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.     

Isoelectric focusing of the chick eye ciliary neuronotrophic factor

A procedure is presented in which a crude extract from selected chick embryo intraocular tissues is submitted to analytical polyacrylamide slab gel isoelectric focusing. The extract contains a protein, ciliary neuronotrophic factor (CNTF) which can be eluted in active form from focused gels in a region occupied by only two protein bands. A "slot" technique is presented in which we demonstrate that the eluted CNTF activity focuses in the very restricted region between the two visible bands and is not associated with either band. Silver stain-densitometry is used to correlate staining intensity with protein concentration and from such an analysis it is concluded that the CNTF protein represents an extremely low proportion of total extract protein and that the minimum CNTF specific activity eluted from gel slices is 10(6) trophic units per mg protein. This one-step procedure will be used in the future to prepare highly purified CNTF for antibody generation.     

The ultrastructural localization of alpha-bungarotoxin binding sites in relation to synapses on chick ciliary ganglion neurons

The distribution of alpha-bungarotoxin binding sites on chick ciliary ganglion neurons was examined at the ultrastructural level by incubating ganglia with horseradish peroxidase-conjugated toxin and examining the peroxidase-stained and thin-sectioned ganglia with the electron microscope. Both in embryonic and in adult ganglia heavy labeling was restricted to the surface membrane of short processes emerging from the ciliary and choroid cell somata in the region of preganglionic innervation. Less dense labeling occasionally was present on the smooth surface membrane of the soma in the same region. In contrast, the pre- and postsynaptic membranes of most synapses were clearly not labeled even in the immediate vicinity of heavily labeled processes. The labeling represented specific binding of the toxin conjugate since it could be prevented by d-tubocurarine and hexamethonium or by unconjugated toxin. The conjugated toxin was not excluded from the synaptic cleft on the basis of size because a substantially larger protein conjugate, a horseradish peroxidase-labeled monoclonal antibody, was able to enter the cleft and heavily label synaptic membranes as well as soma membranes. Even neurons in adult ganglia had very little synaptic labeling after exposure to the conjugated toxin. These results strongly suggest that the high affinity alpha-bungarotoxin binding sites on chick ciliary ganglion neurons are different from the synaptic ACh receptors which would be expected to be concentrated in the postsynaptic membrane. Clustering of the alpha-bungarotoxin binding sites in the vicinity of synapses, however, may reflect a related synaptic function.     

Localized survival of ciliary ganglionic neurons identifies neuronotrophic factor bands on nitrocellulose blots

A novel and sensitive method has been developed to identify ciliary neuronotrophic factors (CNTFs) from tissue extracts after blotting to nitrocellulose paper. The CNTF proteins are required for the in vitro survival of embryonic chick ciliary ganglionic neurons. Tissue extracts containing such CNTFs are electrophoresed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose paper. Purified ciliary ganglionic neurons are seeded on the surface of the nitrocellulose blot, and the culture is incubated for 24 hr in medium lacking CNTF. CNTF can be localized on the blot because it retains its ability to support the survival of the neurons cultured on the nitrocellulose. A band of viable neurons, easily visualized by staining with a vital dye, is supported by the blotted CNTF polypeptide. The number of neurons surviving on the blotted CNTF is related to the amount of CNTF originally loaded on the electrophoretic gel. As little as 2 ng (16 trophic units) of CNTF protein contained in crude tissue extracts can be loaded on the sodium dodecyl sulfate gel and still be recognized by the cultured neurons. This method was used to identify CNTF polypeptides from extracts of adult rat nerve (24,000 and 19,000 daltons) and from tissue found near experimentally induced adult rat brain lesions (24,000 daltons). The electrophoretic mobilities of these peptides are distinct from the previously purified chick eye CNTF polypeptide (20,400 daltons).     

