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G Pilar

Publications and source records attributed to G Pilar.

At least 37 records · Page 2Linked to original sources
PubMed1981

Functional maturation of motor nerve terminals in the avian iris: ultrastructure, transmitter metabolism and synaptic reliability.

1. The transformation of easily fatigued embryonic neuromuscular junctions into highly reliable mature terminals was examined by studying functional and morphological changes during development of the avian iris. The mature ability to follow repetitive electrical nerve stimulation was correlated with the rate of acetylcholine (ACh) synthesis and choline uptake, and with the fine structure of the nerve terminals and the post-synaptic elements.2. The terminals of the ciliary nerve of the chick initially form functional synaptic contacts with the iris muscle at embryonic St. 34-40. At the onset of this period, no Na(+)-dependent high affinity choline uptake can be demonstrated, and the low level of ACh synthesis present is sensitive to Na(+) removal. At St. 36 [(3)H]ACh synthesis begins to increase, the increment being Na(+)-dependent.3. ACh synthesis in the embryonic iris was insensitive to a conditioning [K(+)](o) depolarization even as late as St. 43. Just before hatching, depolarization elicits some augmentation in synthesis, but by 2 days ex ovo this release-induced response has increased by an order of magnitude.4. Concurrently with the acquisition of the ability to respond to depolarization with accelerated synthesis, neuromuscular transmission in the iris becomes reliable and secure during stimulation at 20 Hz. Embryonic junctions rapidly block during such stimulation, and the failure is shown to be presynaptic in origin, resulting most probably from failure to sustain adequate levels of transmitter release.5. Ultrastructural examination of the developing ciliary terminals revealed few synaptic vesicles at early stages, and a dearth of other specializations. The sequence of development from these small structurally undistinguished endings to large en plaque junctions completely filled with vesicles was reconstructed and compared to other neuromuscular junctions. Morphological maturation appears progressive with little evidence of discontinuity signalling functional status, but it is only after the terminals enlarge and become closely packed with vesicles that mature synaptic reliability is found.6. The temporal correlation between responsiveness of transmitter synthesis to depolarization and reliable neuromuscular transmission suggests that modulation of neurotransmitter metabolism in response to demand signals the achievement of junctional maturity.

Acetylcholine↗
PubMed1980

The release of acetylcholine from post-ganglionic cell bodies in response to depolarization.

1. Acetylcholine (Ach) release from parasympathetic ganglia cell somata was investigated in denervated avian ciliary ganglia. Three days after the input to the ganglion (the oculomotor nerve) was sectioned, all presynaptic nerve terminals had degenerated. 2. Denervated ganglia were shown to contain endogenous ACh and to be capable of synthesizing [3H]ACh from [3H]choline added to the incubation medium. 3. In response to depolarization induced by incubation in 50 mM-[K+]o, denervated ganglia released [3H]ACh into bath effluents in amounts approximately 15% of the non-denervated contralateral control. This release was shown to be Ca2+ dependent in both intact and denervated ganglia. 4. Antidromic electrical stimulation of ciliary nerves also elicited [3H]ACh release. Nicotine (1 microgram/microliter.) depolarized denervated ciliary ganglion cells and evoked release of the transmitter and this release was antagonized by curare. 5. It is concluded that the ganglionic cell bodies sysnthesized ACh and released the transmitter in response to K+ depolarization, antidromic stimulation and cholinergic agonists, despite the lack of morphological specializations usually associated with stimulus-induced release of neurotransmitter. The evidence suggests the existence of a mechanism of transmitter release which is Ca2+ dependent, probably from a cytoplasmic pool and therefore distinct from the usual vesicular release at the nerve terminal.

Acetylcholine↗
PubMed1980

Competition for survival among developing ciliary ganglion cells.

