Calcium signals activated by arachidonic acid in embryonic chick ciliary ganglion neurons

Arachidonic acid (AA, 20:4) has been reported to modulate a variety of calcium-permeable ionic channels, both in the plasma membrane and in the endoplasmic reticulum. We have studied the effects of AA on calcium signaling in a well-characterized model of developing peripheral neurons, embryonic chick ciliary ganglion neurons in culture. When given at low non-micellar concentrations (5 microM), in the majority of cells AA directly activated a delayed and long-lasting increase in [Ca2+]i, involving both the cytoplasm and the nucleoplasm, that was completely reversed by abolition of extracellular calcium. Other fatty acids (FAs), either saturated like arachidic acid (20:0), or unsaturated like linoleic (18:2) and docosahexaenoic acid (22:6), shared its ability to activate calcium influx. This entry was not suppressed by voltage-dependent calcium channel inhibitors omega-conotoxin and nifedipine, by the voltage-independent calcium channel antagonist LOE-908, by pre-treatment with blockers of AA metabolic pathways or with pertussis toxin. The arachidonate-activated calcium pathway was permeable to Mn2+ and blocked by La3+, Gd3+ and Ni2+. In a neuronal subpopulation, AA at the same concentration was also able to elicit calcium release from thapsigargin-sensitive intracellular stores; we provide evidence that cytochrome P450 epoxygenase is involved in this process.     

Nonselective cation conductance activated by muscarinic and purinergic receptors in rat spiral ganglion neurons

The present studycharacterizes the ionic conductances activated by acetylcholine (ACh)and ATP, two candidate neuromodulators, in isolated spiral ganglionneurons (SGNs). Brief application (1 s) of ACh evoked in adose-dependent manner (EC₅₀ = 4.1 µM) a reversibleinward current with a long latency (average 1.3 s), at holdingpotential (V_h) = 50 mV. This current wasreversibly blocked by atropine and mimicked by muscarine. Applicationof ATP also evoked a reversible inward current atV_h = 50 mV, but the current showed twocomponents. A fast component with a short latency was largely reducedwhen N-methyl-D-glucamine (NMDG) replaced extracellular sodium, implying a P2X-like ionotropic conductance. Thesecond component had a longer latency (average 1.1 s) and waspresumably activated by metabotropic P2Y-like receptors. The secondcomponent of ATP-evoked current shared similar characteristics with theresponses evoked by ACh: the current reversed near 0 mV, displayedinward rectification, could be carried by NMDG, and was insensitive toextracellular and intracellular calcium. This ACh-/ATP-evokedconductance was reversibly inhibited by preapplication of ionomycin.These results suggest that muscarinic receptors and purinergicmetabotropic receptors activate a similar large nonselective cationconductance via a common intracellular pathway in SGNs, a candidatemechanism to regulate neuronal excitability of SGNs.

     

Effects of preganglionic denervation and postganglionic axotomy on acetylcholine receptors in the chick ciliary ganglion

The regulation of nicotinic acetylcholine receptors (AChRs) in chick ciliary ganglia was examined by using a radiolabeled anti-AChR mAb to quantitate the amount of receptor in ganglion detergent extracts after preganglionic denervation or postganglionic axotomy. Surgical transection of the preganglionic input to the ciliary ganglion in newly hatched chicks caused a threefold reduction in the total number of AChRs within 10 d compared with that present in unoperated contralateral control ganglia. Surgical transection of both the choroid and ciliary nerves emerging from the ciliary ganglion in newly hatched chicks to establish postganglionic axotomy led to a nearly 10-fold reduction in AChRs within 5 d compared with unoperated contralateral ganglia. The declines were specific since they could not be accounted for by changes in ganglionic protein or by decreases in neuronal survival or size. Light microscopy revealed no gross morphological differences between neurons in operated and control ganglia. A second membrane component of cholinergic relevance on chick ciliary ganglion neurons is the alpha-bungarotoxin (alpha-Bgt)-binding component. The alpha-Bgt-binding component also declined in number after either postganglionic axotomy or preganglionic denervation, but appeared to do so with a more rapid time course than did ganglionic AChRs. The results imply that cell-cell interactions in vivo specifically regulate both the number of AChRs and the number of alpha-Bgt-binding components in the ganglion. Regulation of these neuronal cholinergic membrane components clearly differs from that previously described for muscle AChRs.     

