Ciliary Neurons Research Articles

Oncostatin M regulates VIP expression in a human neuroblastoma cell line.

Oncostatin-M (OM), a recently described glycoprotein cytokine, is structurally and functionally related to cholinergic differentiation factor/leukemia inhibitory factor (CDF/LIF) and ciliary neurotrophic factor (CNTF). To determine whether OM, like CDF/LIF and CNTF, possesses trophic or differentiative functions for neurons we examined the effects of recombinant human OM on ciliary neuron survival and neurotransmitter expression in sympathetic neurons. Like CDF/LIF, but in contrast to CNTF, OM had no effect on ciliary neuronal survival at any concentration tested. OM produced small but reproducible increases in choline acetyl transferase (ChAT) activity and vasoactive intestinal peptide (VIP) levels in rat sympathetic neuron cultures, but this effect was significantly less than that of CNTF or CDF/LIF. To determine if human OM would elicit a more robust response from human cells, we utilized a human neuroblastoma cell line, NBFL, that responds to CNTF and CDF/LIF by altering vasoactive intestinal peptide (VIP) levels. OM specifically elevated VIP and c-fos mRNA levels in NBFL cells and was as potent as CDF/LIF in this assay. Our data provides evidence that OM acts on neurons and identifies a neural cell line responsive to OM, CNTF, CDF/LIF.

Membrane-associated neurotransmitter stimulating factor is very similar to ciliary neurotrophic factor

Membrane-associated neurotransmitter stimulating factor (MANS) can modulate sympathetic neurotransmitter expression and promote ciliary neuron survival in cell culture. Previous studies have shown that its biological effects and biochemical properties are similar to those of ciliary neurotrophic factor (CNTF). In addition, CNTF is present in spinal cord, the source of MANS. These observations raised the possibility that MANS preparations contain CNTF. We find that partially purified MANS fractions contain a 24-kD protein that is recognized in Western blots by an antiserum generated against recombinant rat CNTF (rCNTF). This antiserum immunoprecipitates virtually all the cholinergic-inducing and the ciliary neurotrophic activities present in MANS preparations. When iodinated rCNTF is incubated with spinal cord membranes, a significant proportion of the labeled CNTF segregates with the membrane pellet. The membrane-associated exogenous CNTF can be eluted from the membrane fraction by treatment with high-salt solutions, similar to that used to solubilize MANS from spinal cord membranes. Our data suggest that a substantial portion of the cholinergic differentiation and ciliary neurotrophic activities present in MANS preparations can be attributed to CNTF or a CNTF-like molecule.

Preparation and biological properties of native and recombinant ciliary neurotrophic factor.

CNTF (ciliary neurotrophic factor), purified from rabbit sciatic nerves by a relatively simple procedure, is bioactive in tissue culture at low picomolar concentration and appears as a doublet on polyacrylamide gel electrophoresis (PAGE). In these nerves, CNTF accounts for more than one-half of the survival-promoting activity on ciliary neurons. The concentration of CNTF in rabbit sciatic nerves is estimated to be 5 nmol/kg, more than 1000 times higher than would seem to be required to support neurons if the neurotrophic factor were homogeneously distributed. With recombinant DNA technology, rat CNTF has been synthesized in Escherichia coli, purified without denaturating agents, and found to be bioactive at a slightly lower concentration than CNTF extracted from rabbit sciatic nerves. After radioiodination, CNTF retains biological activity but is not specifically internalized and retrogradely transported in motor and sensory axons. In peripheral nerves, ciliary neurotrophic factor differs biologically from nerve growth factor (NGF) by its much higher tissue concentration and apparent lack of internalization by peripheral nerve axons.