Blockade of ganglionic transmission during synaptogenesis decreases alpha-bungarotoxin binding in the chick ciliary ganglion and iris

The role of normal synaptic activity in the biochemical development of the nervous system has been examined in the chick embryo. Chlorisondamine, a ganglionic blocking drug, was administered in ovo during the period of synaptogenesis in the parasympathetic ciliary ganglion. Following treatment with chlorisondamine, nicotinic binding sites (as measured with [125I] alpha-bungarotoxin) were significantly reduced in both the ganglion and its end organ, the striated iris muscle. While the number of [125I] alpha-bungarotoxin binding sites eventually approached control levels in the iris, binding in the ciliary ganglion remained below normal values through hatching.     

Purification of adult rat sciatic nerve ciliary neuronotrophic factor

The ciliary neuronotrophic factor (CNTF), a protein required for the survival of cultured avian embryonic parasympathetic ciliary ganglionic neurons, was recently purified from extracts of selected chick intraocular tissues. Here we report the purification of a mammalian CNTF activity from extracts of adult rat sciatic nerve using a fractionation procedure similar to that employed for isolating chick eye CNTF. About 2 micrograms of CNTF protein can be obtained from each 1.5 g batch of nerve tissue. Like the chick CNTF, the mammalian factor displays trophic activity for dorsal root and sympathetic as well as ciliary ganglionic neurons. The nerve CNTF activity differs from its chick counterpart in molecular weight and chromatographic behavior on ion-exchange columns. Unlike purified nerve growth factor (NGF), nerve CNTF activity is insensitive to anti-NGF antibodies and is unable to support the survival of 8-day chick embryo dorsal root ganglion neurons.     

Immunolocalization of ciliary neuronotrophic factor in adult rat sciatic nerve.

Two rabbit polyclonal antibodies were raised against synthetic peptides corresponding to residue numbers 45-59 and 181-200 of rat ciliary neuronotrophic factor (CNTF). The resulting antibodies were purified by affinity chromatography and both purified antibodies reacted by enzyme-linked immunoassay (ELISA) and immunoblotting with rat sciatic nerve CNTF. The anti-CNTF peptide antibodies were used to immunostain sections of adult rat sciatic nerve, previously known as the richest tissue source of CNTF. By light microscopy both antibodies appeared to stain exclusively Schwann cells and axons and both did so with the same pattern of specific staining. Immunostaining was eliminated by absorption of the anti-peptide antibodies with either their corresponding peptide or with purified rat nerve CNTF or by using purified nonspecific IgG. Schwann cells were stained and in semi-thin sections this staining appeared to be in the Schwann cell cytoplasm. Axons could be stained in addition to Schwann cells providing higher concentrations of antibodies were used. Epineurial, endoneurial and endothelial cells appeared unstained. Since all Schwann cells and axons appear to contain CNTF and since CNTF is known to act in vitro to support sensory and sympathetic ganglionic and motor neurons, we suggest that Schwann cells may normally provide CNTF to those neurons contributing axons to the peripheral nerve.     

Neuronal survival and calcium influx induced by basic fibroblast growth factor in chick ciliary ganglion neurons

Basic fibroblast growth factor (bFGF/FGF2) exhibits widespread biological activities in the nervous system. However, little is known about the cascade of intracellular events that links the activation of its tyrosine kinase receptors to these effects. Here we report that, in ciliary ganglion neurons from chick embryo, this trophic factor significantly enhanced neuronal survival. The percentage of surviving neurons was reduced when intracellular calcium was chelated by adding a membrane-permeable BAPTA ester to the culture medium, while antagonists of L- and N-type voltage-dependent calcium channels were ineffective. The ionic signals in response to bFGF stimulation have been studied using cytofluorimetric and patch-clamp techniques. In single-cell Fura-2 measurements, bFGF elicited a long lasting rise of the cytosolic calcium concentration that was dependent on [Ca²⁺]_o. In whole-cell experiments, we observed a reversible depolarization of the membrane resting potential and an inward cationic current. Single channel experiments, performed in the cell-attached configuration, provide evidence for the activation of two families of Ca²⁺-permeable cationic channels. Moreover, inositol 1,4,5-trisphosphate opens channels with similar properties, suggesting that this cytosolic messenger can be responsible for the calcium influx induced by bFGF.     