Variation among acetylcholine receptor clusters induced by ciliary ganglion neurons in vitro

We have examined the variation in receptor density and area among neurite-associated acetylcholine receptor patches (NARPs) induced by chick ciliary ganglion neurons on nearby myotubes in vitro. Quantitative analysis of rhodamine-alpha-bungarotoxin (RBTX) NARPs revealed that about 15% of the NARPs were "outstanding" in terms of size (greater than 60 micron 2) and fluorescence intensity (greater than 100 units on a 0-255 scale). The total number of receptors at different NARPs ranged over 3 orders of magnitude. It is likely that variation in NARP size and intensity reflects regional variation in the ability of myotubes to respond to the neuronal influence because (1) no gradient in NARP size or intensity with distance from the soma was evident; (2) the intensities and areas of uninnervated receptor clusters (hot spots) were similar to those of NARPs; (3) acetylcholinesterase was present at the same proportion of hot spots and NARPs at all times examined. We found no physiological or morphological evidence that outstanding NARPs were more effective sites of transmitter release. Outstanding NARPs were restricted to the longest neurite of individual neurons, so they may signal trophic interactions of the sort that promote neurite outgrowth and survival.     

Single channel studies of inward rectifier potassium channel regulation by muscarinic acetylcholine receptors

Negative regulation of the heartbeat rate involves the activation of an inwardly rectifying potassium current (I(KACh)) by G protein-coupled receptors such as the m2 muscarinic acetylcholine receptor. Recent studies have shown that this process involves the direct binding of G(betagamma) subunits to the NH(2)- and COOH-terminal cytoplasmic domains of the proteins termed GIRK1 and GIRK4 (Kir3.1 and Kir3.4/CIR), which mediate I(KACh). Because of the very low basal activity of native I(KACh), it has been difficult to determine the single channel effect of G(betagamma) subunit binding on I(KACh) activity. Through analysis of a novel G protein-activated chimeric inward rectifier channel that displays increased basal activity relative to I(KACh), we find that single channel activation can be explained by a G protein-dependent shift in the equilibrium of open channel transitions in favor of a bursting state of channel activity over a long-lived closed state.     

Surface AChE in the chick ciliary ganglion neurons: Ultrastructural localization and possible relations to α-bungarotoxin receptors

The localization of acetylcholinesterase activity in the chick ciliary ganglion was investigated by ultrastructural cytochemistry. Both ganglionic cell populations, i.e. the ciliary and the choroid neurons, showed similar distribution patterns of the enzymic activity in the cytoplasm as well as at the neuronal surface. As indicated by specific inhibition tests, the whole enzymic activity was attributable to specific acetylcholinesterase. While the endocellular activity was mainly localized in the rough endoplasmic reticulum, the surface activity occurred at postsynaptic level and at extrasynaptic areas, where the neuronal membrane comes into contact with the plasma membrane of the satellite cell (boundary neuron-satellite cell). Enzymic activity also uniformly occurred at the surface of preganglionic nerve terminals. The surface localization of specific acetylcholinesterase recalls that recently described for α-bungarotoxin receptors, which suggests that acetylcholinesterase and α-bungarotoxin receptors can be distributed together, not only at postsynaptic level but also in extrasynaptic neuronal areas and at presynaptic level. The possibility that α-bungarotoxin receptors and acetylcholinesterase form a ·receptive’ system not engaged in ganglionic transmission and not exclusively confined to postsynaptic level is discussed in relation to the electrophysiological data existing in literature.     

3H]acetylcholine synthesis in cultured ciliary ganglion neurons: effects of myotube membranes

Avian ciliary ganglion neurons in cell culture were examined for the capacity to synthesize acetylcholine (ACh) from the exogenously supplied precursor, choline. Relevant kinetic parameters of the ACh synthetic system in cultured neurons were found to be virtually the same as those of the ganglionic terminals in the intact iris. Neurons were cultured in the presence of and allowed to innervate pectoral muscle; this results in an capacity for ACh synthesis. In particular, the ability to increase ACh synthesis upon demand after stimulation is affected by interaction with the target. This effect is shown to be an acceleration of the maturation of the cultured neurons. Lysed and washed membrane remnants of the muscle target were able to duplicate, in part, this effect of live target tissue on neuronal transmitter metabolism. Culture medium conditioned by muscle, and by the membrane remnants of muscle, was without significant effect. Thus, substances secreted into the medium do not play a major role in this interaction. Neurons cultured with either muscle or muscle membrane remnants formed large, elongate structures on the target membrane surface. These were not seen in the absence of the target at the times examined. This morphological difference in terminal-like structures may parallel the developmental increases in size and vesicular content of ciliary ganglion nerve terminals in the chick iris, and may relate to the increased ACh synthetic activity. The results suggest that direct contact with an appropriate target membrane has a profound, retrograde influence upon neuronal metabolic and morphological maturation.     