Tyrosine hydroxylase and neuropeptide Y are increased in ciliary ganglia of sympathectomized rats

We have examined the expression of tyrosine hydroxylase and neuropeptide Y in ciliary ganglia of normal adult rats and of adult rats in which the environment of these neurons was altered by sympathectomy at birth. Following neonatal 6-hydroxydopamine treatment, the proportion of tyrosine hydroxylase-immunoreactive and neuropeptide Y-immunoreactive neurons in ciliary ganglia was significantly increased. In ciliary neurons of both control and sympathectomized rats, neuropeptide Y immunoreactivity was preferentially co-localized with tyrosine hydroxylase. Immunoblot analysis confirmed the presence of tyrosine hydroxylase and its increase following sympathectomy. In situ hybridization studies revealed that many ciliary neurons contain mRNA for tyrosine hydroxylase and for neuropeptide Y. Like tyrosine hydroxylase immunoreactivity, the number of ciliary neurons containing tyrosine hydroxylase mRNA and the amount of mRNA per cell were increased in 6-hydroxydopamine-treated rats. In contrast, neuropeptide Y mRNA levels were the same in control and 6-hydroxydopamine-treated rats. Nerve growth factor is a candidate for mediating the effects of sympathectomy and most ciliary neurons in control and sympathectomized rats expressed immunoreactivity for the low-affinity nerve growth factor receptor. In addition, ciliary neurons from 6-hydroxydopamine-treated animals possessed increased nerve growth factor receptor immunoreactivity. These studies indicate that both tyrosine hydroxylase and neuropeptide Y in the ciliary ganglion are regulated by alterations in their environment. Their expression was enhanced by chemical sympathectomy which does not affect ciliary neurons directly but, rather, removes sympathetic innervation of shared targets, including the iris. In situ hybridization analysis suggests that the increased tyrosine hydroxylase and neuropeptide Y levels result from different mechanisms and provides evidence that neuropeptide levels can be regulated without changes in mRNA levels.

Screening of adrenal medullary neuropeptides for putative neurotrophic effects

Chromaffin granules, the secretory organelles of the neuron-like adrenal medullary chromaffin cells, have previously been shown to store and liberate neurotrophic activities that support in vitro survival of several neuron populations including those innervating the adrenal medulla. Molecules resembling fibroblast growth factor and ciliary neurotrophic factor have been identified among these activities. Since chromaffin granules store a variety of neuropeptides and many neuropeptides can have pleiotropic effects on neuronal growth and maintenance we have tested 24 different neuropeptides for their capacities to promote survival of embryonic chick ciliary, dorsal root and sympathetic ganglionic neurons. Peptides tested included several derivatives of proenkephalin (Leu- and met-enkephalin, fragments BAM 22, B, F and E), somatostatin, substance P, neuropeptide Y, neurotensin, VIP, bombesin, secretin, pancreastatin, dynorphin B, dynorphin 1–13, β-endorphin, alpha-, beta-, and gamma-MSH. Control cultures received saturating concentrations of ciliary neurotrophic or nerve growth factor (CNTF; NGF), or no trophic supplements. At 1 × 10−5 M leu- and met-enkephalin as well as somatostatin supported sympathetic neurons to the same extent as NGF. At the same concentrations, leuenkephalin, the proenkephalin fragments BAM 22 and E, and somatostatin maintained about half of the dorsal root ganglionic neurons supported by NGF, but were not effective on ciliary neurons. VIP promoted the survival of approximately 50% of the ciliary and embryonic day 10 dorsal root ganglionic neurons as compared to saturating amounts of CNTF, but required the presence of non-neuronal cells in the cultures to be effective. Neurotensin (1 × 10−5M) had a small effect on ciliary neurons. All other peptides had no effect on any neuron population tested. Our results suggest that neuropeptides that occur in the adrenal medulla may play a role for the maintenance of cholinergic, sensory, and sympathetic neurons innervating the adrenal medulla.

Changes in the electrical properties of chick ciliary ganglion neurones during embryonic development.