Effects of nerve growth factor on autonomic neurons in the chick embryo: A stereological study

Quantitative effects of nerve growth factor (NGF) on the sympathetic, Remak and ciliary ganglia in chicken embryos were investigated. Purified mouse beta NGF was injected (80 micrograms per day for three or four consecutive days) into the yolk sac at different stages (starting on days 6, 8, 10 and 13) of embryonic development. Ganglia were taken for fixation and embedding one day after the last NGF injection. The number of neurons belonging to the different size classes was determined by a computer aided stereological method based on unfolding of cell diameter frequencies. The volume of sympathetic ganglia was increased at all stages with a maximum of 8-fold occurring on day 10. The ganglion of Remak showed a 3-fold volume increase up to embryonic days 10 and 12. Ciliary ganglia did not exhibit any differences in volume or neuron size between the controls and the embryos injected with NGF. The number of neurons was increased in younger sympathetic and Remak ganglia in response to NGF, as was the recruitment of neurons to the larger size classes.     

Polysialic acid and the formation of oculomotor synapses on chick ciliary neurons

The polysialic acid (PSA) moiety of the neural cell adhesion molecule (NCAM) participates in a variety of developmental processes, including axonal guidance and cell migration. PSA's function in these contexts stems from its ability to reduce cell interactions. The present study examines the regulation of PSA expression during formation of the calyciform synapse by the oculomotor axons on chick ciliary neurons. Prior to synaptogenesis, PSA is abundantly and uniformly expressed on the surface of the ciliary neuron body. However, at the time synaptic bonds start to form, as reflected in the localized accumulation of synaptic vesicles, PSA is lost from the point of synaptic contact. Thereafter, PSA is progressively lost from the ciliary neuron surface as the calyx grows. The dense mats of pseudodendritic-like somatic spines, which extend from the postsynaptic cell body, form an exception. These spines, which are known to undergo morphological remodeling, retain PSA expression until the end of embryogenesis. The experimental removal of PSA did not affect synaptogenesis itself, in that no significant changes were observed in the surface covered by the calyx, the number of spine aggregates, the size of acetylcholine receptor clusters, the cell surface area covered by these receptors, or the ultrastructure of the calyx, spine mats, and active zones. Together, these observations suggest that the synapse eliminates PSA as a part of its normal development and that the loss of PSA from the site of axon-target interaction may serve to stabilize structures formed during synaptogenesis.     

Effects of ciliary neuronotrophic factor on rat spinal cord neurons in vitro: survival and expression of choline acetyltransferase and low-affinity nerve growth factor receptors.

We have studied the effects of ciliary neuronotrophic factor (CNTF) and nerve growth factor (NGF) on cultures of E14 rat spinal cord cells maintained for 7 days. The trophic factors were supplied at the day of seeding and every other day thereafter. Treatments with CNTF (human recombinant or purified from rat sciatic nerve, 100 TU/ml) resulted after 7 days in an increase, relative to control cultures, of: (i) the total number of neurons (identified by neurofilament protein and neuron-specific enolase immunostaining) that were not stained with choline, acetyltransferase (ChAT) and low affinity nerve growth factor receptor (LNGFR) antibodies; (ii) the number of motoneurons (0.5% of the neuronal population) as identified by size (greater than 25 microns), morphology and immunostaining for ChAT and LNGFR; and (iii) a population of small- to medium-sized (less than 25 microns), ChAT- and LNGFR-positive neurons, representing 5-10% of the total neuronal population. NGF treatments (mouse submaxillary beta NGF; 10-3000 TU/ml) were without effect on all 3 neuronal populations. Experiments in which CNTF administration was delayed revealed that the population of ChAT- and LNGFR-negative neurons and the population of motoneurons, were both dependent on CNTF for their survival. The third population, small ChAT and LNGFR-positive neurons, was not dependent on CNTF for survival but was induced by CNTF to express its two markers. These observations indicate that CNTF is a neuronotrophic factor for motoneurons, but that the effect of CNTF is not restricted to that cell population. In addition to its survival promoting effect, CNTF has also a regulatory role on the expression of ChAT and LNGFR for some spinal cord neurons.     

