outward currents were present only when there were clear indications of incomplete voltage clamp. 4. Single‐electrode whole‐cell recordings from ciliary‐ganglion neurones showed that transient tetrodotoxin (TTX)‐sensitive outward currents were present, but only when partial TTX blockade produced significant alterations in the kinetics of INa. In cells that were properly voltage clamped, there was no effect of TTX on the kinetics of INa or on voltage‐evoked outward currents. 5. Examination of the relationship between peak INa and the command potential showed that transient outward currents were only present in neurones that showed sharp deviations from the behaviour expected of a cell that is adequately voltage clamped. Transient outward currents were not present in cells that were adequately voltage clamped. 6. Application of TTX to isolated outside‐out patches obtained from ciliary ganglion neurones eliminated voltage‐evoked inward currents but had no effect on outward currents. 7. Isolated inside‐out patches obtained from ciliary‐ganglion neurones did not contain IK(Na) channels. These patches usually contained Ca(2+)‐activated K+ channels (IK(Ca] with a unitary conductance of around 200 pS when [K+]o = 150 mM and [K+]i = 75 mM. 8. Two‐electrode whole‐cell recordings from cultured brain stem neurones showed that transient outward currents were present only when there were clear indications of incomplete voltage clamp. 9. Application of TTX caused blockade of inward but not outward currents in brain stem neurones voltage clamped with a single whole‐cell pipette. TTX had no effect on the kinetics of INa. Application of TTX to outside‐out patches isolated from the same cells blocked only the inward currents. 10. Isolated inside‐out patches obtained from brain stem neurones contained IK(Na) channels that could be activated by exposure of the cytoplasmic face of the patch membrane to 75 mM‐Na+. These channels had a predominant conductance state of around 100 pS when [K+]o = 150 mM and [K+]i = 75 mM. The IK(Na) channels were not activated by 1 mM‐Ca2+.(ABSTRACT TRUNCATED AT 400 WORDS)

Target tissues and innervation regulate the characteristics of K+ currents in chick ciliary ganglion neurons developing in situ

The expression of appropriate ensembles of ionic channels is necessary for the differentiation and normal function of vertebrate neurons. Cell- cell interactions may regulate the expression and properties of ionic channels in embryonic neurons. Previous studies have shown that the expression of A-type K+ channels (IA) and Ca2+-activated K+ channels (lK[Ca]) is abnormal in chick ciliary ganglion neurons developing in vitro in the absence of normal cell-cell interactions. Other voltage- activated currents develop normally under these conditions. The present studies were designed to establish the role of the target tissues and the preganglionic innervation in regulating the expression of these currents in embryonic chick ciliary ganglion neurons developing in situ. Surgical manipulations were used to remove the developing optic vesicle, which contains the target tissues, the mid-dorsal region of the midbrain primordium, which contains the preganglionic nucleus, or both, all prior to the formation of the ciliary ganglion. IA and IK[Ca] were then examined in acutely isolated neurons that developed in ovo in the presence (OV+) or absence (OV-) of the normal target tissues, in the presence (MB+) or absence (MB-) of preganglionic innervation, and in the absence of both preganglionic innervation and target tissues (OV- /MB-). The amplitude of IA was unaffected by the operations. However, the activation and inactivation kinetics of IA were two- to threefold faster in OV- or OV-/MB- cells compared to neurons isolated from control OV+ ganglia at embryonic days 11–14 (E11-E14). There were no changes in the voltage dependence of activation or steady-state inactivation, or in the time course of recovery from inactivation. By contrast, neurons isolated from MB- ganglia expressed an IA with amplitude, voltage dependence, and kinetics that were indistinguishable from those of control MB+ and OV+ ganglia. Therefore, interactions with target tissues in the eye play a role in determining the characteristics of IA in developing ciliary ganglion neurons, whereas preganglionic innervation does not. Furthermore, the amplitude of IK[Ca] was reduced by 90–100% in OV-, MB-, and OV-/MB- neurons isolated at E12-E14 as compared to MB+ and OV+ controls. Voltage-activated Ca2+ currents were present at normal amplitudes in all of these neurons. Thus, the expression of IK[Ca] in chick ciliary ganglion neurons is regulated by both target tissue interactions and preganglionic innervation. Therefore, cell-cell interactions are necessary for the expression of a normal ensemble of ionic channels in chick ciliary ganglion neurons developing in situ.

