The mechanical and pharmacological characteristics of the cholinergic activation of the smooth muscle in the choroidal coat of the chick eye have been assessed in tissues isolated from birds 1 d posthatching using histological, electrophysiological, and immunological techniques. The choroidal coat is innervated by a dense network of cholinergic nerves that make en passant synapses with smooth muscle. Thirty-hertz stimulation of these nerves initiates red blood cell (RBC) movement in the vessels of the choroidal coat, and this activation is blocked by muscarinic ACh receptor (AChR) antagonists. Force-transducer recordings of nerve-induced contractions of this tissue have a slow onset and relaxation time course similar to those of smooth muscle contractions. Furthermore, since nearly half the cholinergic neurons innervating the choroid die within a defined period during development, the onset and pharmacology of this innervation were studied during embryogenesis. With a neural cytoskeletal-like immunostain, we demonstrated that choroid axons are present in peripheral tissue by stage (St) 29. Extracellular electrical recordings made after choroid nerve stimulation allowed us to distinguish axon from muscle responses. These procedures permitted us to examine the time course of the innervation of the smooth muscle. However, to visualize the postsynaptic smooth muscle response, it was necessary to treat the isolated preparation with tetraethylammonium chloride (TEA). Accordingly, TEA-enhanced electrical smooth muscle responses to single-nerve stimuli could be recorded only after St 39. Treatment of the nerve-muscle preparation with prostigmine allowed the recording of TEA-enhanced electrical activity as early as St 36 (1 d after the beginning of the normal choroid neuron death period). This synaptic activation was completely blocked by atropine or quinuclidinyl benzylate (QNB), and was not affected by alpha bungarotoxin (alpha BTX), indicating that, as in the posthatching tissue, neuromuscular transmission is mediated by muscarinic receptors. These results show that cholinergic muscarinic activation of the choroidal coat can occur as early as St 36, but that it is not as efficient as transmission later in embryogenesis.
The development of the mechanical characteristics of contraction and the pharmacology of synaptic activation in chick iris and ciliary body were examined from embryonic day 9 through posthatching. The ciliary ganglion-target muscle system has proven to be a useful model for both in vivo and in vitro studies of neuron-target interactions; one such interaction is involved in neuronal cell death, which in the ciliary ganglion occurs from Stage (St) 34 to 40. To understand the mechanism by which cholinergic blocking agents prevent naturally occurring neuronal death in the chick ciliary ganglion (see the following paper, Meriney et al., 1987), it was necessary to determine the effect of these agents on synaptic transmission at target structures during the cell death period. Initially (St 34-36), iris muscle contraction are synaptically mediated via muscarinic ACh receptors (AChRs) on myoepithelial cells, which have the contractile and structural characteristics of smooth muscle. Close apposition of synaptic terminals, similar to that described for mature synapses, was observed on these myoepithelial cells. Subsequently (St 37), the striated muscle fibers that appear are activated by nicotinic receptors, although muscarinic AChRs are also present. Mechanically, this can be seen as gradually changing from a slow-onset contraction, elicited only by 30 Hz stimulation, to a fast-twitch response (St 37-44). Dilator fibers that develop later in the iris (at about St 39) also possess nicotinic and muscarinic receptors. The ciliary body musculature, although not extensively studied, also appears to have dual cholinergic activation during development. The mature iris has predominately striated muscle fibers that have both junctional nicotinic and muscarinic (mostly extrajunctional) AChRs. The dual presence of both receptor types in the same muscle fiber was confirmed with intracellular recordings, in which only the initial portion of the ACh-elicited depolarization was sensitive to alpha bungarotoxin (alpha BTX). In addition, specific muscarinic binding sites were described in the developing, as well as in the mature, iris. The developing chick iris was also shown to contract directly in response to light, this response disappearing after hatching. This unique dual-receptor pharmacology (nicotinic-muscarinic) and light response of a striated muscle may be due to the neural crest origin of these cells.