Overexpression of ciliary neurotrophic factor in vivo rescues chick ciliary ganglion neurons from cell death

Ciliary ganglion (CG) neurons undergo target-dependent cell death during embryonic development. Although ciliary neurotrophic factor (CNTF) was identified in vitro by its ability to support the survival of chick CG neurons, its function as a target-derived neurotrophic factor has been questioned by those working on mammalian-derived forms of CNTF. We have purified and cloned a chicken CNTF [chCNTF; formerly growth-promoting activity (GPA)] that is expressed in CG targets during the period of cell death and is secreted by cells transfected with chCNTF. In the present study we used a retroviral vector, RCASBP(A), to overexpress chCNTF in CG target tissues. Elevation of chCNTF biological activity three- to fourfold in the embryonic eye rescued an average of 31% of the neurons that would have normally died in vivo. In some individuals, nearly all of the neurons were rescued. ChCNTF had no effect on the number of neurons observed prior to cell death, nor were there any deleterious effects of either viral infection or overexpression of CNTF. These results show that chCNTF is able to function in vivo as a trophic factor for CG neurons, and suggest that limited availability of trophic support is one of the factors regulating CG neuron survival during development. © 1998 John Wiley & Sons, Inc. J Neurobiol 34: 283–293, 1998     

Disulfide bonds in acetylcholine receptors of frog sympathetic ganglion neurons

Changes in responses of frog sympathetic ganglion neurons to perfusion with cholinomimetics were studied during modification of acetylcholine receptors by dithiothreitol and ferricyanide. Perfusion with dithiothreitol suppressed responses to carbachol, suberyldicholine, and 5-methylfurmethide, whereas subsequent perfusion with ferricyanide partly restored responses to suberyldicholine but suppressed responses to 5-methylfurmethide. Acetylcholine and tetramethylammonium, used as protectors, protected nicotinic and muscarinic receptors against the action of dithiothreitol, but acetylcholine was more effective than tetramethylammonium for nicotinic acetylcholine receptors. It is suggested that disulfide bonds, some of them located in the anionic centers of the receptors, are present in the recognition sites of acetylcholine receptors of the frog sympathetic ganglion.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 11, No. 6, pp. 593–600, November–December, 1979.     

Regulation of axon arborization pattern in the developing chick ciliary ganglion: Possible involvement of caspase 3

During a certain critical period in the development of the central and peripheral nervous systems, axonal branches and synapses are massively reorganized to form mature connections. In this process, neurons search their appropriate targets, expanding and/or retracting their axons. Recent work suggested that the caspase superfamily regulates the axon morphology. Here, we tested the hypothesis that caspase 3, which is one of the major executioners in apoptotic cell death, is involved in regulating the axon arborization. The embryonic chicken ciliary ganglion was used as a model system of synapse reorganization. A dominant negative mutant of caspase‐3 precursor (C3DN) was made and overexpressed in presynaptic neurons in the midbrain to interfere with the intrinsic caspase‐3 activity using an in ovo electroporation method. The axon arborization pattern was 3‐dimensionally and quantitatively analyzed in the ciliary ganglion. The overexpression of C3DN significantly reduced the number of branching points, the branch order and the complexity index, whereas it significantly elongated the terminal branches at E6. It also increased the internodal distance significantly at E8. But, these effects were negligible at E10 or later. During E6–8, there appeared to be a dynamic balance in the axon arborization pattern between the “targeting” mode, which is accompanied by elongation of terminal branches and the pruning of collateral branches, and the “pathfinding” mode, which is accompanied by the retraction of terminal branches and the sprouting of new collateral branches. The local and transient activation of caspase 3 could direct the balance towards the pathfinding mode.     

Nicotinic synapses formed between chick ciliary ganglion neurons in culture resemble those present on the neurons in vivo