Characteristics of multiple Ca(2+)‐activated K+ channels in acutely dissociated chick ciliary‐ganglion neurones.

1. Whole‐cell and single‐channel recordings were used to characterize Ca(2+)‐activated K+ channels (IK(Ca)) in acutely dissociated chick‐ganglion neurones. 2. Application of depolarizing voltage steps resulted in outward currents that could be separated according to their dependence on external Ca2+ and/or holding potential. IK(Ca) was the only outward current that could be evoked from holding potentials of ‐50 mV or less. IK(Ca) was eliminated by bath application of Ca(2+)‐free salines. A voltage‐dependent outward current (IK(V)) could be evoked from more negative holding potentials in Ca(2+)‐free salines. IK(V) was only partially blocked by as much as 30 mM‐tetraethylammonium (TEA). 3. Tail currents associated with IK(Ca) reversed close to the K+ equilibrium potential (EK). IK(Ca) tail currents appeared sigmoidal, but the falling phase of the tail currents could be fitted with exponential curves that decayed faster at more negative membrane potentials. 4. IK(Ca) was blocked completely and reversibly by 10 mM‐TEA. IK(Ca) was substantially reduced (80‐90%) by as little as 1 mM‐TEA. 5. Total IK(Ca) was reduced but not eliminated by saturating concentrations of apamin (200 nM). This blockade was not reversible with up to 30 min of washing. Application of 100 microM‐d‐tubocurare (dTC) also produced a partial blockade of total IK(Ca). 6. Whole‐cell current‐clamp recordings showed that IK(Ca) contributed to the late phases of spike repolarization and was the dominant current flowing during the spike after‐hyperpolarization (AHP). Application of 200 nM‐apamin caused a reduction in the duration of the AHP. This reduction was best seen when multiple spikes were evoked by prolonged (20‐50 ms) injections of depolarizing current. 7. Three distinct types of IK(Ca) channels could be observed in inside‐out patches in the presence of free Ca2+ concentrations of 2 x 10(‐7) M, but not in the presence of free Ca2+ at concentrations of less than 10(‐9) M. These had unitary chord conductances of 190 pS (i1), 110 pS (i2), and 45 pS (i3) with [K+]o = 150 mM and [K+]i = 75 mM. Each of these three channels had distinct kinetic properties. The 45 pS channel was most sensitive to activation by Ca2+ and could be detected at free Ca2+ concentrations as low as 10(‐8) M. 8. All three IK(Ca) channels could be observed in inside‐out patches held at membrane potentials where IK(V) was fully inactivated. Application of 10 mM‐TEA caused a complete block of IK(Ca) channels in outside‐out patches.(ABSTRACT TRUNCATED AT 400 WORDS)

Regulation of A-currents by cell-cell interactions and neurotrophic factors in developing chick parasympathetic neurones

1. The developmental regulation of ion channel expression was studied in parasympathetic neurones isolated from the chick ciliary ganglion. Whole‐cell patch clamp recordings were made from ciliary ganglion neurones that were removed from the embryo on the ninth embryonic day (E9) and maintained in dissociated cell culture for an additional 4 days. Previous studies have shown that the expression of a transient voltage‐activated K+ current (IA) is regulated by unidentified environmental stimuli during these developmental stages. 2. The effect of interactions between neurones and target tissue on the expression of IA was tested by co‐culturing ciliary ganglion neurones with chick striated muscle cells. Neurones from the nerve‐muscle co‐cultures expressed normal amplitudes of IA, but the neurones did not express normal levels of IA when they were plated onto lysed muscle fibres. 3. The effect of interactions between ganglionic neurones and non‐neuronal ganglionic cells was tested by culturing ganglia as explants rather than as dissociated cells. Neurones isolated from the explant cultures did not express normal levels of IA. Similarly, when dissociated ganglionic neurones were co‐cultured with fibroblasts isolated from embryonic chick skin, they did not express normal amplitudes of IA. 4. Chronic depolarization caused by growing ciliary ganglion neurones in the presence of elevated K+ concentrations did not allow for the normal expression of IA, although it did promote the survival of these neurones in vitro. 5. Addition of 40 ng ml‐1 of recombinant human ciliary neurotrophic factor (CNTF) or basic fibroblast growth factor (bFGF) to the cell culture medium had no effect on IA expression in developing chick ciliary ganglion neurones. However, 40 ng ml‐1 of acidic fibroblast growth factor (aFGF) stimulated the expression of IA. All trophic factors promoted the growth and survival of ciliary ganglion neurones in vitro. 6. Dissociated ciliary ganglion neurones were maintained in a culture medium containing an aqueous extract of the whole brain. Neurones developing under these conditions expressed normal levels of IA. The stimulatory activity of the brain extract was destroyed by heating. 7. The expression of IA in chick ciliary ganglion neurones developing in vitro can be regulated by soluble growth factors and by interactions with certain other cell types. Similar interactions may regulate the expression of IA in ciliary ganglion neurones developing in situ.

