Characterization of a snake venom neurotoxin which blocks nicotinic transmission in the avian ciliary ganglion

Bungarus multicinctus venom was fractionated by ion exchange chromatography and the various fractions were assayed for their ability to block synaptic transmission through the chick ciliary ganglion. alpha-Bungarotoxin purified from this venom failed to block transmission at 50 micrograms/ml. A second neurotoxin, which we designate Toxin F, blocked transmission at 1-3 micrograms/ml and also blocked ganglionic depolarizations induced by carbachol. Toxin F was clearly distinguishable from alpha-bungarotoxin on the basis of molecular weight (estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and isoelectric point. Binding assays revealed that 125I-labeled toxin F bound to two sites in the ciliary ganglion: one site that was shared by alpha-bungarotoxin and toxin F and another site that was recognized solely by toxin F. Carbachol and d-tubocurarine displaced only that [125I]toxin F bound to the shared site and had no effect on [125I]toxin F bound to the site recognized by toxin F alone. The results suggest that toxin F blocks synaptic transmission in the chick ciliary ganglion by a postsynaptic mechanism. Further study is required to determine whether this effect of toxin F is mediated through a direct interaction with ganglionic nicotinic receptors.     

The onset and development of transmission in the chick ciliary ganglion

1. The onset and development of transmission has been studied electro-physiologically in the isolated chick ciliary ganglion from Stage 25 (Hamburger & Hamilton, 1951) until 28 days after hatching. Ultrastructure of the synapses was concomitantly investigated.2. Synaptic transmission began at Stage 26(1/2) and was 100% in both cell groups, ciliary and choroid, by Stage 33. It was initially chemical until Stage 41 when effective electrical coupling first appeared in the ciliary population. The proportion of electrically transmitting synapses increased to 80% by 1-2 days post-hatching.3. Few morphological synapses were present at Stage 33(1/2) when all ganglion cells were transmitting. A scarcity of synaptic vesicles persisted until late in embryonic development when all ciliary cells possessed calyces. At hatching the calyces were filled with synaptic vesicles.4. Initial synaptic contacts were by fine terminal branches often on the intricate processes of early ganglion cells. Calyces formed from Stage 36(1/2) and there was a concomitant retraction of ganglion cell processes, so that by Stage 40 all ciliary cells had simple calyces. The calyx was a transitory structure, which from the first week post-hatching began to break up into a cluster of boutons.5. Chemical post-synaptic potentials (PSPs) were at Stage 40 long (30 x the membrane time constant) and further prolonged by eserine. By Stage 43, PSPs had become markedly shortened and were unaffected by eserine. No simple explanation can be offered for the changes in PSP time course and sensitivity to anticholinesterases during development.6. Intracellular records from Stage 40 ciliary cells, which all possess calyces, showed 1-2 mV amplitude, diphasic, fast decaying electrical coupling potentials (CPs). Later in development the CPs became 20-40 mV amplitude, more slowly decaying and monophasic. This seemed to be correlated with faster presynaptic conduction velocities and myelination of the cell soma. Such changes in CPs may reflect a shift from capacitative to more resistive coupling and point to several factors contributing in varying degrees to the electrical transmission.7. Presynaptic fibres innervating ciliary cells were from the start of lower threshold and faster conduction velocity than those innervating ciliary cells, as occurred in the adult. It is concluded that these preganglionic fibres were probably specified by the time transmission starts and that they selectively innervated the proper post-synaptic cells.     

Analysis of quantal content and quantal conductance in two populations of neurons in the avian ciliary ganglion

The avian ciliary ganglion contains two populations of parasympathetic cells, termed the ciliary and choroid neurons. We have estimated the quantal contents of nicotinic excitatory postsynaptic potentials in both populations of neurons by several methods. The singly innervated ciliary neurons have quantal contents of 15–30. In contrast, the multiply innervated choroid cells have quantal contents of 4–7. Quantal conductance was also determined, using a parallel conductance model which takes into account the capacitance of the cell membrane. This analysis indicates that in both populations of neurons one quantum activates approximately 100 postsynaptic receptors.

It is concluded that in autonomie ganglia singly innervated cells demonstrate a larger quantal content, consistent with a higher safety factor for neurotransmission, while quantal content in multiply innervated cells is generally much lower, allowing for considerable summation of presynaptic inputs. Further, in autonomic neurons many fewer postsynaptic receptors are activated by a single quantum than is the case at the neuromuscular junction.     

A VIP-like peptide co-occurs with substance P and enkephalin in cholinergic preganglionic terminals of the avian ciliary ganglion

Using single-label and double-label immunohistochemical techniques, many of the substance P-enkephalin containing preganglionic terminals of the avian ciliary ganglion were found to also contain a VIP-like peptide. Since these terminals are also known to be cholinergic, these results suggest that neurotransmission at many synapses of the avian ciliary ganglion involves 3 different neuropeptides and a conventional neurotransmitter, acetylcholine.     