Nicotine and alpha-bungarotoxin modify the dendritic growth of cholinoceptive neurons in the developing chick tectum

Nicotinic acetylcholine receptors have been suggested to participate in morphogenetic processes during development of the nervous system. In this study, nicotine applied both in ovo and in vitro produced a reduction of the neuritic length of cholinoceptive neurons of the developing chick tectum, whereas alpha-bungarotoxin produced the opposite effect. Taken together with previous data, our results are indicative of a role of the alpha-bungarotoxin-sensitive nicotinic receptors in neural development.     

Synthesis, purification, and characterization of human ciliary neuronotrophic factor from E. coli

The cDNA for human ciliary neuronotrophic factor (CNTF) has been cloned into an expression vector under the control of the T7 promoter. The BL21 strain of E. coli was transformed with this vector. Human CNTF accounted for about 30% of the total bacterial protein after induction with isopropyl-B-D-thiogalactopyranoside. This human CNTF was purified to homogeneity from inclusion bodies by a combination of ion exchange chromatography and reverse-phase high performance liquid chromatography. The amino-terminal amino acid sequence of the purified protein was identical to the deduced amino acid sequence; however, the methionyl residue has been removed. On SDS-PAGE gels, human CNTF displayed a molecular weight of about 24 kDa, in accord with its deduced molecular mass; a pI of 5.8 indicates the acidic nature of the molecule. A proposed structure for human CNTF includes major alpha helical regions. The ED50 of purified human CNTF was approximately 30 pM, using cultured embryonic day 10 chicken dorsal root ganglion neurons; no activity was observed with neurons from embryonic day 8 ganglia. Polyclonal antibodies prepared against both a synthetic peptide of CNTF and the entire human CNTF protein recognized a single 24 kDa band on Western blots, corresponding to human CNTF. However, only the antibodies against intact CNTF blocked its biological activity. This represents the first molecular expression and purification of human CNTF.     

Regulation of acetylcholine receptors on chick ciliary ganglion neurons by components from the synaptic target tissue.

Chick ciliary ganglion neurons have nicotinic acetylcholine receptors (AChRs) that mediate synaptic transmission through the ganglion. A soluble component of about 50 kDa from embryonic eye tissue, the synaptic target of the ganglion, increases the development of ACh sensitivity by the neurons 10-fold over a 1-week period in culture. The increased sensitivity does not arise from a change in agonist affinity or esterase activity. Both the basal ACh response obtained in the absence of the 50-kDa component and the elevated responses obtained with it can be inhibited by neuronal bungarotoxin (nBgt) but not by alpha-bungarotoxin (alpha Bgt). Increases of less than twofold are observed for the binding of anti-AChR monoclonal antibody 35 (mAb 35), nBgt, and alpha Bgt to the neurons under these conditions. Extract fractions containing the 50-kDa component also enable the neurons to enhance their ACh responses through a cAMP-dependent mechanism. Either the 50-kDa fraction induces the appearance of a new type of AChR regulated by cAMP, or it alters the function of existing AChRs. The 50-kDa fraction produces no change in neuronal growth but can increase GABA responses sixfold, indicating that its effects are not confined to AChRs. It is not clear whether a single molecular species is responsible for the diverse regulatory effects or whether several types of active components are present in the fraction. The component which enhances ACh sensitivity is trypsin-sensitive and heat-labile, as expected for a protein. The component may be widely distributed since the 50-kDa fraction from a number of tissues can increase the ACh response. The fraction from eye tissue, however, has a specific activity 5-10 times greater than that of the liver fraction. A wide distribution would suggest multiple targets and roles for the component during development.     

