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)     

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.     

Effect of ciliary neuronotrophic factor on somatostatin expression in chick ciliary ganglion neurons

The normal development of somatostatin (SOM) expression in neurons of the chick ciliary ganglion and the effects of ciliary neuronotrophic factor (CNTF) on SOM induction in cultured ciliary ganglion neurons, were studied by immunocytochemical techniques. SOM immunoreactivity was first detectable in some neurons of the ganglion at embryonic day (E)8 and between E14 to hatch, 44–46% of the neuronal population contained the peptide. It was inferred that essentially all choroid neurons, which constitute 50% of the neuronal population, contain SOM. Culture studies indicated that CNTF supported both the SOM positive choroid neurons and the SOM negative ciliary neurons. Although CNTF was necessary for the survival and maturation of cultured ciliary ganglion neurons, it did not influence either the induction or maintenance of SOM expression in these neurons. CNTF may instead act as a permissive factor, allowing the induction of SOM in neurons of the ciliary ganglion by other, more specific, factors.     

Enhancement of naturally occurring cell death in the sympathetic and parasympathetic ganglia of the chicken embryo following blockade of ganglionic transmission

Both presynaptic and postsynaptic blockade of ganglionic transmission during the period of naturally occurring ganglion cell death reduced the number of surviving neurons in the sympathetic ganglia (SG) and ciliary ganglion (CG). The CG was chosen for analysis because there was a temporal separation between cell proliferation and death in the CG but not in the SG. Ganglion cell proliferation and migration were unaffected by ganglionic blockade. The increased ganglion cell loss that followed ganglionic blockade was accompanied by an increased number of degenerating cells. These results indicate that the decreased number of healthy ganglion cells that followed ganglionic blockade was the result of enhanced naturally occurring cell death.     

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.     

Intrinsic choroidal neurons in the duck eye express galanin

Recently, it has been shown that the choroid of the duck eye harbours approximately 1,000 intrinsic choroidal neurons positive for vasoactive intestinal polypeptide and neuronal nitric oxide synthase. Their connections and functional significance are largely unknown. This study was performed to establish a typical chemical code for these neurons and to define their targets by using immunocytochemistry and confocal laser scanning microscopy. Almost all intrinsic choroidal neurons coexpressed galanin (GAL), vasoactive intestinal polypeptide (VIP), and neuronal nitric oxide synthase (nNOS)/NADPH-diaphorase. A few stained for GAL and/or nNOS only. Among extrinsic ganglia, GAL/VIP/nNOS coexpressing neurons were only found in the pterygopalatine ganglion where they accounted for approximately 30% of the neuronal population. Thus, GAL/VIP/nNOS-positive nerve fibres around branches of the ciliary artery and within the nonvascular smooth muscle stroma of the choroid may originate mainly from intrinsic neurons and to some extent in a subpopulation of pterygopalatine ganglionic neurons exhibiting the same chemical coding. Close contacts of GAL-positive fibres upon intrinsic choroidal neurons may indicate reciprocal connections between them. Thus, intrinsic choroidal neurons may represent peripherally displaced pterygopalatine ganglion neurons forming a local network for regulation of vascular and nonvascular smooth muscle tone in the duck choroid. They may be integrated in the neuronal circuitry controlling intraocular pressure, choroidal thickness, accommodation, and axial bulbus length.     

Maturation of postsynaptic nicotinic structures on autonomic neurons requires innervation but not cholinergic transmission

Postsynaptic development at the neuromuscular junction depends on nicotinic transmission and secreted components from the presynaptic motor nerve terminal. Similarly, secreted components and synaptic activity are both thought to guide development of glutamatergic synapses in the CNS. Nicotinic synapses on chick ciliary neurons are structurally complex: a large presynaptic calyx engulfs the postsynaptic neuron and overlays a series of discrete mats of receptor-rich somatic spines tightly interwoven and folded against the soma. We used fluorescence imaging of alpha 7-containing nicotinic receptors and the spine constituent drebrin to monitor postsynaptic development. The results show that surgical disruption of the preganglionic input or removal of the ganglionic synaptic target tissue after synapses form in the ganglion does not disrupt the receptor-rich spine mats. Similarly, removal of the target tissue even prior to synapse formation in the ganglion does not prevent subsequent formation of the receptor clusters and associated spine constituents. Postsynaptic development is arrested, however, if normal innervation is prevented by ablating the preganglionic neurons prior to synapse formation. In this case the neurons express reduced levels of nicotinic receptors and cytoskeletal components and organize them only into early-stage clusters. Even low levels of residual innervation, however, can restore much of the normal postsynaptic receptor patterns. Chronic pharmacological blockade of cholinergic synaptic activity fails to replicate the effects of ablating the preganglionic nucleus. The results indicate that ciliary neurons are programmed to express postsynaptic components and can initiate clustering of alpha 7-containing receptors but need presynaptic guidance for maturation of the postsynaptic structure.     