Microglia-like cells in the chicken ciliary ganglion

Using light and electron microscopy we have identified in the ciliary ganglion of the chick a class of small cells characterized by the electron-dense appearance of both nucleus and cytoplasm, slender cell processes, paucity of mitochondria and microtubules, presence of elongated cisterns of granular endoplasmic reticulum with narrow lumina, free ribosomes, dense bodies of varying size and coated vesicles. The small, elongated electron-dense cells lack a basal lamina and occur within the satellite sheath surrounding ganglionic perikarya as well as in the endoneurial connective tissue. Occasionally, the small electron-dense cells within the ganglion cell capsule contain phagocytized cell debris.The small electron-dense cells of the ciliary ganglion differ substantially from satellite cells and other types of cell in the endoneurial connective tissue, except those of hematic origin. On the other hand, in their fine structure they closely resemble resting microglial cells of the CNS. We suggest that small electron-dense cells in the ganglion and microglial cells in the CNS belong to the same cell category.     

Reciprocal synapses between cholinergic axons and small granule-containing cells in the rat cardiac ganglion

Electron microscopy of the rat cardiac ganglion shows occurence of small granule-containing cells that form reciprocal synaptic junctions with cholinergic terminals. At the synaptic junctions which are from axon to granule-containing cell, the intraaxonal vesicles are clustered against the junctional axolemma, but dense-cored vesicles in the postynaptic cell do not cluster towards the membrane densities in these synapses. By contrast, the synaptic zone polarized in the opposite direction shows an absence of axonal vesicles in close proximity to the postsynaptic axolemma, but there is a marked aggregation of dense-cored vesicles towards the presynaptic specializations of granule-containing cells. The synaptic zones are multifocal rather than bifocal, and the minimal distance separating each synaptic zone is about 0.3 μ. These findings may indicate that cholinergic excitation of some or all granule-containing cells causes a reciprocal inhibition of one or more cholinergic terminals.     

The formation of synapses between chick embryo skeletal muscle and ciliary ganglia grown in vitro.

1. Chick embryo ciliary ganglia (explanted) and skeletal muscle (dissociated) were grown together in vitro for up to 3 weeks. Nerve processes sprouted from the ganglia and contacted neighbouring myotubes and striated muscle fibres. 2. Spontaneous action potentials and subthreshold e.p.p.s. were recorded from muscle fibres with intracellular micropipettes. Similar potentials could be evoked by electrical stimulation of the ganglion. The pharmacological effects of curare and tetrodotoxin were identical to those observed at adult vertebrate neuromuscular junctions. 3. The amplitude, but not the frequency, of the spontaneous potentials was affected by changing the muscle fibre membrane potential. The reversal potential of evoked synaptic potentials occurred at a membrane potential of about 0 mV.     

Polysialylated neural cell adhesion molecule is involved in the neuroplasticity induced by axonal injury in the avian ciliary ganglion

We demonstrated previously in the quail ciliary ganglion, that the immunoreactivity for the neural cell adhesion molecule labeling the postsynaptic specializations of intraganglionic synapses decreases when synaptic remodeling is induced by crushing the postganglionic ciliary nerves. Here we show, in the same experimental conditions, that the immunolabeling for its polysialylated non-stabilizing isoform, which promotes cell plasticity, increases at these subcellular compartments. In control ganglia, poor immunolabeling for the polysialylated neural cell adhesion molecule was occasionally observed surrounding the soma of the ciliary neurons, in correspondence with the calyciform presynaptic ending and the perineuronal satellite cells sheath. At the electron microscope, several neuronal compartments, including some postsynaptic specializations, somatic spines and multivesicular bodies, were immunopositive. Three to six days after ciliary nerve crush, both the number of ciliary neurons labeled for the polysialylated neural cell adhesion molecule and the intensity of their immunolabeling increased markedly. Electron microscopy revealed that, in parallel to the injury-induced detachment of the preganglionic boutons, numerous postsynaptic specializations were found to be immunopositive. Twenty days later, when intraganglionic connections were re-established, polysialylated neural cell adhesion molecule immunoreactivity was comparable to that observed in control ganglia. The increase in immunolabeling also involved the other neuronal compartments mentioned above, the perineuronal satellite cells and the intercellular space between these and the ciliary neurons.From these results we suggest that the switch, at the postsynaptic specializations, between the neural cell adhesion molecule and its polysialylated form may be among the molecular changes occurring in axotomized neurons leading to injury-induced synaptic remodeling. Moreover, from the increase in polysialylated neural cell adhesion molecule immunolabeling, observed at the somatic spines and at the interface between neurons and perineuronal satellite cells, we suggest that this molecule may be involved not only in synaptic remodeling, but also in other more general aspects of injury-induced neuronal plasticity.     

