Guillermo Pilar

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.

SELECTIVE LOCALIZATION OF A HIGH AFFINITY CHOLINE UPTAKE SYSTEM AND ITS ROLE IN ACh FORMATTON IN CHOLINERGIC NERVE TERMINALS

The avian iris‐ciliary nerve preparation exhibits two distinct choline uptake systems. One component, a sodium dependent, high affinity system Km‐2 am and Vmax ‐ 0.5 pmolpin per preparation is confined to nerve terminals. The other component is localized in muscle cells. It is sodium independent and low affinity system (Km ‐ 200 am and Vmax ‐ 16 pmol/min per muscle). The high affinity uptake of choline and the synthesis of ACh in the nerve terminals are coupled. Vmax Ach formation ‐0.5 pmol/min. is the same as Vmax for choline transport; however. with the external choline concentration equal to that of avian plasma only ‐50% of choline taken up is converted to ACh. In contrast to the nerve terminals, the cell bodies of the same neurons are deficient in the high affinity uptake‐ACh synthesis coupled system. This indicates a nerve terminal membrane specialization related to neuro‐transmitter synthesis.

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.

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.

CHOLINE UPTAKE BY CHOLINERGIC NEURON CELL SOMAS

The cellular compartments of ciliary ganglia take up choline by a single, saturable process with Km=7.1 × 10−5 M and Vmax= 4.66 pmol/min per ganglion: Denervation of the ganglia and the resultant degeneration of nerve terminals caused no significant decrease of the rate of accumulation of choline by the ganglia. This indicates that the measured uptake is by the postganglionic ncurons and nonneural elements (NNE: glial and connective tissue cells) in the ganglia. This uptakc is not dependent on metabolic energy and is not affectcd by lowcring Na+ or raising K+ concentrations in the incubating mcdia but is depressed in the presence of ouabain and hemicholinium‐3. The presence or Na+‐dependent. rapidly saturable uptake in the preganglionic nerve terminals which is not detectablc kinetically is, however, inferred from a decrease in ACh synthesis in dcncrvatcd prcparations and a similar decrcasc in intact ganglia incubated in low Na+ solution.

Specific in vitro biological activity of snake venom myotoxins.

Abstract: Some snake venoms contain toxins that are reported to be selective for damaging muscle. This specificity can be used to design experiments intended to eliminate muscle. We studied the small myotoxins and fractions IV and V of Bothrops nummifer venom to evaluate their direct effect on cultured muscle cells, neurons, macrophages, and a fibroblast cell line. The small myotoxins, at 100 μg/ml for 2 h, had no effect in vitro, contrary to the in vivo applications. Fractions IV and V were both myotoxic and, at 100 μg/ml, destroyed all cell types. However, at 10 μg/ml the effects of fraction IV were more selective for muscle. Vacuolation of the sarcoplasmic reticulum and T‐tubules was first seen in the poisoned muscles, without initial lesions in the nuclei, sarcolemma, mitochondria, and rough endoplasmic reticulum. Fractions IV and V have different toxic activity in cells other than muscles and are a mixture of two basic proteins (i and ii). Protein ii is predominant in fraction IV and protein i is predominant in fraction V. The toxic effects may be mediated by the formation of nonspecific ionic pores in the sarcolemma and/or T‐tubule muscle membrane.

Selective reinnervation of two cell populations in the adult pigeon ciliary ganglion.

1. Presynaptic fibres innervating the adult pigeon ciliary ganglion were cut 2 mm proximal to the ganglion. The modification of transmission in the ciliary and choroid cell populations was studied after periods of 6 hr—100 days.

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.

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.

Enhanced chemosensitivity of chick parasympathetic neurones in co‐culture with myotubes.

1. The influence of target interaction upon the electrophysiological properties of dissociated ciliary ganglion cells was investigated by testing the sensitivity of the neuronal somal membrane to ionophoretically applied acetylcholine (ACh). Variations in the percentage of cells responsive to the transmitter were measured with time in culture. 2. Twenty‐four hours after plating, all cells respond to an ionophoretic pulse of ACh with a depolarization. However, 1 week after plating (between 7 and 14 days) most of the neurones are unresponsive, and highly responsive cells (greater than 100 mV peak depolarization/nC) are extremely rare. At even later times in culture, neurones sensitive to the transmitter are again more frequent. 3. When neurones are plated onto pre‐formed pectoral myotubes, however, ACh sensitivity is maintained throughout a 3 week culture period. Neuromuscular junctions are formed by the neurones, and when sufficient neurones are present, all the muscle fibres tested show evidence of functional synaptic transmission. Chemosensitivity to ACh is not maintained by neurones in muscle‐free microcultures are present on the same cover‐slip. 4. Interneuronal synaptic contacts, defined by ultrastructural criteria, are formed in cultures of neurones alone, but evidence of widespread functional synaptic interaction between cells was not found at 7‐14 days in culture. 5. It is concluded that the maintenance of ACh sensitivity of cultured ciliary ganglion cells is enhanced by the presence of muscle in co‐culture. The interneuronal synaptic contacts observed are apparently not as potent a stimulus as co‐culture with muscle for the full expression of the cholinergic phenotype under these culture conditions.