1. Whole-cell recording techniques were used to examine the expression of ionic currents in chick ciliary ganglion neurones dissociated acutely at various stages of embryonic development. Currents were also examined in dissociated cells that had been maintained in vitro for several days. 2. Voltage-activated, tetrodotoxin (TTX)-sensitive Na+ currents (INa) could be detected in all cells tested between stage 25 and stage 40 (embryonic days 4.5-14). INa increased in both amplitude and density throughout development, but no obvious changes in kinetics or sensitivity to TTX were observed. 3. High-threshold Ca2+ currents (ICa) were also detectable between stage 25 and stage 40. ICa increased in both amplitude and density throughout this time. No obvious changes in kinetics or voltage dependence were observed. 4. Delayed rectifier K+ currents (IDR) and A-currents (IA) could be detected in Ca(2+)-free salines, and distinguished on the basis of differences in kinetics, voltage dependence, and sensitivity to tetraethylammonium (TEA). IA was either absent, or present at very low densities at stages 26-30, but showed a sharp increase in density thereafter. In contrast, IDR was detectable as early as stage 25, and did not display a significant increase in density during development. 5. Ca(2+)-activated K+ currents (IK(Ca)) were either undetectable or present at very low density between stage 26 and stage 30 (embryonic days 5-9) but showed a large increase in amplitude and density thereafter. 6. Ionic currents were examined in age-matched cells dissociated acutely on embryonic day 13, or isolated on embryonic day 9 and maintained in vitro for an additional 4 days. Most of the cells maintained in culture for 4 days did not express detectable IK(Ca), and had significantly reduced IA compared to acutely isolated controls. The cultured cells expressed normal densities of IDR, ICa and INa. 7. All ionic currents increased in amplitude during normal embryonic development, and all but IDR increased in density. The largest change in density generally occurred between stages 30 and 40, during which time ciliary ganglion neurones form synapses with target tissues. 8. Isolation of ciliary neurones from the in ovo environment prevented the normal development of IA and IK(Ca), suggesting that the expression of these channels is controlled by one or more extrinsic environmental factors. In contrast, the normal expression of INa, ICa and IDR is not dependent upon extrinsic factors.

An inward rectifier is present in presynaptic nerve terminals in the chick ciliary ganglion

Inwardly rectifying voltage-sensitive channels have been detected in the cell bodies and axons of a number of excitable cells. The question of whether similar channels exist at axon terminals has been a matter of speculation for some time. We now report the first direct evidence for the existence of inward rectifiers in vertebrate presynaptic nerve terminals. Following impalement with intracellular electrodes, the large calyciform nerve terminals innervating chick ciliary ganglion neurons exhibit pronounced inward rectification upon hyperpolarization that increases with increasing current strength. The response is blocked by 2 mM Cs +, but is insensitive to Ba 2+, tetraethylammonium and tetrodotoxin. The inward rectifier exhibits dependence on both Na + and K +, but is unaffected by altering extracellular Ca 2+. Ciliary neurons innervated by these nerve terminals display inward rectification with similar properties. We conclude that the inward rectifier present in these presynaptic nerve terminals resembles the H-current previously described in sensory ganglion neurons and the Q-current found in hippocampal pyramidal neurons. The presence of channels that are activated by hyperpolarization may serve to enhance the excitability of the calyciform nerve terminals, which are capable of relatively high frequencies (> 100 Hz) of discharge.

Class 1 heparin binding growth factor promotes the differentiation but not the survival of ciliary neurones in vivo

Molecules isolated from target tissues of various neuronal populations have been shown to enable the survival of those innervating neurones. A molecule derived from bovine heart which can support the survival of parasympathetic neurones of the chick embryo ciliary ganglion in vitro has been sequenced and identified as a member of the class 1 heparin binding growth factors (HBGF-1). When injected into developing chick embryos during the period of naturally occurring cell death in the ciliary ganglion, HBGF-1 failed to rescue those neurones which normally die. There was, however, a premature increase in the activity of choline acetyltransferase within the ganglion and a shift to the right in the size distribution of the neurones. We conclude that HBGF-1 may promote the differentiation of some subpopulation of neurones of the ciliary ganglion of the chick embryo but does not act in vivo as a survival molecule.

Expression of recombinant rat ciliary neurotrophic factor in Escherichia coli and its synergy with basic fibroblast growth factor

Rat CNTF was expressed in Escherichia coli using a T7 RNA polymerase vector system. rCNTF was obtained as an insoluble aggregate at levels approaching 70% of total E. coli protein. After extraction, renaturation, and purification by anion-exchange chromatography and gel filtration the yield of biologically active rCNTF was nearly 0.5 mg/ml of E. coli culture broth. The effects of rCNTF and bFGF on cultured chick ciliary neurons were compared. bFGF was primarily neuritogenic and did not support ciliary neuron survival beyond 2 days in culture. rCNTF induced neuritogenesis more slowly and supported ciliary neuron survival in culture for a longer period. bFGF strongly potentiated the effect of rCNTF. Without bFGF, the ED 50 for rCNTF was 102 ± 25 pg/ml. With bFGF, the potency of rCNTF increased to an ED 50 of 42 ± 6 pg/ml. Chick nodose neurons also responded to rCNTF and their response was potentiated by bFGF. Thus, bFGF modulates the response of ciliary and nodose neurons to CNTF in vitro and may have a similar effect in vivo.

