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)     

Kappa-Bungarotoxin: a probe for the neuronal nicotinic receptor in the avian ciliary ganglion

The interaction of snake alpha-neurotoxins with neuronal membranes has been examined in the chick ciliary ganglion. Some, but not all, alpha-neurotoxins block nicotinic transmission in this ganglion. alpha-Bungarotoxin (ABgT), the major alpha-neurotoxin in the venom of Bungarus multicinctus, does not block transmission at high concentrations (1.2 microM) although it binds (Kd = 1 nM) to a pharmacologically nicotinic site in the ganglion. A toxin (kappa-bungarotoxin, KBgT) has been purified from the venom of Bungarus multicinctus. KBgT has a molecular weight of 6500 daltons and a pI of 9.1. KBgT is a potent inhibitor of nicotinic transmission in the ciliary ganglion, producing a reversible (overal several hours) blockade at 75 nM. Pre-exposure of ganglia to 1.2 microM ABgT does not prevent the effects of KBgT, indicating that the blockade occurs at a site distinct from that recognized by ABgT. Binding of [125I]KBgT to ciliary ganglia reveals two binding sites: one which has previously been characterized by [125I]ABgT and one which is not identified by [125I]ABgT. Both of these [125I]KBgT binding sites are blocked following pre-treatment of ganglia with the irreversible nicotinic affinity agent bromoacetylcholine. A two-site model is proposed to account for these observations. One site (the ABgT binding site) is seen by both ABgT and KBgT, and has as yet no physiological function associated with it. The second site is recognized only by the physiologically active KBgT, and may represent binding of the toxin to the physiologically detected nicotinic receptor.     

Nicotinic acetylcholine receptor agonists promote survival and reduce apoptosis of chick ciliary ganglion neurons

The abundance, diversity, and ubiquitous expression of neuronal nicotinic acetylcholine receptors (AChRs) suggest that many are involved in functions other than synaptic transmission. We now report that a major AChR class promotes neuronal survival. The 10-day survival of ciliary ganglion neurons in basal culture medium (MEM) was approximately 35%, but increased to approximately 75% in MEM containing nicotine (MEM/Nic) or carbachol, an effect similar to that achieved by chronic depolarization with KCl. Pharmacological experiments revealed that agonist-enhanced survival requires activation of AChRs sensitive to alpha-bungarotoxin (alphaBgt). alphaBgt-AChRs partly support neuronal survival by limiting apoptosis since fewer apoptotic neurons were observed in MEM/Nic compared to MEM. Moreover, nicotinic survival support was not further enhanced by fibroblast growth factor, as seen for KCl, but increased to 100% by adding PACAP, a trophic neuropeptide present in the ganglion. These results indicate that alphaBgt-AChR activation regulates neuronal survival and suggest a mechanism involving reduced apoptosis and interaction with an endogenous neuropeptide growth factor.     

F-spondin regulates neuronal survival through activation of disabled-1 in the chicken ciliary ganglion

The extracellular membrane-associated protein F-spondin has been implicated in cell-matrix and cell-cell adhesion and plays an important role in axonal pathfinding. We report here that F-spondin is expressed in non-neuronal cells in the embryonic chicken ciliary ganglion (CG) and robustly promotes survival of cultured CG neurons. Using deletion constructs of F-spondin we found that the amino-terminal Reelin/Spondin domain cooperates with thrombospondin type 1 repeat (TSR) 6, a functional TGFβ-activation domain. In ovo treatment with blocking antibodies raised against the Reelin/Spondin domain or the TSR-domains caused increased apoptosis of CG neurons during the phase of programmed cell death and loss of about 30% of the neurons compared to controls. The Reelin/Spondin domain receptor - APP and its downstream signalling molecule disabled-1 are expressed in CG neurons. F-spondin induced rapid phosphorylation of disabled-1. Moreover, both blocking the central APP domain and interference with disabled-1 signalling disrupted the survival promoting effect of F-spondin. Taken together, our data suggest that F-spondin can promote neuron survival by a mechanism involving the Reelin/Spondin and the TSR domains.     

