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G Pilar

Publications and source records attributed to G Pilar.

At least 19 recordsLinked to original sources
PubMed2001

Restoration of mitochondrial function reverses developmental neuronal death in vitro.

In a previous study characterizing morphological and functional features of cell death in trophically deprived chick ciliary ganglion neurons (Pena and Pilar [2000] J. Comp. Neurol. 424:377-396), we hypothesized that early cell death events might be targets for reversal, allowing for rescue of dying neurons. To test this hypothesis, ciliary ganglion (CG) neurons were cultured with or without trophic support (choroid, iris, and pigment epithelium soluble extract [CIPE]), or without trophic support for 11 or 18 hours and then exposed to trophic support. Prior to and at the onset of cell death commitment (11 hours) CIPE-deprived neurons exhibited increased membrane permeability, blebbing, cytoplasmic vacuolization, swollen mitochondria, low adenosine triphosphate levels, and release of cytochrome c from mitochondria. CIPE readdition at 11 hours reversed these changes. Between 11 and 18 hours, irreversible DNA fragmentation increased in CIPE-deprived neurons. Cyclosporin A and bongkrekic acid (inhibitors of mitochondrial transition permeability pores) prevented membrane permeability increases and delayed the progression to death in trophically deprived neurons by 12 hours; however, by 48 hours all neurons had died. BOC-Asp-CH2F (BAF), a pan-caspase inhibitor, did not prevent early events of cell death including increased membrane permeability and Cyto c release, but it inhibited DNA fragmentation and prolonged neuronal survival to 48 hours. We conclude that mitochondria changes occur early, prior to commitment and that the suppression of these changes can prevent all the downstream events of death, whereas caspase inhibitors have no effect on the early mitochondria/plasma membrane changes. Mitochondria thus play a critical role in the transition from reversible to irreversible commitment to developmental neuronal death. Furthermore, neuronal death is brought about by activation of one of two distinct pathways, one localized in mitochondria and the other dependent on activation of caspases.

Adenosine Triphosphate↗
PubMed2000

Early morphologic alterations in trophically deprived neuronal death in vitro occur without alterations in cytoplasmic Ca(2+).

Morphologic and functional parameters altered during neuronal death were investigated in chick ciliary ganglion (CG) neurons in vitro isolated from embryonic day 8 (E8, stage 34). Neurons were separated from nonneuronal cells to investigate their inherent cell death program and were cultured with or without trophic support (choroid, iris, pigment epithelium) from their appropriate target tissue. The cell death process was characterized with investigations focused on the earliest events at the onset of commitment to cell death at 11 hours after plating. Initial morphologic changes in the process of cell death were cytoplasmic; swelling, dendritic retraction, blebbing, vacuolization, which are all characteristics of necrosis. Later, nuclear chromatin condensation occurred, a characteristic of apoptosis. An increase in membrane permeability was measured earlier at 8 hours. During these alterations (associated with the initiation of cell death) single cell analysis was performed to evaluate mobile Ca(2+) changes in the same trophically deprived neurons during the course of the death process; Ca(2+) levels remained at 50 nM. Transient Ca(2+) entry was buffered in control and deprived cells at 13 hours but with different parameters. During the execution stage of death mobile Ca(2+) levels were variable. In this final stage of cell death, neurons demonstrated nuclear damage, cytosol disintegration, or morphology sharing both characteristics. These observations define embryonic CG cell death in vitro as neither purely apoptotic nor necrotic but a form of that exhibits features of both. These results also demonstrate a disassociation of changes in cytosolic Ca(2+) from both CG neuronal survival and trophically deprived cell death in vitro.

Animals↗
PubMed1999

kappa- and mu-opioids reverse the somatostatin inhibition of Ca2+ currents in ciliary and dorsal root ganglion neurons.

Neuromodulators, including transmitters and peptides, modify neuronal excitability. In most neurons, multiple neuromodulator receptors are present on a single cell. Previous work has demonstrated either occlusive or additive effects when two neuromodulators that target the same ion channel are applied together. In this study, we characterize the modulation of Ca2+ and K+ channels in embryonic chick ciliary ganglion neurons by somatostatin (Som) and opioids, including the effects of these neuromodulators when applied in combination. We report a modulation of calcium current by kappa- or mu-opioids that can prevent Som effects when applied before Som and can replace Som effects when applied after Som. We term these effects demodulation because they do not have the characteristics of simple occlusion but rather represent a dominant effect of opioid-mediated modulation of calcium channels over Som-mediated modulation. These opioid effects persist in the presence of kinase and phosphatase inhibitors, as well as after alteration of the intracellular Ca2+ concentration. Furthermore, they are present in both whole-cell and perforated-patch recording configurations. These effects of opioids on Som-mediated modulation do not seem to be mediated by a general uncoupling of Som receptors from G-protein-coupled signaling systems because K+ current modulation by Som can persist in the presence of opioids. Demodulation by opioids was also observed in dorsal root ganglion neurons on the modulation of calcium current by GABA and norepinephrine (NE). In both preparations, this demodulatory interaction occurred between voltage-independent (opioids) and voltage-dependent (Som, GABA, and NE) modulatory pathways.