Recombinant Human and Rat Ciliary Neurotrophic Factors

The human ciliary neurotrophic factor (CNTF) gene was identified and cloned, based on homology with the recently cloned rat cDNA. The gene encodes a protein of 200 amino acids, which shares about 80% sequence identity with rat and rabbit CNTF and, like these homologues, lacks an apparent secretion signal sequence. The human CNTF gene, like the rat gene, appears to contain a single intron separating two protein coding exons. An intronless human CNTF gene was constructed by the use of polymerase chain reactions and introduced into vectors designed for expression of foreign proteins in E. coli. The rat CNTF gene was also introduced into similar vectors. Both the human and rat proteins were expressed at exceptionally high levels, at 20-40% and 60-70% of total protein, respectively. Extraction of the recombinant proteins from inclusion bodies by guanidinium chloride, followed by two column chromatography steps, produced high yields of pure CNTF that supported survival and neurite outgrowth from embryonic chick ciliary neurons in culture. The biological activity of both recombinant proteins was comparable to that of native rat CNTF.

Probabilistic secretion of quanta from nerve terminals in avian ciliary ganglia modulated by adenosine.

1. The effects of adenosine on the probability of secretion of acetylcholine quanta and on presynaptic and postsynaptic action potentials was examined in the post-hatched avian ciliary ganglion. 2. Adenosine (20 microM) reduced the average size of the excitatory postsynaptic potential (EPSP) by 33%. This was due to a decrease in quantal content of the EPSP (m). The effect was blocked by theophylline (50 microM). 3. Adenosine deaminase (2.5 i.u./ml) increased the size of the EPSP by 70%, suggesting that endogenous adenosine modulates synaptic transmission in the ciliary ganglion. However, theophylline (20-100 microM) did not affect the EPSP in a low [Ca2+]o of 1 mM and high [Mg2+]o of 6 mM. 4. Plateau-type action potentials with a large calcium component were generated in the ciliary neurones by bathing the ganglion in tetraethylammonium ions (TEA, 10 mM). Adenosine (20 microM) reduced the duration of these action potentials on short exposures (less than 20 min) but increased the duration on longer exposure (greater than 30 min). Adenosine did not affect the normal action potential recorded in the absence of TEA. 5. Adenosine (20 microM) hyperpolarized the nerve terminal and as a consequence increased the size of the presynaptic action potential and reduced its after-hyperpolarization. 6. Plateau-type action potentials with a large calcium component were generated in the nerve terminals using TEA (10 mM). The duration of these action potentials was significantly reduced by adenosine (20 microM). 7. Adenosines action on nerve terminals, to hyperpolarize the membrane and reduce calcium influx, may contribute to its effect in reducing m of the EPSP.

A ciliary neuronotrophic factor from peripheral nerve and smooth muscle which is not retrogradely transported

We have found that a CNTF-like molecule which supports ciliary and sympathetic neurons is not retrogradely transported in either sympathetic or parasympathetic nerves. The factor has an apparent Mr of 21 kDa, a pI of 4.9, and is present in peripheral nerves and smooth muscle of the chick. Our experiments indicate that CNTF-like activity does not accumulate on the distal side of ligated chick expansor nerves. In contrast, there is a clear accumulation of NGF. The activity further differs from NGF in that it is not removed from a smooth muscle of the chick wing by innervating sympathetic fibers. Transection of these fibers does not lead to an accumulation of ciliary activity in the expansor secundariorum muscle, suggesting that neurons do not actively deplete the muscle of factor by retrograde transport. Finally, recombinant CNTF or semi-purified preparations of CNTF-like activity labelled with 125I were not transported to the ciliary ganglion of chicks following injection of biologically active material into the eye. Our results suggest either that endogenous CNTF does not act as a survival factor in vivo, or that retrograde transport is not a property inherent to all neuronotrophic molecules.