1. Functionally different subgroups, each innervating a different part of the peripheral target, were defined within the ciliary population of the avian ciliary ganglion by electrical stimulation of the various ciliary nerve branches. 2. Although neurons innervating defined parts of the peripheral target consistently sent their axons through certain nerves, the technique of retrograde horseradish peroxidase (HRP) transport showed that the ganglion cell bodies were not spatially grouped but distributed throughout the ganglion, both before and after the period of naturally occurring cell death. However, such neurons tended to be clustered into groups of two or greater. 3. Ciliary and choroid populations, however, were found to be for the most spatially separate and recognizable by location and soma size before the period of cell death. Choroid cells did not project out the ciliary nerves even prior to the cell death period, confirming previous observations of selective axon outgrowth in the two populations. 4. Competition for survival was demonstrated within the ciliary population by experimentally removing approximately two-thirds of the neurons by axotomy-induced cell death at stage 32-34 just prior to the normal cell death period. This reduction in the number of competing neurons resulted in rescue of approximately 40% of the neurons that would have died, as assessed both by the number of axon profiles in the remaining intact nerve branch, as well as the number of somata that could be retrogradely labeled from this nerve. 5. It was concluded that many of the neurons that are normally removed during the cell death period are not destined to die, but can be rescued by reducing the number of neurons competing for a limited supply of some aspect of the peripheral target. Further, the postulated interaction with the target was shown to occur relatively late, just prior to the onset of cell death. 6. At the time of the peripheral interaction, the target was found to consist primarily of myoepithelial cells, which had migrated into the target region following the arrival of the ciliary axons. The target per se, therefore, cannot be involved in the selective growth of ciliary axons to the appropriate region. Well-defined synapses were rare, although many axonal endings were observed in close contact with both myoepithelial cells and the sparser differentiated muscle fibers, which increased to account for 60% of the target by the end of the cell death period. 7. Competition was also found to retard the rate of neuronal maturation because intact axons in the partially axotomized ganglion developed more rapidly than control axons, as assessed by axon diameter, conduction velocity, and degree of glial ensheathment. 8. Finally, at least some of the neurons in the partially axotomized ganglion expanded to innervate the peripheral territory of the axotomized branches, suggesting that competition between neurons is involved in the establishment of the observed peripheral innervation pattern.

Animals↗
PubMed1979

Mechanisms controlling choline transport and acetylcholine synthesis in motor nerve terminals during electrical stimulation.

Electrical stimulation of the chick ciliary nerve leads to a frequency-dependent increase in the Na+-dependent high affinity uptake of [3H]choline (SDHACU) and its conversion to acetylcholine (ACh) in the nerve terminals innervating the iris muscle. The forces that drive this choline (Ch) uptake across the presynaptic membrane were evaluated. Depolarization with increased [K+] out or veratridine decreases Ch accumulation. In addition to the electrical driving force, energy is provided by the Na+ gradient. Inhibition of the Na,K-ATPase decreased the Ch taken up. Thus, changes in the rate of Ch transport are dependent on the electrochemical gradients for both Ch and Na+. Ch uptake and ACh synthesis were increased after a conditioning preincubation with high [K+] out or veratridine. As is the case for electrical stimulation, this acceleration of Ch uptake and ACh synthesis was strongly dependent on the presence of Ca++ in the incubation medium. Na+ influx through a TTX-sensitive channel also contributed to this acceleration. Inasmuch as membrane depolarization reduces the initial velocity of Ch uptake and ACh synthesis, their increases during electrical stimulation therefore cannot be the direct effect of the depolarization phase of the action potential. Instead they are the result of the ionic fluxes accompanying the presynaptic spike. It is concluded that stimulation of Ch uptake and ACh synthesis by nerve activity depends first, on the ACh release elicited by Ca++ influx after depolarization and second, on the activation of the Na,K-ATPase due to Na+ entry. Furthermore, it is suggested that the release of ACh after stimulation drives translocation of cytoplasmic ACh into a protected compartment (probably vesicular). This recompartmentation of intraterminal ACh stimulates ACh synthesis by mass action, allowing further accumulation of Ch.