Concanavalin A inhibits nicotinic acetylcholine receptor function in cultured chick ciliary ganglion neurons

The effects of various lectins and toxins on neuronal nicotinic acetylcholine receptor function have been studied in primary cultures of chick ciliary ganglion neurons. Neuronal response to acetylcholine receptor activation was measured by a cation flux method at 4 degrees C in a high potassium-low sodium medium designed to stabilize membrane potential near zero, with acetylcholine as the agonist and cesium-137 as the tracer ion. Exposure to 1 mM acetylcholine for 30 s produced a 5-10-fold stimulation of cesium-137 influx. Acetylcholine-stimulated influx was inhibited more than 95% by 10 microM D-tubocurarine, but was insensitive to both 1 microM tetrodotoxin and 1 microM alpha-bungarotoxin. Concanavalin A (50 micrograms/ml) inhibited agonist-induced ion flux by 80% at 4 degrees C. Succinyl-concanavalin A was ineffective at concentrations up to 250 micrograms/ml, and could not protect against the concanavalin A inhibition. However, inhibition by concanavalin A was eliminated by prior incubation of the lectin with 0.2 M alpha-methyl-D-mannoside and subsequent co-incubation with the sugar. Wheat germ agglutinin, lentil lectin, cholera toxin and tetanus toxin were without effect at either 4 degrees C or 37 degrees C. These results suggest a specific interaction between concanavalin A and neuronal nicotinic acetylcholine receptors.     

Distribution of acetylcholine in the normal and denervated pigeon ciliary ganglion

In the normal rat the axons of the parvicellular neurosecretory system form terminals which are characterised by dense core vesicles of 80–160 nm diameter and which lie against the fenestrated portal capillaries in the upper neurohypophysis (median eminence). The magnocellular neurosecretory axons traverse the interanal zone of the median eminence and form terminals against the fenestrated capillaries of the distal neurohypophysis (neural lobe). In the neural lobe it is assumed that the more electron dense vesicles (of around 200 nm diameter) characterise the vasopressin containing terminals of neurones in the supraoptic nucleus and that the less numerous terminals with the more electron lucent vesicles (also of around 200 nm diameter) belong to the axons of the paraventricular neurones and contain oxytocin.For the first few days after hypophysectomy, the proximal stumps of the cut neurosecretory axons in the internal zone of the median eminence are distended with neurosecretory material while axonal sprouts which are rich in neurotubules and completely ensheathed in satellite cell cytoplasm gradually penetrate the external zone. Subsequently there is a massive hypertrophy of fenestrated capillaries and the newly formed axonal sprouts progressively lose their satellite cell cover and thus acquire neurohaemal contacts with the outer basement membrane of the perivascular space. The development of these contacts is associated with the appearance of increasing numbers of synaptic vesicles and prejunctional dense projections. This reconstitution of the morphological features of the normal neurohaemal contacts is associated with recovery of antidiuretic function.In the hypertrophic median eminence the neurosecretory axon terminals with the electron lucent type of vesicles far outnumber those with the electron dense vesicles. It is suggested that the greater degree of cell loss from the supraoptic as opposed to the paraventricular nuclei puts the paraventricular axons at an advantage in competing for space on the basement membrane around the perivascular space of the newly formed capillaries.It is suggested that an important factor in promoting the regeneration of the neurosecretory axons is the remarkable hypertrophy of the fenestrated capillaries against which they come to terminate.     

Differential co-localization of nicotinic acetylcholine receptor subunits with calcium-binding proteins in retinal ganglion cells

Immunohistochemistry was used to examine the co-occurrence of nicotinic acetylcholine receptor subunits with calcium-binding proteins in ganglion cells of the chick retina. The α3 subunit was rarely observed in ganglion cells containing calbindin, calretinin, or parvalbumin. On the other hand, the α8 subunit was more often co-localized with all calcium-binding proteins studied. These results may be related to the high calcium permeability of nicotinic receptors that contain the α8 subunit.     

On the neuronal origin of the afferents to the ciliary ganglion in cat

The histological constitution of the oculomotor nuclear complex and the surrounding structures of cat was re-examined, and it was found that the so-called Edinger-Westphal (EW) nucleus and the anteromedian nuclei consisted of oval cells of small sizes and polygonal cells of medium sizes. The latter cells were scattered in the peripheral portions of the EW and the anteromedian nuclei, and were frequently intermingled with cells of the same type scattered in the central gray and the medial portion of the ventral tegmental area (VTA). Horseradish peroxidase (HRP) was injected into the ciliary ganglion, in order to locate the cells of origin of afferents to this ganglion. The cells labeled by an injection into the ganglion were these polygonal cells of medium sizes scattered in the injected side of midbrain. They weremainly distributed in the VTA, the central gray, and the caudal half of the anteromedian nucleus. The peripheral portion of the EW and the anteromedian nuclei also contained some labeled cells, but these were considered to be cells which had migrated from the central gray and the VTA. Most of the cells in the EW and the anteromedian nuclei were labeled by the injection into the spinal cord. Hence, it was concludedthat the main sources of the afferents to the ciliary ganglion were polygonal cells of medium sizes scattered in the VTA and the central gray, and some of these intermingled with the cells of peripheral boundaries of the EW and the anteromedian nuclei. By contrast, the greater part of the EW and the anteromedian nuclei had a connection with the spinal cord, but not with ciliary ganglion.     