Blot and culture analysis of neuronotrophic factors in nerve regeneration chamber fluids

The fluid accumulating in silicone nerve regeneration chambers implanted between the cut ends of rat sciatic nerve contains neuronotrophic activities towards embryonic chick ciliary and sympathetic neurons. The blot and culture technique of Carnow et al. was used to determine if part of the neuronotrophic activities is due to ciliary neuronotrophic factor, which supports the survival of both types of neurons in vitro. The technique involves separating the fluid proteins by SDS-polyacrylamide gel electrophoresis, Western transfer, and then culturing of purified neurons on the nitrocellulose blots. After 24 hr surviving neurons are restricted to regions of the blot where neuronotrophic factor is present. Analysis of 1 and 2 day fluids showed that a multitude of factors are present, particularly in the 19-30 kD molecular weight range, with discrete peaks of activity at molecular weights consistent with those reported for ciliary neuronotrophic factor. There were several other peaks of activity present in the fluids in addition to these.

Neurotransmitter organization of the nucleus of Edinger–Westphal and its projection to the avian ciliary ganglion

Two morphologically distinct types of preganglionic endings are observed in the avian ciliary ganglion: boutonal and cap-like. Boutonal endings synapse on ciliary ganglion neurons (called choroidal neurons) innervating choroidal blood vessels, while cap-like endings synapse on ciliary ganglion neurons (called ciliary neurons) controlling the lens and pupil. Some of both types of preganglionic endings contain the neuropeptides substance P (SP) and/or leucine-enkephalin (LENK). Although both types of preganglionic terminals are also known to be cholinergic, there has been no direct evidence that SP and LENK are found in cholinergic endings in the ciliary ganglion. The present studies in pigeons, which involved the use of single- and double-label immunohistochemical techniques, were undertaken to examine this issue, as well as to (1) determine the relative percentages of the boutonal and cap-like endings that contain SP, LENK, or both SP and LENK; and (2) determine if the two different types of terminals in the ciliary ganglion arise from different subdivisions of the nucleus of Edinger-Westphal (EW). Single- and double-label immunohistochemical studies revealed that all neurons of EW, regardless of whether they contained immunohistochemically detectible amounts of SP or LENK, are cholinergic. In the medial subdivision of EW (EWM), which was found to contain approximately 700 neurons, 20.2% of these neurons were observed to contain both SP and LENK, while 11.6% were observed to contain SP only and 10.7% were observed to contain LENK only. In contrast, in lateral EW (EWL), which was found to contain approximately 500 neurons, 16.2% of the neurons were observed to contain both SP and LENK, while 19.2% of the neurons were observed to contain SP only and 12.6% were observed to contain LENK only. Retrograde-labeling studies involving horseradish peroxidase injections into the ciliary ganglion revealed that EW was the sole source of input to the ciliary ganglion and all, or nearly all, neurons in EW innervate the ciliary ganglion. Immunohistochemical labeling of the ciliary ganglion neurons with an antiserum against choline acetyltransferase revealed that approximately 900 choroidal neurons and approximately 600 ciliary neurons are present in the ganglion, all of which receive cholinergic preganglionic endings. Of the choroidal neurons, 94% receive butonal terminals containing both SP and LENK, while only 2% receive SP+ only boutonal endings and 2% receive LENK+ only butonal endings. Of the ciliary neurons, 25% receive cap-like endings containing both SP and LENK, 30% receive cap-like endings containing only SP and 3% receive cap-like endings containing only LENK.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of a calcium current in a vertebrate cholinergic presynaptic nerve terminal

Calcium currents were recorded from a cholinergic presynaptic nerve terminal in the chick ciliary ganglion using the whole-cell voltage-clamp technique. The presynaptic element of this synapse is in the form of a calyx that envelops the postsynaptic ciliary neuron. A method was developed to isolate the ciliary neuron, expose the calyx, and apply patch-clamp electrodes under visual control. The presynaptic Ca current activated at +30 mV with a fast time constant of about 1.5 msec and deactivated at -80 mV with a time constant of about 0.5 msec, values that are consistent with a role in action-potential-dependent transmitter release. The calyx Ca current was blocked by 0.1 mM Cd or 2 microM omega-conotoxin and was resistant to voltage-dependent inactivation. The presynaptic Ca channel exhibits similarities to the N-type group but differs from these by the minimal voltage-dependent inactivation. This type of channel, designated CaN-PT (N-like, presynaptic terminal), may play a key role in transmitter release at many vertebrate fast-transmitting synapses.