Acetylcholine↗
PubMed1979

Normal distribution and denervation changes of neurotransmitter related enzymes in cholinergic neurones.

1. The activities of choline acetyltransferase (CAT) and acetylcholinesterase (AChE) were assayed in adult pigeon ciliary ganglia, in the post-synaptic ciliary and choroid nerves, and in ciliary nerve iris terminals isolated from control birds and from animals from which the oculomotor nerve was previously transected. Enzyme activity levels were also measured in the iris terminals after surgical section of the ciliary nerves. From differences in enzyme activity between control and 3-day denervated tissues, the localization of CAT and AChE in pre- and post-synaptic elements of the ganglia and at the iris neuromuscular junctions was estimated. The fate of the preganglionic nerve terminals after denervation was investigated by electron microscopic examination of ganglia after surgical section of the oculomotor nerve.2. The CAT activity in the ganglion was distributed as follows: 60% in presynaptic elements, 31% in cell somas, and 9% in intraganglionic post-synaptic axons; in the iris junctions, 98% of the activity was present in the ciliary nerve terminals. For AChE: 20% was present in the preganglionic terminals, 69% in ganglion cell somas and the remaining 11% in post-ganglionic axons; at the neuromuscular iris junctions, 20% was found in the ciliary nerve terminals and 80% in the iris striated muscle.3. The first changes in the fine structure of the nerve terminals were observed 14 hr after surgery, and by 24 hr marked alteration of the synaptic structure were clearly recognized. No preganglionic endings were found in 3 day-old denervated ganglia.4. There was a positive correlation between CAT activity in the control iris nerve terminals and in ganglia. After denervation, when the activity of the enzyme decreased in ganglion cell somas, there was a corresponding decrease in the post-synaptic nerves. These two findings suggest that CAT slow axoplasmic transport is related to its perikarial concentration.5. There was a 60% reduction of CAT activity in the post-synaptic elements, assayed in the 10-day denervated ganglia, which was accompanied by a 30% decrease in activity in the iris nerve terminals. Similarly, post-synaptic AChE decreased approximately 30% in the ganglion and approximately 30% in the iris 10 days after section of the oculomotor nerve. At the same time, CAT activity also decreased in the nerve trunks, 70% at the ciliary nerve and 40% at the choroid; for AChE there were smaller changes.6. In contrast to CAT and AChE, there were no differences in ganglionic protein content, or lactate dehydrogenase (LDH), co-enzyme A (CoA) and monoamine oxidase (MAO) levels between short-term (3 days) and long-term (10 days) denervated ganglia.7. The later decrease of CAT and AChE activity in the cell somas, axons and nerve terminals after long-term preganglionic transection suggests that the activity of these enzymes is regulated across the synapses. It is postulated that the AChE regulation is part of a general ;trophic interaction' between neurones, but that the trans-synaptic modulation of CAT is specific for cholinergic cells.

Acetylcholinesterase↗
PubMed1978

Interactions between neurons and their targets during in vivo synaptogenesis.

In vivo synaptogenesis is described in a simple vertebrate system, the chick ciliary ganglion, a parasympathetic autonomic ganglion. An attempt is made to integrate anatomical, physiological and biochemical observations during synapse formation in the ganglion and in the peripheral target structures; the iris, ciliary muscle, and smooth muscle of the choroid coat. The relationship between synaptogenesis and neuron survival is explored, and it is shown that a critically timed interaction between the neuron and target organ is necessary for full neuronal maturation and survival. The existence of an active competition between neurons for survival is documented, and the possible relationship between neuronal cell death and specificity of connections is discussed.

Animals↗
PubMed1976

Ultrastructural differences during embryonic cell death in normal and peripherally deprived ciliary ganglia.