Invited Article: Autonomic ganglia: target and novel therapeutic tool

Nicotinic acetylcholine receptors (AChR) are ligand-gated cation channels that are present throughout the nervous system. The muscle AChR mediates transmission at the neuromuscular junction; antibodies against the muscle AChR are the cause of myasthenia gravis. The ganglionic (alpha 3-type) neuronal AChR mediates fast synaptic transmission in sympathetic, parasympathetic, and enteric autonomic ganglia. Impaired cholinergic ganglionic synaptic transmission is one important cause of autonomic failure. Pharmacologic enhancement of ganglionic synaptic transmission may be a novel way to improve autonomic function. Ganglionic AChR antibodies are found in patients with autoimmune autonomic ganglionopathy (AAG). Patients with AAG typically present with rapid onset of severe autonomic failure. Major clinical features include orthostatic hypotension, gastrointestinal dysmotility, anhidrosis, bladder dysfunction, and sicca symptoms. Impaired pupillary light reflex is often seen. Like myasthenia, AAG is an antibody-mediated neurologic disorder. The disease can be reproduced in experimental animals by active immunization or passive antibody transfer. The patient may improve with plasma exchange treatment or other immunomodulatory treatment. Antibodies from patients with AAG inhibit ganglionic AChR currents. Other phenotypes of AAG are now recognized based on the results of antibody testing. These other presentations are generally associated with lower levels of ganglionic AChR antibodies. A chronic progressive form of AAG may resemble pure autonomic failure. Milder forms of dysautonomia, such as postural tachycardia syndrome, are associated with ganglionic AChR in 10-15% of cases. Since ganglionic synaptic transmission is a common pathway for all autonomic traffic, enhancement of autonomic function through inhibition of acetylcholinesterase is a potential specific therapeutic strategy for autonomic disorders. Increasing the strength of ganglionic transmission can ameliorate neurogenic orthostatic hypotension without aggravating supine hypertension. Recent evidence also suggests a potential role for acetylcholinesterase inhibitors in the treatment of postural tachycardia syndrome.     

gamma-Aminobutyric acid receptors on chick ciliary ganglion neurons in vivo and in cell culture

In the chick ciliary ganglion, preganglionic terminals maintain cholinergic synapses on the choroid neurons and both cholinergic and electrical synapses on the ciliary neurons. The preganglionic terminals also contain enkephalin- and substance P-like immunoreactivity, suggesting that transmission through the ganglion is more complicated than is indicated by the known synaptic connections. We report here that embryonic chick ciliary ganglion neurons also have gamma-aminobutyric acid (GABA) receptors and that GABA applied to the ganglion can block transmission elicited by preganglionic stimulation. Studies on the neurons in cell culture indicate that the GABA response is mediated by GABAA receptors: GABA activates a Cl- conductance, and the response can be mimicked by muscimol and blocked by bicuculline or picrotoxin. The GABA receptors are regulated independently from acetylcholine (ACh) receptors on the neurons since the levels of ACh and GABA sensitivity are influenced differently by culture age and by chronic exposure to GABA or elevated K+ concentrations. Application of GABA to intact ciliary ganglia increases the membrane conductance of ganglionic neurons (as in culture), reduces to subthreshold the amplitude of excitatory postsynaptic potentials in the neurons elicited by preganglionic stimulation and completely blocks transmission through the ganglion. A native source of ligand for the receptors in vivo has yet to be identified.     