The structural basis for electrotonic coupling in the avian ciliary ganglion. A study with thin sectioning and freeze-fracturing.

Each "ciliary" neurone in the ciliary ganglion of adult birds receives its innervation from a single myelinated fibre of the oculomotor nerve by means of a dual mode of synaptic action, electrical and chemical. The preganglionic fibre branches repeatedly around the postganglionic axon but the extra-cellular compartment is large. The preterminal fibres, most of which are unmyelinated, end with large boutons on the axon hillock, a few on short dendrites and on the portion of the perikaryon of the ciliary neurone from which the axon emerges. This synaptic apparatus is enveloped by a glial sheath, mainly consisting of satellite cell bodies and loose myelin lamellae. The nonsynaptic portion of the ciliary perikaryon is covered by a sheath consisting mainly of compact myelin. The ciliary neurone has an initial axon segment like that of C.N.S. neurones. The area of each neurone apposed to boutons measures about 16,000 mum2. Approximately 9 percent is specialized for chemical transmission and 0.17 percent for electrical transmission. Each neurone has about 280,000 gap junctional particles. Assuming that each particle represents one channel, the electrical resistance provided by these junctions is estimated to be of the order of 100 k omega. The electrical coupling between the preganglionic fibre and the ciliary neurone may therefore be of resistive nature.     

Ca(2+)-independent and Ca(2+)-dependent stimulation of quantal neurosecretion in avian ciliary ganglion neurons.

1. Although it is generally agreed that Ca2+ couples depolarization to the release of neurotransmitters, hypertonic saline and ethanol (ETOH) evoke neurosecretion independent of extracellular Ca2+. One possible explanation is that these agents release Ca2+ from an intracellular store that then stimulates Ca(2+)-dependent neurosecretion. An alternative explanation is that these agents act independently of Ca2+. 2. This work extends previous observations on the action of ETOH and hypertonic solutions (HOSM) on neurons to include effects on [Ca2+]i. We have looked for Ca(2+)-independent or -dependent neurosecretion evoked by these agents in parasympathetic postganglionic neurons dissociated from chick ciliary ganglia and maintained in tissue culture. The change in concentration of free Ca2+ in the micromolar range inside neurons ([Ca2+]i) was measured with indo-1 with the use of a Meridian ACAS 470 laser scanning microspectrophotometer. 3. Elevated concentration of extracellular KCl increased [Ca2+]i and the frequency of quantal events. Also, a twofold increase in osmotic pressure (HOSM) produced a similar increase in quantal release and a significant rise in [Ca2+]i; however, the Ca2+ appeared to come from intracellular stores. 4. In contrast, ETOH stimulated quantal neurosecretion without a measurable change in [Ca2+]i. It appears the alcohol exerts its influence on some stage in the process of exocytosis that is distal to or independent of the site of Ca2+ action. 5. The effects of high [KCl]o and osmotic pressure were occlusive. This is explained in part by the observation that hypertonicity reduced Ca2+ current, but an action on Ca2+ stores is also likely.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative anatomy of the accessory ciliary ganglion in mammals

Summary The orbits of 13 mammalian species (Pig, sika deer, domestic sheep, horse, cat, fox, racoon dog, marten, rat, rabbit, crab-eating macaque, japanese macaque and man) were stained with silver nitrate and dissected under a dissecting microscope with special attention to the presence and location of the accessory ciliary ganglion. Some preparations were stained with thionin and examined as whole-mounts in a transmission microscope. The accessory ciliary ganglion was present in all 13 species, although the number and degree of development varied greatly from species to species. The accessory ciliary ganglion could readily differentiated from the main ciliary ganglion in the following respects: it was located on the short ciliary nerve, and it had no root derived directly from the inferior trunk of the oculomotor nerve and it never attaches to this nerve. In many species, ganglion cells were also scattered in the short ciliary nerves in the stained whole preparations. In a few species, there were one or more small ganglia on the nerve to the inferior oblique muscle.     

Correlation between ciliary beat frequency and metachronal wave disorder using image analysis method

Ciliary beating and metachronal waves are fundamental to effective mucociliary transport. The ciliary beat frequencies (CBFs) and metachronal wave directions of multiple cilia beating in culture media were measured simultaneously using digital microscopic images. The degree of synchronisation between ciliary beats was determined by the correlation between ciliary signals at two different locations. The wave propagation directions of cilia were determined from a two-dimensional correlation map by a principal axis method. The standard deviation of measured wave directions in a region of interest was defined as a measure of metachronal wave disorder (MWD). Considerable variation was found in the beat frequencies and metachronal wave directions of cilia beating on epithelium. The pooled mean of MWDs was 23.4±8.8°, and the pooled mean of CBFs was 10.1±1.9 Hz on 120 cells from five healthy subjects. The means of the MWD and the CBF from subjects were highly correlated (correlation=−0.83). The higher the CBF, the lower the level of the MWD.