Normally occurring neuron death and that brought about by prior removal of the peripheral target organ was studied ultrastructurally in embryonic chick ciliary ganglion in order to better understand the mechanism of cell death in this system. Before the period of cell death, all neurons in the normal ganglion developed a well-organized rough endoplasmic reticulum (RER) which coincided with peripheral synapse formation. None of the peripherally deprived neurons underwent this change, suggesting that some interaction with the periphery, possibly synapse formation, triggered them into the secretory state. Cell death in peripherally deprived neurons was signalled by nuclear changes followed by freeing of ribosomes from polysomes and RER and presumably cessation of protein synthesis. In contrast, normal cell death was brought about by dilation of the RER with eventual cytoplasmic disruption, nuclear changes appearing only secondarily. It is suggested that failure to form or maintain peripheral synapses could result in the accumulation of transmission-related proteins with consequent cisternal dilation, and eventual cell death.

Animals↗
PubMed1976

Fate of ganglionic synapses and ganglion cell axons during normal and induced cell death.

In order to understand the significance of cell death in the formation of neural circuits, it is necessary to determine whether before cell death neurons have (a) sent axons to the periphery; (b) reached the proper target organs; and (c) have established synaptic connections with them. Axon counts demonstrated that, after sending out initial axons, ciliary cells sprouted numerous collaterals at the time of peripheral synapse formation. Subsequently, large numbers of axons were lost from the nerves, slightly later than the onset of ganglion cell death. A secondary loss of collaterals later occurred unaccompanied by cell death. Measurements of conduction velocity and axon diameters indicated that all ganglion cell axons grew down the proper pathways from the start, but it was not possible to determine whether all axons had actually formed proper synapses. This was ascertained, however, in the ganglion itself where preganglionic fibres were shown to synapse selectively with all ganglion cells before cell death. During this period, degenerating preganglionic synapses were observed on normal cells. It can therefore be inferred that at least some preganglionics established proper synapses before dying and that a single synapse is not sufficient to prevent cell death. In this system neither preganglionic nor ganglionic cell death seems designed to remove improper connections but rather to remove cells that have not competed effectively for a sufficient number of synapses, resulting in a quantitative matching up of neuron numbers.

Animals↗
PubMed1976

Induction of cholinergic enzymes in chick ciliary ganglion and iris muscle cells during synapse formation.

1. In chick ciliary ganglia and irises, cholineacetyltransferase (ChAc) and acetylcholinesterase (AChE) activities were measured from the fifth day of incubation until 1 week after hatching. The changes in enzyme activity were correlated in time with previous electrophysiological and morphological findings of synapse formation in these tissues. 2. At Stage 26 (Hamburger & Hamilton, 1951; before synapse formation in the ganglia) low activities of ChAc (12 +/- 4 [mean +/- S.E.] p-mole of ACh synthesized/hr) were measured in the iris nerve terminals, indicating that ganglion cells are biochemically differentiated, immediately after cell migration is completed. The specific acitivities of ChAc and AChE rose during development and these increases were closely related to the onset and maturation of ganglionic and iris synaptic transmission. These increases in enzyme activities can be used in cholinergic synapses as an index of synapse formation. 3. The 200-fold specific increase of ChAc in iris nerve terminals which occurs at Stage 34 probably reflects an increase in synthesis of the enzyme in ganglion cells and suggests that the formation of the iris neuromuscular junction triggers the enzyme induction. It is implied that the cell responds to a signal ascending the axon from the terminal. 4. The initial increase of AChE specific activity in the ganglion occurs after transmission is established in all cells between Stage 30 and 34 and is mainly due to enzyme synthesis by the ganglion cells. In the iris there is a twofold increase in specific activity after the formation of neuromuscular junctions which probably reflects enzyme induction in the muscle subneural region. It is concluded that the specific induction of AChE in post-junctional cells is due to an influence of the prejunctional element. 5. During synaptic formation in the ciliary ganglion, reciprocal interactions between the neurones and their targets result in the induction of ChAc in the prejunctional elements and AChE in the post-junctional cells.