Myasthenia gravis with autoimmune autonomic neuropathy

The autoantibodies that impair neuromuscular junction transmission in myasthenia gravis are specific for the nicotinic acetylcholine receptor (AChR) of muscle. Antibodies specific for AChRs in ganglionic neurons are found in a majority of patients with subacute autonomic neuropathy. Dysautonomia is not a recognized feature of myasthenia gravis, but there have been rare reports of myasthenia gravis coexisting with autonomic failure, usually in association with thymoma. Here we report seven patients who had myasthenia gravis with subacute autonomic failure. Their autonomic dysfunction ranged from isolated gastroparesis to severe panautonomic failure. Gastrointestinal dysmotility was a common feature. All had antibodies against muscle AChR, and three (all of whom had thymoma) had antibodies against neuronal ganglionic AChRs. In several patients, gastrointestinal function improved clinically after administration of an acetylcholinesterase inhibitor. These observations support a rare but definite clinical association between myasthenia gravis and autonomic failure and strengthen the concept that subacute autonomic neuropathy is an autoimmune disorder.     

Expression of a chicken ciliary neurotrophic factor in targets of ciliary ganglion neurons during and after the cell-death phase

Ciliary ganglion (CG) neurons, like other neuronal populations, become dependent on their targets for survival during development. We have previously purified and cloned a secreted ciliary neurotrophic factor that was called growth-promoting activity (GPA). We report here the expression and purification of a highly active form of recombinant GPA, the preparation of GPA-specific polyclonal and monoclonal antibodies, and the use of these antibodies to investigate the cellular location and timing of GPA expression in tissues innervated by CG neurons. Virtually all of the trophic activity in extracts of embryonic eyes could be depleted by GPA-specific antibodies. GPA-like immunoreactivity was found in both targets of the CG: the arterial vasculature of the choroid layer and the ciliary body of the eye. In the choroid layer, GPA was localized to smooth muscle cells surrounding the choroid arteries. Staining in the choroid layer was first detectable at embryonic day (E) 10, or about 2 days after cell death has begun in the ganglion, then increased in intensity through E19. Quantification of trophic activity from whole eye extracts at various ages showed a small increase in activity observed between E9 and E12 and at least a 10-fold increase between E12 and E18. The presence of GPA protein in target cells of CG neurons during the specific developmental period when these neurons undergo cell death is consistent with its proposed function as a target-derived ciliary neurotrophic factor. © 1996 Wiley-Liss, Inc.     

The distribution of acetylcholine receptors in chick ciliary ganglion neurons following disruption of ganglionic connections

Chick ciliary ganglion neurons have nicotinic acetylcholine receptors (AChRs) that mediate primary chemical synaptic transmission through the ganglion. Previous studies have shown that preganglionic denervation reduces the total number of AChRs in the ganglion about 3-fold in 10 d, while postganglionic axotomy reduces AChR levels about 10-fold in 5 d. Since the neurons contain large numbers of intracellular AChRs in addition to the surface AChRs, the present studies were undertaken to determine whether either surface or internal AChR pools are changed selectively by the operations. An anti-AChR monoclonal antibody followed by an HRP-conjugated secondary antibody was used to visualize AChR distributions on neurons in ciliary ganglia 5 d after postganglionic axotomy and 10 d after preganglionic denervation. Ganglia were permeabilized by treatment with saponin to obtain access to intracellular receptors. The results show that the operations do not qualitatively change the subcellular localization of AChRs, but they do alter the levels relative to control ganglia. Axotomy produces substantial declines both in the number of synaptic AChRs and in the number of intracellular AChRs. Denervation produces a significant, though less extensive decline in the number of intracellular receptors while having no detectable effect on the number of synaptic AChRs. Small alterations in receptor distribution would have gone undetected by the present techniques. Regulation of neuronal AChRs differs in several respects from that described for muscle AChRs: presynaptic input appears to be less important for controlling neuronal AChRs, while signals from the postsynaptic target tissue may be essential for maintaining synaptic receptors on the neurons.     

Stimulation of somatostatin expression in developing ciliary ganglion neurons by cells of the choroid layer.