Functional blockade of neuronal acetylcholine receptors by antisera to a putative receptor from brain

Antisera to a putative acetylcholine receptor purified from chick brain specifically inhibit the acetylcholine response of chick ciliary ganglion neurons in cell culture. The putative brain receptor and a similar membrane component previously identified on ciliary ganglion neurons appear to be functional nicotinic acetylcholine receptors in the nervous system and are clearly distinct from membrane components in the tissues that bind alpha-bungarotoxin.     

Acetylcholine receptor expression in developing chick ciliary ganglion neurons

Little is known about the levels of nicotinic ACh receptors (AChRs) in neurons prior to innervation and whether the distribution and number of receptors change in response to innervation. In the present study, AChR levels were examined in developing chick ciliary ganglion neurons in situ at stages preceding and during early and late phases of synaptogenesis. AChRs were localized in surface and intracellular pools of intact and saponin-permeabilized ganglionic neurons, respectively, by using a highly sensitive immunocytochemical approach that included the binding of an anti-AChR monoclonal antibody (mAb) followed by a biotinylated secondary antibody and an avidin-biotinylated HRP complex. At older stages of development, embryonic day (ED) 7-7.5 and ED 11, when all of the neurons are known to be receiving synaptic contacts, AChRs were present in both internal and surface pools. Within the neurons, AChRs were associated with organelles that function in the biosynthesis, processing, and transport of integral plasma membrane proteins. On the surface of the neurons, AChRs were predominantly localized in the specialized postsynaptic membrane, with low levels of AChRs being present in extrasynaptic regions. The earliest stage at which synapses could be detected in the ganglion was ED 4.5. Synapses were detected by light microscopic immunocytochemical labeling with anti-SV2, an mAb to a synaptic vesicle protein, and by ultrastructural analysis. At this stage, most of the neurons were not labeled by the anti-AChR mAb, while a few neurons had dense deposits of reaction product on the rough endoplasmic reticulum and portions of the nuclear envelope. Low levels of reaction product were also found on the surface of a small number of neurons, being localized predominantly on the specialized postsynaptic membrane of the few immature synapses present. Occasionally, small patches of labeling were observed in extrasynaptic regions. In contrast, little internal and no surface anti-AChR immunolabeling was detected in ciliary ganglion neurons prior to innervation, at ED 3.5-4. The finding of a large increase in both internal and surface AChR levels in the neurons at the time of innervation suggests that signals from the presynaptic input play an important role in the induction of AChR expression in neurons.     

Presynaptic calcium currents evoking quantal transmission from avian ciliary ganglion neurons

Using whole-cell patch clamp techniques, we simultaneously recorded presynaptic Ca++ current and excitatory postsynaptic currents (EPSCs) from avian neuromuscular junctions in culture. Quantal synaptic transmission was proportional to evoked presynaptic Ca++ current except with large stimuli, which evoked bursts of quanta, reflecting a shift to synchronized release. Synaptic delay, measured from the onset of presynaptic depolarization to the appearance of the first postsynaptic quantal response, was often greater than 100 msec for weak depolarizations but declined as stimulus intensity was increased. Quantal events evoked by Ca++ tail currents had a mean synaptic delay of 1.67 msec. The single type of presynaptic Ca++ current observed displayed an inactivation time constant of greater than 100 msec and tail currents well fit by a single exponential function.     

Non-cholinergic excitation in neurons after a chronic glutamate receptor blockade

Previous experiments revealed a dramatic increase in excitatory acetylcholine transmission in hypothalamic cultures during a chronic decrease in glutamate activity. Data suggested that in the absence of glutamate excitation, acetylcholine becomes the major excitatory neurotransmitter. However, non-cholinergic excitatory activity was also detected in some neurons. Here, using calcium imaging in hypothalamic cultures chronically subjected to the glutamate receptor blockade, we demonstrate the contribution of metabotropic glutamate receptors, P2-purinoreceptors, histamine receptors, adrenoreceptors, and gap junctions, but not nitric oxide to this non-cholinergic excitation. We also show that the sensitivity of neurons to receptor agonists is increased following the blockade. Data suggest that multiple components contribute to the excitatory activity in hypothalamic neurons during a long-term decrease in glutamate activity.     