We studied nicotinic synapses between chick ciliary ganglion neurons in culture to learn more about factors influencing their formation and receptor subtype dependence. After 4--8 days in culture, nearly all neurons displayed spontaneous excitatory postsynaptic currents (sEPSCs), which occurred at about 1 Hz. Neurons treated with tetrodotoxin displayed miniature EPSCs (mEPSCs), but these occurred at low frequency (0.1 Hz), indicating that most sEPSCs are actually impulse driven. The sEPSCs could be classified by decay kinetics as fast, slow, or biexponential and, reminiscent of the situation in vivo, were mediated by two major nicotinic acetylcholine receptor (AChR) subtypes. Fast sEPSCs were blocked by alpha-bungarotoxin (alpha Bgt), indicating dependence on alpha Bgt-AChRs, most of which are alpha 7 subunit homopentamers. Slow sEPSCs were unaffected by alpha Bgt, and were blocked instead by the alpha 3/beta 2-selective alpha-conotoxin-MII (alpha CTx-MII), indicating dependence on alpha 3*-AChRs, which lack alpha 7 and contain alpha 3 subunits. Biexponential sEPSCs were mediated by both alpha Bgt- and alpha 3*-AChRs because they had fast and slow components qualitatively similar to those comprising simple events, and these were reduced by alpha Bgt and blocked by alpha CTx-MII, respectively. Fluorescence labeling experiments revealed both alpha Bgt- and alpha 3*-AChR clusters on neuron somata and neurites. Colabeling with antisynaptic vesicle protein antibody suggested that some alpha 3*-AChR clusters, and a few alpha Bgt-AChR clusters are associated with synaptic sites, as is the case in vivo. These findings demonstrate the utility of ciliary ganglion neuron cultures for studying the regulation of nicotinic synapses, and suggest that mixed AChR subtype synapses characteristic of the neurons in vivo can form in the absence of normal inputs or targets.     

Development of receptors for alpha-bungarotoxin in chick embryo sympathetic ganglion neurons in vitro

The development of receptors for α-bungarotoxin was examined in neurons in dissociated cultures of cells derived from chick embryo sympathetic ganglia. Neurons from 12 day embryos showed a marked increase in receptor numbers per cell over 3–4 days in culture. The increase was less marked in neurons from 14 day embryos and absent in 19 day embryos. The incidence of cholinergic synapses in cultures from 12 day and 19 day embryos was also examined. Evidence for synapse formation was found only in cultures from older embryos.     

Effect of d-sparteine on nicotinic acetylcholine receptors in rat superior cervical ganglion neurons

Effects of d-sparteine (d-SP), a ganglionic blocking agent, on membrane currents evoked by iontophoretic applications of acetylcholine to rat superior cervical ganglion neurons, were studied using a whole-cell patch-clamp recording technique. Blocking effects of d-SP were enhanced by membrane hyperpolarization to potentials more negative than –50 mV. Analysis of the d-SP effect on the dose—response relationship suggests that d-SP at concentrations of 0.5–5.0 µM exerts both voltage-independent and voltage-dependent competitive actions on nicotinic acetylcholine receptors. No use-dependence of the d-SP-induced blockade was found using paired ACh applications at interpulse intervals longer than 0.5 sec. Inhibitory constantK _i estimated by the Dixon method was equal to 0.62±0.15 and 0.28±0.08 µM at membrane potential levels –30 and –90 mV, respectively. These characteristics of the d-SP blocking effects are compatible with a voltage-dependent competitive blocking mechanism. The possibility remains that an open channel-blocking mechanism with a comparatively fast kinetics contributes to the d-SP-induced blockade, but its contribution is small.Neirofiziologiya/Neurophysiology, Vol. 25, No. 4, pp. 266–272, July–August, 1993.     

G-protein-gated TRP-like cationic channel activated by muscarinic receptors: effect of potential on single-channel gating

There is little information about the mechanisms by which G-protein-coupled receptors gate ion channels although many ionotropic receptors are well studied. We have investigated gating of the muscarinic cationic channel, which mediates the excitatory effect of acetylcholine in smooth muscles, and proposed a scheme consisting of four pairs of closed and open states. Channel kinetics appeared to be the same in cell-attached or outside-out patches whether the channel was activated by carbachol application or by intracellular dialysis with GTPgammaS. Since in the latter case G-proteins are permanently active, it is concluded that the cationic channel is the major determinant of its own gating, similarly to the K(ACh) channel (Ivanova-Nikolova, T.T., and G.E. Breitwieser. 1997. J. Gen. Physiol. 109:245-253). Analysis of adjacent-state dwell times revealed connections between the states that showed features conserved among many other ligand-gated ion channels (e.g., nAChR, BK(Ca) channel). Open probability (P(O)) of the cationic channel was increased by membrane depolarization consistent with the prominent U-shaped I-V relationship of the muscarinic whole-cell current at negative potentials. Membrane potential affected transitions within each closed-open state pair but had little effect on transitions between pairs; thus, the latter are likely to be caused by interactions of the channel with its ligands, e.g., Ca(2+) and Galphao-GTP. Channel activity was highly heterogeneous, as was evident from the prominent cycling behavior when P(O) was measured over 5-s intervals. This was related to the variable frequency of openings (as in the K(ACh) channel) and, especially, to the number of long openings between consecutive long shuttings. Analysis of the underlying Markov chain in terms of probabilities allowed us to evaluate the contribution of each open state to the integral current (from shortest to longest open state: 0.1, 3, 24, and 73%) as P(O) increased 525-fold in three stages.     