Control of Embryonic Motoneuron Survival in Vivo by Ciliary Neurotrophic Factor

During development of the nervous system, neurons in many regions are overproduced by proliferation, after which the excess cells are eliminated by cell death. The survival of only a proportion of neurons during normal development is thought to be regulated by the limited availability of neurotrophic agents. One such putative trophic agent is ciliary neurotrophic factor (CNTF), a polypeptide that promotes the survival of ciliary, sensory, and sympathetic neurons in vitro. In contrast to the results of in vitro studies, however, the daily treatment of chick embryos in vivo with purified human recombinant CNTF failed to rescue any of these cell populations from cell death, whereas CNTF did promote the in vivo survival of spinal motoneurons. Thus, CNTF may not act as a neurotrophic agent in vivo for those embryonic neurons (especially ciliary neurons) on which it acts in vitro. Rather, CNTF may be required for in vivo survival of motoneurons.

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.

Retinoic acid potentiates the neurotrophic but not the mitogenic action of the class 1 heparin-binding growth factor (HBGF-1)

Retinoic acid promotes the neuronal survival properties of the class 1 heparin-binding growth factor (HBGF-1) on 8-day-old chick ciliary and sensory neurones. It has little or no effect on the survival promoting properties of class 2 heparin-binding growth factor (HBGF-2) on these cell types. Nerve growth factor (NGF) promoted survival of sympathetic and sensory neurones, and was unaffected at any concentration of retinoic acid. The mitogenic effect of both HBGF-1 and HBGF-2 was unaltered at any concentration of retinoic acid. It is suggested that naturally occurring gradients of retinoic acid, for example, as occur in the developing limb bud, could have a role in the development of normal innervation patterns through selective interaction with neurotrophic molecules.

Co‐activation of insulin‐like growth factor‐I receptors and protein kinase C results in parasympathetic neuronal survival

We have studied the interaction between several growth factors to promote parasympathetic neuronal survival. Neither insulin nor insulin-like growth factor-I (IGF-I) had any effect on the survival of embryonic day 8 chick ciliary neurons in culture. Similarly, the protein kinase C activator phorbol dibutyrate (PdBu) had only a minor survival-promoting activity. In combination with PdBu, however, IGF-I or insulin, at concentrations sufficient to act through the IGF-I receptor, were highly synergistic. In a similar fashion, acidic fibroblast growth factor (aFGF)-induced neuronal survival was greatly enhanced by PdBu, as well as by insulin or IGF-I. When added alone, aFGF-induced cell survival required the presence of 1% serum. However, addition of aFGF, IGF-I, or insulin with PdBu under serum-free conditions replaced the serum requirement. That is, these agonist combinations could apparently induce the second messenger requirement for ciliary neuronal survival. Therefore, IGF-I must now be included in the list of candidate molecules responsible for directing parasympathetic nerve formation. The synergy between agonists observed in these experiments highlights the possibility that combinations of growth factors, rather than sole molecules, may dictate parasympathetic nervous system development in vivo.

Quantitative study of neuronal degeneration induced by Ricinus toxin and crush of postganglionic nerves in the ciliary ganglion of quail

The effects of Ricinus toxin on the neurons of the ciliary ganglia were investigated in the quail. The neuronal death and the morphological alterations of the ganglionic cells were assessed following injection of the toxin in the anterior chamber of the eye or after application of the toxin on the postganglionic nerves at a crush site. A 45% loss of choroid neurons without loss of ciliary neurons was observed after postganglionic nerve crush alone. Injection of the toxin in the anterior chamber of the eye led to a selective loss of ciliary neurons (38%). Application of the toxin to the crushed postganglionic nerves led to a loss from both neuronal populations (40% of total neurons). This work indicates that different procedures result in selective lesion of the different neuronal populations in the ciliary ganglion.