We have described in the preceding 2 papers the development of the pharmacological and contractile properties of all targets of the ciliary ganglion: the iris and ciliary body (Pilar et al., 1987), and the choroidal coat (Meriney and Pilar, 1987). In this paper, we examine the chronic effects of ACh receptor (AChR) blockade on ciliary ganglion neuron survival. Nicotinic or muscarinic AChR blockers were administered daily to developing chicken embryos during the normal neuronal death period in the ciliary ganglion. The effects of the blockers on ganglionic and neuromuscular transmission were assessed, and neuronal survival was assayed by counting both the total number of ganglion neurons and the selectively HRP-labeled ciliary neurons after the normal neuronal death period. Blockade of ganglionic transmission decreases survival in both populations of neurons. Blockade of neuromuscular muscular transmission increases survival in the ciliary population, which innervates the striated iris and ciliary body muscle. In contrast, blockade of synaptic activity has various influences on the survival of the choroid population, which innervates the smooth muscle of the choroid coat. Smooth muscle muscarinic receptor blockade with atropine does not influence survival. At higher doses (which block ganglionic transmission), atropine decreases choroid survival. Survival of the choroid population is increased by nicotinic blockade with 75 micrograms alpha bungarotoxin (alpha BTX), but decreased by 12.5 micrograms alpha BTX. Two main conclusions arise from these studies. Activation of postsynaptic AChRs in both the ganglion and the periphery are important in the regulation of neuronal survival. These effects usually occur in opposite directions: Blockade of ganglionic transmission decreases neuronal survival, while paralysis of neuromuscular transmission increases neuronal survival. This embodies the "balance" hypothesis (Cunningham, 1982) for neuronal survival, which states that motoneurons must balance afferent and target interactions during a critical period after synapses are formed in both regions. The present observations support this hypothesis. However, although both ciliary and choroid neurons have been shown to depend on the presence of the periphery for survival, target muscle paralysis via AChR blockade rescues the ciliary neurons but does not influence survival in the choroid population. Target-dependent regulation of choroid neuron survival during the normal neuronal death period is clearly different from the regulation of ciliary neuron survival.
Most studies on the trophic regulation of the normal neuronal competition for survival have focused on interactions between neurons and their target environment. However, it is also likely that trophic modulators are released from premotor inputs onto motoneurons. We have examined the developmental distribution of endogenous enkephalin-like immunoreactivity and the role that these endogenous opioid peptides play in normal neuronal degeneration. During the early portion of the normal cell death period, enkephalin-like immunoreactivity is highest within preganglionic cell bodies in the midbrain and their nerve terminals in the ciliary ganglion. Exogenous daily morphine administration to the chick embryo has previously been shown to delay most of the normal neuronal death in the ciliary ganglion (see Meriney et al., 1985). We hypothesized that opiate receptor activation increases the probability that ciliary ganglion neurons will survive their developmental competition and, further, that the endogenous opioid peptides in the ciliary ganglion normally modulate this competition. However, in our previous report (Meriney et al., 1985), we noted that daily administration of the antagonist naloxone to the chorioallantoic membrane did not significantly alter neuronal survival, as would have been expected if endogenous opioids were involved in regulating cell death. In contrast, in this report we show that three times daily application of naltrexone (a long-lasting opiate antagonist) significantly decreased neuronal survival among the ciliary ganglion cells, and that the surviving cells were not ultrastructurally different than neurons from controls of the same developmental stage. To control for toxic effects of naltrexone, we performed cell counts following naltrexone, we performed cell counts following naltrexone treatment in another population of cholinergic motoneurons (lumbar spinal motoneurons). In this population of cells, the total number of motoneurons remains unchanged following naltrexone treatment. To test for a specific toxic effect on the neurons of the ciliary ganglion, we generated a dose-response curve for toxicity in vitro and determined that naltrexone was not toxic over concentration ranges that are likely to exist in vivo. It appears, therefore, that a multiple daily antagonist application protocol blocks opiate receptors sufficiently in the ciliary ganglion to decrease an endogenous opiate influence significantly. We tested the possibility that endogenous opioids exert their effect by modifying transmission at peripheral and ganglionic synapses. In the generally accepted hypothesis, paralysis at the peripheral nerve-striated muscle synapse would rescue cells, while paralysis of ganglionic synapses would decrease survival. Iris neuromuscular junctions onto striated muscle cells were not blocked by opioids, but neuromuscular transmission in the smooth muscle of the choroid coat was blocked.(ABSTRACT TRUNCATED AT 400 WORDS)
Repeated administration of morphine in increasing doses delayed normal cell death in the ciliary ganglion of the chick embryo; the effect was completely blocked by naloxone. Survival of spinal motoneurons was not affected. Morphine also inhibited potassium-stimulated synthesis of acetylcholine in ganglion cells cultured with muscle, suggesting that morphine can influence neurotransmission. Morphine's effect on cell death may be due to an inhibition of transmission at the neuromuscular junction, but opiates may also directly affect cell death. Although it is now known whether the endogenous opiates in the ciliary ganglion influence neuronal survival during embryogenesis, exogenous opiates can affect normal cell death in the autonomic nervous system.
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