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.

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.

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.

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)

Properties of Ca2+ currents in acutely dissociated neurons of the chick ciliary ganglion: inhibition by somatostatin-14 and somatostatin-28.

Whole-cell voltage-clamp recordings were made from acutely dissociated neurons obtained from the embryonic chick ciliary ganglion. Recording pipettes were filled with salines containing 120 mM CsCl or 120 mM tetraethylammonium-Cl. Application of depolarizing voltage commands evoked L-type Ca2+ currents and, at voltages positive to 0 mV, an unidentified cationic conductance. The unidentified cationic conductances made the Ca2+ currents appear to undergo voltage-dependent inactivation and made a large contribution to tail currents present during repolarizing voltage steps. Ca2+ Ca2+ currents showed little or no sign of inactivation and did not reverse at voltages up to +60 mV. Application of somatostatin-14 or somatostatin-28 produced a reversible inhibition of Ca2+ currents in virtually all cells, regardless of size. Somatostatin-28 (1-14) was inactive. The effects of somatostatin-14 and somatostatin-28 were attenuated by pretreatment with pertussis toxin, suggesting a role for G-proteins in mediating the response. Somatostatin-14 and somatostatin-28 had no effect on voltage-dependent K+ currents. The results suggest that somatostatin peptides modulate the motor output of the chick ciliary ganglion.