Acetylcholinesterase↗
PubMed1975

Size and shape of transverse tubule openings in frog twitch muscle fibers.

The openings of transverse tubules in frog twitch fibers are described. The tubules open to the extracellular space by a narrow neck, with an inner diameter of 20 nm. The most peripheral portion of the tubules is tortuous and has a variable diameter. The similarity in size of the openings of T tubules and caveolae and the meandering path of the tubules are sufficient to account for the paucity of observed openings.

Animals↗
PubMed1974

Synapse formation during embryogenesis on ganglion cells lacking a periphery.

1. The development of transmission was studied in chick ciliary ganglia that had been deprived of their periphery during early embryonic development.2. Peripherally deprived neurones in the ganglion differentiate in normal numbers and send functional axons into the post-ganglionic nerve.3. Ganglion cells lacking a periphery follow the normal developmental sequence sending out transient dendrites at the time ganglion cell synapses are formed, and later retracting them when calyces appear.4. Synapses, which appear functionally and ultrastructurally normal, form on all ganglion cells at the normal time and transmission is normal until Stage 34. Therefore information from the periphery is not required for ganglion cell synapse formation per se.5. From Stages 35 to 38 most cells die, so that only 8% of the original number of cells remain in the operated ganglion. Transmission fails in many cells during this same time, but precedes cell loss by only a short time, so that deafferentation probably does not contribute substantially to cell death.6. Both ciliary and choroid cells achieve full cytologic differentiation and are distinct from each other, indicating that the periphery is not required for the elaboration of the distinctive characteristics of these cells. Presynaptic fibres also differentiate into typical bouton as well as calyciform endings. Therefore, the type of preganglionic ending does not depend on ganglion cells establishing proper peripheral contacts.7. It has not been possible to ascertain whether ganglion cell specificity is affected by the periphery.8. Peripheral removal affects ganglion cell migration, so that two ganglia are formed. Approximately half of the cells migrate into the remnant optic cup forming a second misplaced ganglion. Ciliary and choroid cells occur in both ganglia and these cells go through the typical sequence of events described above.

Action Potentials↗
PubMed1974

Synaptic transmission and cell death during normal ganglionic development.

1. During normal embryonic development of the chick ciliary ganglion, cell death over a 4-day period (Stages 35-39) reduces the number of ganglion cells by half, from 6500 to 3200. Both ciliary and choroid populations are affected by approximately the same amount.2. Previous to cell death, preganglionic fibres form functional synapses on all ganglion cells, indicating that synapses form on cells which are destined to die.3. Shortly before the period of cell death, there is a failure of transmission in approximately half the cells. Some evidence suggests that transmission failure in at least some of the cells is of preganglionic origin.4. Cell death is nearly synchronous with the establishment of peripheral connexions by ganglion cells, at least with respect to the ciliary population which forms functional synapses with iris muscle. This implies that those cells which die do so because they have failed to form adequate peripheral connexions.5. It is suggested that many of the cells in which transmission has failed die, bringing transmission through the ganglion back to 100%. However, transmission failure appears to be a transitory phenomenon in other cells which survive and probably results from death of their preganglionic elements. Restoration of transmission would then be brought about by the formation of new or more effective synapses by surviving preganglionic fibres.

Action Potentials↗
PubMed1972

Axotomy mimicked by localized colchicine application.

Comparable depression of synaptic transmission in the avian ciliary ganglion resulted from either section or localized colchicine treatment of the ciliary nerves. Both colchicine treatment and axotomny produced similar changes in RNA distribution in the cell bodies as well. Colchicine did not directly affect transmission, and action potential propagation along the ciliary nerves was normal. Interference with axoplasmic transport of material in both cases is postulated to signal the observed chromatolytic changes.

Action Potentials↗
PubMed1972

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.

Animals↗