An important component of neuronal development is the matching of neurotransmitter expression with the appropriate target cell. We have examined how peptide transmitter expression is controlled in a simple model system, the avian ciliary ganglion (CG). This parasympathetic ganglion contains 2 distinct types of neurons: choroid neurons, which project to vasculature in the eye's choroid layer and use somatostatin as a co-transmitter with ACh, and ciliary neurons, which innervate the ciliary body and iris and use ACh but no known peptide co-transmitter. We have found that the earliest developmental stage in which neurons with somatostatinlike immunoreactivity (SOM-IR) are consistently found in vivo is stage 30 (embryonic day 6.5), a time shortly after the extension of neurites to targets in the eye's choroid layer. In cell culture, CG neurons expressed SOM-IR in co-culture with choroid cells, but not when cultured with striated muscle myotubes or with ganglion non-neuronal cells. No significant differences in neuronal survival or in ChAT activity were observed under these different co-culture conditions, which suggests that somatostatin expression is independently regulated. The stimulation of somatostatin expression was also specific in that other neuropeptides commonly found in autonomic neurons [neuropeptide Y (NPY), substance P (SP), vasoactive intestinal polypeptide (VIP)] were not induced in the presence of choroid cells. The ability to stimulate SOM-IR was not contact dependent because a macromolecule of greater than or equal to 10 kDa in choroid-conditioned medium (ChCM) was found to stimulate somatostatin expression in a dosage-dependent fashion. The somatostatin-stimulating activity induced SOM-IR in more than 90% of CG neurons, as well as in retrogradely labeled ciliary neurons, which would not normally express SOM-IR. Thus, the expression of somatostatin in cultured CG neurons is regulated by a macromolecule produced by cells in the choroid layer, a target normally innervated in vivo by CG neurons expressing somatostatin.     

The effects of α- and β-neurotoxins from the venoms of various snakes on transmission in autonomic ganglia

We have previously shown that certain commercially available lots of α-bungarotoxin block transmission in ciliary and choroid neurons of both pigeon and chicken ciliary ganglia at a concentration of 10 μg/ml (1.2 μM). The blockade is antagonized by pre-incubation with 100 μM tubocurarine.Further evidence that this blockade is produced by a postsynaptic action, as one would expect of an α-neurotoxin, are our findings that: (a) exposure to the toxin prevents the depolarization of ganglion cells normally seen in response to the cholinergic agonist, carbachol; and (b) the blocking activity of the toxin is removed by treatment with membranes purified from Torpedo electric organ containing an excess of α-neurotoxin binding sites.A high affinity binding site for [¹²⁵I]α-bungarotoxin was characterized in the chicken ciliary ganglion. However, since it is labelled equally well by lots of α-bungarotoxin which block transmission and those that do not, this site does not appear to be involved in the blockade of transmission.α-Cobratoxin (fromNaja naja siamensis), the α-neurotoxin L.s. III (fromLaticauda semifasciata) and certain lots of α-bungarotoxin produce a partial blockade of transmission in ciliary neurons of the pigeon ciliary ganglion at a concentration of 10 μg/ml (1.2 μM), but have no effect on transmission in choroid neurons. Two other α-neurotoxins fromLaticauda semifasciata, erabutoxin a and erabutoxin b, have no effect on transmission in either cell population at this concentration. None of the α-neurotoxins tested had any effect on transmission in either the rat superior cervical ganglion or the rat pelvic ganglion at concentrations up to 100 μg/ml (12 μM). Collagenase treatment of these ganglia, in an attempt to increase access of the toxins to ganglion cells, did not alter these negative results.β-Bungarotoxin (0.5 μg/ml, 0.02 μM) produces a complex blockade of transmission in both avian ciliary ganglia and rat superior cervical ganglia. Unlike the action of α-bungarotoxin, the blockade of ciliary ganglion transmission by β-bungarotoxin is irreversible and is not prevented by pretreatment with tubocurarine.     

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.     