The distribution of acetylcholine receptor clusters and sites of transmitter release along chick ciliary ganglion neurite-myotube contacts in culture

Acetylcholine receptors accumulate along the length of cholinergic neuron-skeletal muscle contacts in vitro. The main purpose of this study was to describe, in a quantitative way, the distribution of acetylcholine receptor clusters induced by ciliary ganglion neurons over a period of time extending from hours to weeks after contacts are established. Neurites were filled with Lucifer Yellow and receptor clusters were identified with rhodamine-bungarotoxin. A cluster located within 5 micron of a nerve process or 10 micron of the base of a growth cone was considered to be a neurite-associated receptor patch (NARP). The first synaptic potentials were evoked 20 min after growth cone-myotube contact, and, after 24 h of co-culture, greater than 60% of the nerve-muscle pairs tested were functionally connected. NARPs appear rapidly; the first clusters were detected approximately 6 h after the neurons were plated. They were composed of several small subclusters or speckles of rhodamine-bungarotoxin fluorescence. The initial accumulation of receptors may occur at the advancing tips of nerve processes because NARPs were found at greater than 80% of the growth cone-muscle contacts examined between 12 and 24 h of co-culture. Over the 3-wk period examined, the mean incidence of NARPs ranged between 1.0 and 2.6 per 100 micron of neurite-myotube contact, with the peak observed on the second day of co-culture. During the first 3 d in culture, when the neurons were multipolar, nearly all of the primary processes induced one or more clusters. With time, as the neurons become unipolar (Role and Fischbach, 1987) NARPs persisted along the remaining dominant process. Measurements made during the third day of co-culture suggest that NARPs disappear along shorter neurites before they retract. Synaptic currents were detected by focal extracellular recording at 55% of the NARPs. The fact that spontaneous or evoked responses were not recorded at 45% suggests that contacts with clusters exhibit two functional states. Two types of presynaptic specialization at identified NARPs observed by scanning electron microscopy appear to be correlated with the functional state.     

Expression of muscarinic acetylcholine receptors and choline acetyltransferase enzyme in cultured antennal sensory neurons and non-neural cells of the developing moth Manduca sexta

Antennal sensory neurons of Manduca sexta emerge from epidermal cells that also give rise to sheath cells surrounding the peripheral parts of the neurons and to glial cells that enwrap the sensory axons in the antennal nerve. Reciprocal interactions between sensory neurons and glial cells are believed to aid in axon growth and guidance, but the exact nature of these interactions is not known. We investigated the possibility of cholinergic interactions in this process by locating muscarinic acetylcholine receptors (mAChRs) and choline acetyltransferase (ChAT) enzyme in cultured antennal sensory neurons and non-neural cells. ChAT and mAChRs were present in the sensory neurons from the first day in culture. Therefore, the sensory neurons are probably cholinergic, as previously suggested, but they may also be controlled by ACh. In 7-day-old cultures a subgroup of small non-neural cells with processes expressed ChAT activity, and in 14-day-old cultures non-neural cells that formed lamellipodia and scaffoldlike structures on the culture substrate were labeled with ChAT antibody. mAChR activity was detected in similar non-neural cells but only in areas surrounding the nuclei. In addition, mAChRs were found in flat lamellipodia and filopodia forming cells that were present in 1-day-old cultures and grew in size during the 2 week investigation period. These findings suggest muscarinic cholinergic interactions between the neural and non-neural cells during the development of Manduca antenna.     

Thyroid hormone receptors of developing chick brain are predominantly in the neurons

The relative concentration of the triiodothyronine (T₃) receptors in the neuronal and glial nuclei of developing chick brain have been studied. Scatchard analysis indicate that the number of T₃ binding sites in the neuronal nuclei increases from 400 to 1600 sites/nucleus between 7–11 day of embryonic development without any concomitant change in the level of glial nuclear receptors (130 – 200 sites/nucleus). Both sites are of high affinity (K_a = 1–3 × 10⁹ M^?1) at all ages examined. The abundance of the T₃-receptors in the neuronal nuclei and the close coincidence of the period of rise in the level of these receptors in these nuclei (7–11 day) with that of maximal neuronal growth and synaptogenesis (7–13 day) suggest that the neurons are the primary site of action of T₃ in the developing brain.