Animals↗
PubMed1996

Membrane delimited and intracellular soluble pathways in the somatostatin modulation of ACh release.

The signal transduction cascade between the activation of the somatostatin (SOM) receptor and modulation of transmitter release was study using Acetylcholine (Ach) release measurements and patch clamp recordings of Ca2+ current from acutely dissociated St 40 ciliary ganglion neurons. As in intact synapses, somal ACh release was blocked by 100 nM SOM or 100 microM dibutyril cGMP, and the SOM-mediated inhibition could be reversed by 10 microM 1-NAME (a selective inhibitor of nitric oxide synthase, NOS) or 100 microM Rp-8p-CPT-cGMPs (a selective inhibitor of a cGMP protein dependent kinase, PKG). In whole cell recordings, SOM inhibition of Ca2+ current rapidly relaxes to control levels but is sustained in perforated patch recordings which decreases cell dialysis. Inhibition of NOS or PKG in perforated patch recordings, however caused SOM effects to become transient again. We hypothesize that PKG alters the characteristics of the membrane-delimited G protein inhibition of Ca2+ current. Therefore SOM receptors trigger a membrane-delimited signal transduction cascade that is modulated by soluble messengers, converging on voltage activated Ca2+ channels. When both pathways are active together, SOM causes a sustained inhibition of neuronal Ca2+ current leading to a decrease in transmitter release.

Acetylcholine↗
PubMed1993

Specific in vitro biological activity of snake venom myotoxins.

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 micrograms/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 micrograms/ml, destroyed all cell types. However, at 10 micrograms/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.

Animals↗
PubMed1993

Choroid tissue supports the survival of ciliary ganglion neurons in vitro.

It is well established that during in vivo development the neurons of the avian ciliary ganglion are dependent for their survival on structures in the eye. Separate neuron populations innervate intraocular smooth and striated muscle targets. All ciliary neurons survive when cocultured with striated muscle. We demonstrate that when ciliary ganglion neurons are plated on explants of the choroid coat (a smooth muscle-containing target tissue) using a defined medium (N2), the neurons survive and grow vigorously into the tissue, forming contacts between axons and target cells identified as smooth muscle. Conditioned medium from choroid explants also rescues all the neurons, as does coculturing ciliary ganglion neurons with dissociated choroid cells. However, the presence of horse serum and chick embryo extract in the medium inhibits the choroid's ability to support ciliary neurons. The effects of these additives on the phenotypic expression of the smooth muscle may explain the inability of previous investigators to demonstrate target-derived support from smooth muscle preparations. Because the choroid contains cell types other than smooth muscle (e.g., fibroblasts and endothelial cells), we could not identify smooth muscle as the only cell type responsible for the release of the soluble trophic factor present in the target tissue. However, indirect evidence using avian primary fibroblast cultures, a fibroblast cell line, and an anatomically simple smooth muscle preparation, the avian amnion, suggests that smooth muscle cells are sufficient to account for the observed trophic activity, and that similar target-derived molecules support the survival of both types of ciliary ganglion cells.

3T3 Cells↗
PubMed1992

Pharmacological and kinetic properties of alpha 4 beta 2 neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes.

1. Co-injection of RNA synthesized from cloned neuronal acetylcholine receptor (nAChR) subunits (alpha 4 and beta 2) in Xenopus oocytes produced functional receptors. In macroscopic voltage-clamp experiments, the agonist-induced current exhibited a strong inward rectification. 2. Voltage jumps from +50 mV to more negative potentials produced relaxations of the agonist-induced current with a single exponential time course. The relaxation rate constant was only weakly voltage dependent. 3. At the single-channel level, three conductances were recorded of 12, 22 and 34 pS. Their burst durations were similar and varied only weakly with voltage (e-fold for 120 to 370 mV), consistent with the poorly voltage-dependent relaxation rate constants. However, the burst durations were less than 10 ms, or less than 1/5 the value expected from voltage-jump relaxations. 4. Hexamethonium (Hex, 0.5 to 8 microM) inhibited the agonist-induced current and produced voltage-jump relaxations characterized by a rapid conductance increase and a slower conductance decrease. Analysis of these relaxations suggested that the Hex-receptor interaction is open-channel blockade characterized by a forward binding rate of 1 x 10(7) M-1 s-1 and a dissociation rate constant of about 25 s-1. 5. For the relaxations produced by QX222, the slowest phase was a conductance increase, suggesting that the dissociation rate constant for QX222 is 10-30-fold greater than for Hex. 6. Hex but not QX222 produced an additional use-dependent blockade that was manifest during repetitive hyperpolarizing pulses. 7. With mouse muscle ACh receptors expressed in oocytes, the blockade by Hex did not depend strongly on voltage. Neither Hex nor QX222 produced appreciable use-dependent block on muscle ACh receptors. 8. Of the four conditions studied (neuronal and muscle receptors, Hex and QX222), only the blockade of the neuronal AChR by Hex is characterized by a residence time longer than the normal open time. 9. It is concluded that the modest differences in primary amino acid sequence between muscle and neuronal receptors lead to profound changes in their interactions with channels blockers.