Properties of choroid and ciliary neurons in the avian ciliary ganglion and evidence for substance P as a neurotransmitter

Intracellular recordings were made from identified choroid and ciliary neurons in the ciliary ganglion of the embryonic chick. Choroid neurons, which were innervated by multiple preganglionic fibers, frequently displayed noncholinergic slow excitatory postsynaptic potentials (EPSPs) following repetitive stimulation of the preganglionic nerve trunk. These slow potentials were blocked by high Mg2+/low Ca2+ buffer and were closely mimicked by bath application of substance P, which is known to be present within both populations of preganglionic nerve terminals. Substance P-induced depolarizations desensitized during prolonged exposure, at which time it was no longer possible to evoke slow synaptic potentials. Following manual voltage clamp to resting membrane potential, parallel increases in input resistance were seen during the slow EPSP and the response to substance P, suggesting that the two responses share common mechanisms. Ciliary neurons, which were innervated by a single preganglionic fiber and displayed dual electrical-chemical synapses, did not exhibit slow synaptic potentials and were unaffected by bath application of substance P. The magnitude and time course of fast nicotinic EPSPs elicited in ciliary neurons by 0.5 Hz presynaptic stimulation were also unchanged in the presence of 1 to 3 microM substance P. Although the ciliary and choroid neurons share a common embryological origin in the neural crest, they are specialized for quite different physiological roles. Integration of multiple presynaptic inputs occurs at choroid synapses, mediated by the presence of both subthreshold fast nicotinic EPSPs and the slow EPSP. In contrast, synapses on ciliary neurons have specializations which preclude any integrative function, including single innervation, a high quantal content, electrical coupling potentials, and a lack of slow synaptic potentials.     

Regulation of muscarinic acetylcholine receptor number in cultured neuronal cells by chronic membrane depolarization

The effect of chronic membrane depolarization on the regulation of muscarinic acetylcholine receptor (mAChR) number was studied in neuroblastoma cells (clone N1E-115). Receptor number was determined by a filter binding assay using 3H-quinuclidinyl benzilate (QNB) in membrane and crude cellular homogenates. Incubation with 50 microM veratridine (VTN), an activator of voltage-sensitive Na+ channels, induced a 50-200% increase in mAChR number at 24 hr, which was inhibited 80% by TTX. Scatchard analysis showed that affinity of the mAChR for 3H-QNB was not affected by VTN. Upon withdrawal of VTN, mAChR number returned to control levels within 20 hr. Chronic membrane depolarization caused by incubation in medium containing 60 mM K+ induced a TTX-insensitive 50% increase in mAChR number at 24 hr. AChE activity was unaffected by chronic membrane depolarization. The VTN-induced increase in mAChR number was not blocked by coincubation with cycloheximide or tunicamycin, both inhibitors of de novo mAChR synthesis. The rate of mAChR degradation was reduced in the presence of 50 microM VTN, with the apparent half-life increased from approximately 18 hr (control) to approximately 40 hr (VTN). Although treatment with either 1 mM 8Br-cAMP or 1 mM 8Br-GMP failed to increase mAChR number, treatment with either the inorganic Ca2+ channel blocker Co2+ (1 mM) or the organic Ca2+ channel antagonist D600 (10-100 microM) produced 40-80% increases in mAChR number. The combination of VTN and either D600 or Co2+ failed to induce a greater increase in mAChR number than incubation with VTN alone.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro identification of dividing neuronal precursors from chick embryonic ciliary ganglion

In chick parasympathetic ciliary ganglion the neuronal birthdate is well defined, between 2.5 and 5.5 days of embryonic development, and neuronal precursor cells that are able to differentiate into neurons in vitro can be isolated from E4.5 ganglia. In this report, using bromodeoxyuridine incorporation and Maplb immunostaining, we demonstrate that these cells can be isolated from E7-E8 chick embryos as well, suggesting that neuronal precursor cells are still present in the ciliary ganglion after the end of the in vivo neurogenesis. These precursor cells retain the ability to divide and generate newly differentiated neurons in vitro when cultured in a chemically defined medium. Such a capacity is highly stimulated by bFGF but not by CNTF.     