Ultrastructural distribution of 125I-toxin F binding sites on chick ciliary neurons: synaptic localization of a toxin that blocks ganglionic nicotinic receptors

Although alpha-bungarotoxin (BGT), a common probe for nicotinic ACh receptors from vertebrate skeletal muscle, binds tightly to many autonomic ganglia, it fails to block nicotinic transmission in most of these ganglia. Recently, we have isolated a second toxin, toxin F, that blocks transmission in several autonomic ganglia, including the chick ciliary ganglion. 125I-Toxin F binds to 2 sites in the ciliary ganglion: one site that is also recognized by BGT and one site that is not. Since the presence of BGT fails to prevent the blocking effect of toxin F, the toxin F site not recognized by BGT most likely represents the neuronal nicotinic receptor. Accordingly, we have localized the binding of 125I-toxin F to both sites, using electron-microscopic autoradiography. After a 45 min incubation, 125I-toxin F binding sensitive to BGT was primarily localized extrasynaptically on the neuronal plasma membranes; however, by 4 hr, much of this site had been internalized. In contrast, the 125I-toxin F binding site not recognized by BGT was highly concentrated near synaptic membranes at both times. Pretreatment of ganglia with the classic nicotinic antagonists dihydro-beta-erythroidine (DHBE) and d-tubocurarine (DTC) significantly reduced 125I-toxin F binding to the toxin F-specific site. The average density of these sites on synaptic membranes was approximately 600 sites/micron 2. We conclude that toxin F binds to 2 pharmacologically distinct sites in the ciliary ganglion and that these sites are distributed differently over the plasma membrane of ciliary neurons. On the basis of the density of the toxin F-specific binding sites at synaptic membranes, we infer that the density of synaptic nicotinic receptors on these neurons is at least 20-fold lower than the density of nicotinic receptors at the vertebrate neuromuscular junction, as determined by BGT binding. These findings are consistent with those of electrophysiological studies, which also suggest low nicotinic receptor densities on ganglionic neurons.     

Synapse formation on trochlear motor neurons under conditions of increased and decreased cell death during development

There is a normally occurring death of about half of the trochlear motor neurons during development. Early removal of the target muscle results in death of almost all neurons whereas neuromuscular blockade prevents neuron death. The present investigation was undertaken to determine whether the number of central afferent synapses on motor neurons is altered under conditions which either accentuate cell loss or rescue neurons. The sole peripheral target of innervation of the trochlear motor neurons, the superior oblique muscle, was extirpated in duck embryos before the motor axon out-growth begins. The neuromuscular blockade was achieved by application of paralyzing dosages of alpha bungarotoxin on to the vascularized chorioallantoic membrane. This treatment began prior to the onset of cell death and embryos were treated daily throughout the period of cell death. Brains were processed for electron microscopy and quantitative observations were made on synapses at the onset, during the period of, and at the end of cell death. It was found that there was no significant difference in the number of synapses on neurons following target removal, following neuromuscular blockade, and those developing normally. This observation indicates that the number of central afferent synapses on cell soma is not altered under conditions which either decrease or increase neuron survival. These results suggest that the synapse number per se may not be directly involved in the process of naturally occurring cell death. The results also suggest that the number of synapses on trochlear motor neurons is independent of interactions with the target.     

Myasthenia, thymoma, presynaptic antibodies, and a continuum of neuromuscular hyperexcitability.

BACKGROUND: Autoantibodies specific for the nicotinic acetylcholine receptor (AChR) of skeletal muscle impair neuromuscular transmission in myasthenia gravis (MG). Autoantibodies specific for alpha3 neuronal AChRs or voltage-gated potassium channels have been reported in patients with Isaacs syndrome, an acquired disorder of continuous muscle fiber activity characterized by neuromyotonia. OBJECTIVE: To report the neuromuscular autoantibody profiles of three patients with a syndrome of MG and neuromuscular hyperexcitability. RESULTS: All three patients reported here had clinical and electrophysiologic evidence of MG and neuromuscular hyperexcitability. None had neuromyotonia. Thymoma was proven in two patients and suspected in the third. One had MG and thymoma and subsequently developed cramp-fasciculation syndrome; MG and rippling muscle syndrome appeared simultaneously in the other two. All patients had muscle and neuronal AChR binding antibodies and striational antibodies. Only one had antibodies reactive with alpha-dendrotoxin-complexed potassium channels. CONCLUSIONS: The coexistence of cramp-fasciculation syndrome and acquired rippling muscle syndrome with MG, thymoma, and neuronal AChR autoantibodies suggests that there is a continuum of autoimmune neuromuscular hyperexcitability disorders related pathogenically to Isaacs syndrome. Manifestations of neuromuscular hyperexcitability may be altered and less apparent in the context of MG because of the coexisting defect of neuromuscular transmission.     

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.