Acetylcholine↗
PubMed1991

Endogenous opioids modulate neuronal survival in the developing avian ciliary ganglion.

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)

Animals↗
PubMed1990

Endogenous modulation of ACh release by somatostatin and the differential roles of Ca2+ channels.

The classical neurotransmitter acetylcholine (ACh) and the potential modulatory peptide somatostatin are colocalized in terminals of avian choroid neurons. We previously showed that exogenous somatostatin inhibits ACh release in the choroid coat (Gray et al., 1989b). In the present work we determine whether endogenous somatostatin plays a role in neuromodulation and what mechanisms are involved. To determine its role and its mode of secretion, voltage-sensitive Ca2+ channels in these terminals were identified pharmacologically using Ca2(+)-dependent K(+)-evoked ACh release. Release of the primary transmitter ACh was triggered in the presence of high K+ by Ca2+ influx primarily via dihydropyridine (DHP)-insensitive channels, while inhibition of ACh release occurred when L-type channels were activated by the DHP agonist Bay K 8644. The somatostatin antagonist cyclo(7-aminoheptanoyl-phe-D-trp-lys-thr (BZL)) (CyCam) blocks the inhibition of ACh release induced by the agonist Bay K 8644 and indicates that endogenous somatostatin may normally modulate ACh release. Additionally, nifedipine, a DHP antagonist, and pertussis toxin, known to antagonize somatostatin's effect on ACh release, both reverse the Bay K 8644 effect on ACh release. Although the release of labeled ACh in the first 3 min collection period was not significantly affected by CyCam or nifedipine alone, release in the first minute was enhanced by 50% in the presence of 10 microM nifedipine. Preincubation with CyCam alone also increased ACh release. These results support the hypothesis that endogenous somatostatin is physiologically released during the initial minute of depolarization in high K+ and that this release is mediated by DHP-sensitive Ca2+ channels.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1987

Muscarinic and nicotinic synaptic activation of the developing chicken iris.

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.

Animals↗
PubMed1987

Differential neuronal survival in the avian ciliary ganglion after chronic acetylcholine receptor blockade.

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.

Animals↗
PubMed1987

Cholinergic innervation of the smooth muscle cells in the choroid coat of the chick eye and its development.

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.

Animals↗
PubMed1985

Morphine-induced delay of normal cell death in the avian ciliary ganglion.

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.

Acetylcholine↗
PubMed1983

Target influences on [3H]ACh synthesis and release by ciliary ganglion neurons in vitro.

The developmental influence of neuron-target interaction upon transmitter synthesis from labeled precursor and the capacity to release labeled transmitter were examined in dispersed cell cultures of embryonic ciliary ganglion neurons by comparing cultures of neurons plated alone and neurons plated upon pectoral myotubes. Of the total ACh synthesized from radiolabeled choline by neurons plated alone, more than half is via a Na+-dependent path, but a larger fraction of the synthesis is Na+ insensitive in culture than in mature neurons in vivo. In addition, at 1 week in culture the neurons lacking target failed to significantly increase ACh synthesis from the labeled choline in response to a previous high [K+]0 depolarization. Synthetic responsiveness to depolarization is a characteristic of mature nerve terminals in this preparation. One week after plating neurons onto myotube cultures, synthesis of ACh from the exogenous precursor is double that of sibling cultures lacking muscle, and prior depolarization with [K+]0 results in an increase in labeled product. Release from the labeled transmitter pool by the neurons with myotubes was also enhanced. [3H]ACh release elicited by depolarization via a Ca2+-dependent mechanism was more than fivefold higher in the cocultures. The influence of coculture with myotubes upon neuronal development is not duplicated by the neurons themselves despite formation of apparent interneuronal synapses (G. Crean, G. Pilar, J. Tuttle, and K. Vaca, 1982, J. Physiol. (London). 331, 87-104), by "fibroblasts" or medium conditioned over myotube cultures. Neurons under these conditions neither increase synthesis of [3H]ACh in response to a prior depolarization nor demonstrate enhanced basal [3H]ACh synthesis and release. Thus, coculture of embryonic ciliary ganglion neurons with a striated muscle target has a somewhat specific inductive effect, enhancing the capacity for neuronal [3H]ACh synthesis and release toward mature levels. This influence of a readily accessible target upon ciliary neuron cholinergic development in vitro may reflect a normal neuromuscular interaction occurring during embryogenesis.

Acetylcholine↗
PubMed1982

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.

Acetylcholine↗