Alteration in the regulation of neuronal muscarinic acetylcholine receptor number induced by chronic lithium in neuroblastoma cells

Because previous studies in whole-animal models have reported inconsistent results regarding the effect of chronic lithium on the regulation of the neuronal muscarinic acetylcholine receptor (mAChR) number, we examined the effect of chronic lithium on the regulation of mAChR in cell cultures of N1E-115, a mouse neuroblastoma clone. Li+ induced a concentration- and time-dependent increase in the mAChR number, with a 30% increase in specific [3H]quinuclidinyl benzilate binding in membrane homogenates induced by a 5-day incubation with 10 mM Li+. Agonist-induced down-regulation of the mAChR number was also inhibited by lithium: chronic treatment with 10 mM Li+ caused a 25-35% reduction in the magnitude of carbachol. In contrast, the decrease in the mAChR number induced by the synergistic action of A23187 (300 nM) and phorbol myristate acetate (300 nM) was unaffected by Li+. These results demonstrate that chronic treatment with Li+ increases the basal mAChR number and dampens the decrease in receptor number induced by a muscarinic agonist in neuroblastoma cells. The implications of these results in understanding the functional regulation of neuronal mAChR number are discussed.     

Cultured smooth muscle targets lack survival activity for ciliary ganglion neurons

Cultured neurons require specific trophic agents in order to survive. This dependence is thought to resemble the neuron-target interdependence that develops in vivo during synaptogenesis and neuronal cell death. The notion that neurons in general derive trophic support from their synaptic targets is based primarily on studies of peripheral neurons and motor neurons. To assess the general applicability of this nerve-target relationship, we tested the ability of vascular smooth muscle (VSM) to support dissociated neurons from the chick ciliary ganglion. The ciliary ganglion contains 2 distinct neuronal populations, one of which innervates striated muscle, the other VSM. Striated muscle cocultures are known to support all of the neurons in the ganglion for extended periods. Dissociated neurons were therefore cocultured in microwells containing VSM derived from the rat or chick aorta and from the choroid coat of the chick eye. Surviving neurons were counted after 1, 2, 5, and 7 d. Striated muscle is able to support full neuronal survival in the same assay. However, in no case was VSM capable of contributing to neuronal survival in vitro. The neurons in the VSM cocultures were able to form neurites and make contacts with their putative targets, as confirmed by scanning electron and light microscopy. The presence of viable and differentiated smooth muscle cells was demonstrated in the cultures by transmission electron microscopy and analysis of smooth muscle alpha-actin. The failure of VSM and even the choroid target tissue to support the survival of their innervating neurons suggests that novel mechanisms may operate to provide trophic support for neurons innervating VSM targets.     

The properties and regulation of functional acetylcholine receptors on chick ciliary ganglion neurons

The properties of acetylcholine receptor (AChR) channels on chick ciliary ganglion neurons in culture were examined using patch-clamp recording techniques. Acetylcholine (ACh) was applied by rapid microperfusion. Whole-cell current noise analysis revealed a single class of functional receptors on the neurons. Dose-response studies indicated a Kd of about 36 microM and a Hill coefficient of 1.5-1.7, predicting 2 ACh binding sites per receptor. Both fast and slow components of receptor desensitization were observed. Single-channel recordings from excised outside-out patches of soma membrane exposed to 2-5 microM ACh indicated a single-channel conductance of 40 pS, a reversal potential of -9 mV, a mean open duration of 1 msec, and an opening probability of 0.34. The kinetic behavior of the channels was provisionally described by a 3-closed, 1-open state model for receptor activation. In all of these properties, AChRs of ciliary ganglion neurons resemble those on skeletal muscle fibers. Growing the neurons in an elevated K+ concentration produced a 2-3-fold decrease in peak whole-cell currents induced by ACh under standard test conditions, without altering any of the single-channel properties described above. Neither changes in cholinesterase activity nor receptor distribution accounted for the decrease. Instead, calculations indicated that elevated K+ reduced the ACh response by decreasing the number of functional AChRs on the neurons. No K+-dependent decrease is observed, however, in the number of total receptors on the neurons detected either by a monoclonal antibody specific for the receptor or by an alpha-neurotoxin that binds to the receptor and blocks its function. Moreover, the number of receptors detected by the 2 probes is at least 10-fold greater than the calculated number of functional receptors. The findings suggest that only a small fraction of the AChRs on the neuronal surface is functional and that the cell can alter the ratio of functional and nonfunctional receptors in response to growth conditions.