Intracellular free Ca2+ in dissociated cells of the chick pineal gland: regulation by membrane depolarization, second messengers and neuromodulators, and evidence for release of intracellular Ca2+ stores

Abstract The regulation of intracellular free Ca 2+ concentration was examined in single dissociated chick pineal cells using the fura-2 technique. ∼ 10% of cells examined exhibited spontaneous Ca 2+ oscillations while the rest were quiescent. Application of salines containing 80 mM KCl evoked large increases in intracellular free Ca 2+ that were dependent upon external Ca 2+ ions. These responses were inhibited by 10 μM nifedipine indicating involvement of L-type Ca 2+ channels. Application of the tumor promoter thapsigargin (2 μM) evoked increases in intracellular free Ca 2+. These responses could be observed in the absence of external Ca 2+ indicating mobilization of internal stores. In the absence of external Ca 2+, the responses to thapsigargin gradually decayed due to depletion of internal Ca 2+ pools. A subsequent exposure to saline containing 5.8 mM CaCl 2 caused a rapid increase in intracellular Ca 2+ that was consistently larger than the peak response to thapsigargin. Applicatio of 100 nM vasoactive intestinal peptide (VIP), a neurohormone that stimulates melatonin secretion from pineal cells, induced a sustained increase in intracellular free Ca 2+ in a subpopulation of cells. In a small number of cells, VIP evoked Ca 2+ oscillations. Approximately half of the cells examined showed no response to VIP. Application of 200 μM norepinephrine, which inhibits melatonin secretion from the chick pineal, had no effect on intracellular free Ca 2+ in any quiescent or spontaneously oscillating cells. Application of 5 mM 8-Br-cAMP evoked sustained increases in intracellular Ca 2+ Similar effects were obtained with the phosphodiesterase inhibitors papaverine (50 μM) or isobutylmethylxanthine (100 μM). Application of 200 nM forskolin, an activator of adenylate cyclase, evoked increases in intracellular free Ca 2+ that could be detected in the presence of 10 μM nifedipine. The responses to forskolin gradually in Ca 2+-free external salines due to depletion of intracellular Ca 2+ stores. Subsequent exposure to external Ca 2+ caused a rapidly developing increase in intracellular Ca 2+ that was larger than the peak response to forskolin. These results indicate that the regulation of intracellular free Ca 2+ in chick pineal cells is complex. These cells exhibit Ca 2+ oscillations and can mobilize both external and internal Ca 2+ pools. Agents that increase intracellular cAMP cause mobilization of internal Ca 2+ stores, possibly secondary messenger systems. Chick pineal cells, like many other cell types, possess mechanisms to allow for refilling of depleted internal Ca 2+ stores. These results suggest New mechanisms for the regulation of melatonin synthesis and secretion and possible sites of action for the intrinsic circadian oscillator.

Melatonin Receptors in the Spinal Cord

The pineal hormone, melatonin, plays an important role in the regulation of diurnal and seasonal rhythms in animals. In addition to the well established actions on the brain, the possibility of a direct melatonin action on the spinal cord has to be considered. In our laboratory, we have obtained data suggesting that melatonin receptors are present in the spinal cords of birds and mammals. Using radioreceptor binding and quantitative autoradiography assays with 2-[125I]iodomelatonin as the specific melatonin agonist, melatonin binding sites have been demonstrated in the rabbit and chicken spinal cords. These sites are saturable, reversible, specific, guanosine nucleotide-sensitive, of picomolar affinity and femtomolar density. The linearity of Scatchard plots of saturation data and the unity of Hill coefficients indicate that a single class of melatonin binding sites is present in the spinal cord membranes studied. The picomolar affinity of these sites is in line with the circulating levels of melatonin in these animals suggesting that these sites are physiologically relevant. Autoradiography studies in the rabbit spinal cord show that melatonin binding sites are localized in the central gray substance (lamina X). In the chicken spinal cord, these binding sites are localized in dorsal gray horns (laminae I-V) and lamina X. As lamina X and laminae I-II have similar functions, melatonin may have comparable roles in the chicken and rabbit spinal cords. Moreover, in the chicken spinal cord, the density of 2-[125I]iodomelatonin binding in the lumbar segment was significantly higher than those of the cervical and thoracic segments. The densities of these binding sites changed with environmental manipulations. When chickens were adapted to a 12L/12D photoperiod and sacrificed at mid-light and mid-dark, there was a significant diurnal variation in the density (maximum number of binding sites; Bmax) of melatonin binding sites in the spinal cord. After constant light treatment or pinealectomy, the Bmax of melatonin receptors in the chicken spinal cord increased significantly in the subjective mid-dark period. Moreover, there was an age-related decrease in the 2-[125I]iodomelatonin binding to the chicken spinal cord. Our results suggest that melatonin receptors in the chicken spinal cord are regulated by environmental lighting and change with development. These receptors may play an important role in the chronobiology of spinal cord function. The biological responses of melatonin on spinal cords have also been demonstrated in vitro. Melatonin decreased the forskolin-stimulated cAMP production in the chicken spinal cord explant. Preincubation with pertussis toxin blocked the melatonin effect. Our results suggest that melatonin receptors in the chicken spinal cord are linked to the adenylate cyclase via a pertussis toxin-sensitive G protein and that melatonin binding sites in spinal cords are melatonin receptors with biological functions. These receptors may be involved in the regulation of spinal cord functions related to sensory transmission, visceral reflexes and autonomic activities.

Functional expression of A-currents in embryonic chick sympathetic neurones during development in situ and in vitro.

1. The functional expression of transient voltage‐activated K+ currents (IA) was examined using whole‐cell recording techniques in embryonic chick sympathetic ganglion neurones that developed in situ and under various growth conditions in vitro. 2. The density of IA increased dramatically during development in sympathetic neurones isolated acutely between embryonic days 7 and 20 (E7‐E20). The time course of IA inactivation became significantly faster between E7 and E13. With these protocols, neuronal differentiation and development occurred entirely in situ. 3. Sympathetic neurones isolated at E9 and maintained in vitro for 4 days did not express a normal IA compared to neurones isolated acutely at E13. Those neurones that were in physical contact with other neurones expressed normal densities of IA, but the resulting inactivation kinetics were abnormally slow. Sympathetic neurones that were cultured on the membrane fragments of lysed neurones expressed normal densities of IA even when they failed to make visible connections with other viable neurones, but the resulting inactivation kinetics were abnormally slow. Those cultured neurones that were not in physical contact with other cells or their membranes had markedly reduced densities of IA with abnormally slow inactivation kinetics. 4. Application of 5‐100 ng ml‐12.5 S nerve growth factor by itself did not promote normal A density of kinetics in E9 sympathetic neurones cultured for 4 days. 5. Sympathetic neurones that developed in vitro in physical contact with ventral spinal cord explants, cardiac myocytes or aortic smooth muscle cells expressed normal densities of IA, but the inactivation kinetics were abnormally slow. Cell culture media conditioned by these tissues failed to promote normal IA expression. Sympathetic neurones cultured as explants or maintained under depolarizing conditions did not express a normal IA. 6. Embryonic chick sympathetic neurones exhibit developmental changes in the density and kinetics of IA that can be regulated independently by extrinsic environmental factors including interactions with insoluble components of the plasma membranes of some cells.