Development of cholinergic pedunculopontine neurons in vitro: comparison with cholinergic septal cells and response to nerve growth factor, ciliary neuronotrophic factor, and retinoic acid

The well-documented role of nerve growth factor (NGF) in the function of cholinergic neurons in the mammalian basal forebrain can be regarded as a paradigm for the action of trophic substances on CNS neurons. Although several growth factors have been identified in recent years, the specificities and importance of such factors for the development of the nervous system are still unknown. In the present study it has been tested whether NGF affects the group of pedunculopontine cholinergic neurons. This population, which has been described in detail only recently, is located more caudally than but resembles, in some aspects, the basal forebrain cholinergic neurons. The cell bodies are located in the metencephalic pedunculopontine and dorsolateral tegmental nuclei. Similar to the forebrain cholinergic neurons, they are medium to large in size and ascend centrally with long axons. Projection areas are widespread throughout the mesencephalon and diencephalon. Dissociated pontine and septal cells of fetal rat brain (embryo ages E14 to E17) were grown in culture for 7 to 14 days in the presence or absence of NGF. Furthermore, a possible action of retinoic acid and ciliary neuronotrophic factor (CNTF) on cholinergic neurons of both the basal forebrain and the pontine area were tested. Differentiation of cultured cholinergic neurons was assessed by biochemical determination of choline acetyltransferase (ChAT) activity and by immunocytochemical staining for ChAT. NGF in concentrations of 1 to 1,000 ng/ml medium increased the number of immunostained cells and the staining intensity in ChAT immunocytochemistry and enhanced ChAT activity by at least 100% above control levels in septal cultures, thus confirming earlier results. In marked contrast, the same concentrations of NGF failed to influence ChAT activity or immunocytochemical staining in cultures of the pontine area. Retinoic acid (10(-8) M to 10(-5) M) and CNTF (0.2 and 2.0 ng/ml, corresponding to 1 and 10 trophic units, as defined in the ciliary ganglion cell assay) failed to enhance ChAT activity in either culture system and did not potentiate the NGF-mediated increase of ChAT activity in septal cultures. Our results, which indicate that pedunculopontine cholinergic neurons do not respond to NGF during development, are in line with those of NGF-receptor visualization studies that failed to demonstrate such receptors on cholinergic pontine cells in postnatal and adult rats. The findings further underline the specificity of NGF action in the central nervous system and, in particular, do not support the idea of transmitter-specific neurotrophic factors.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Regulation of the number of alpha-bungarotoxin binding sites in cultured chick myotubes by a 1,4 dihydropyridine calcium channel antagonist

Calcium has been suggested as the second messenger link between skeletal muscle activity and AChR gene expression and synthesis. We have compared the concentrations of the Ca2+ channel antagonists D600 and nisoldipine needed both to block Ca2+ uptake into cultured myotubes and to increase AChR expression. The good correspondence between these two measurements and the use of the highly specific Ca2+ channel antagonist nisoldipine strengthens the hypothesis that AChR expression is regulated by levels of intracellular Ca2+.     

The developmental appearance of alpha-bungarotoxin binding sites on rodent spinal cord neurons in cell culture

[125I]alpha-Bungarotoxin specifically binds to a subpopulation of rodent spinal cord neurons in vitro. Binding first becomes apparent between 1 and 2 weeks in culture and then increases dramatically after 3 weeks. Similarly, cell suspensions from freshly dissociated embryonic spinal cords do not bind toxin whereas cell suspensions from 1 week old neonates demonstrate specific binding of [125I]alpha-bungarotoxin. In vitro, binding is inhibited more effectively in the presence of nicotinic rather than muscarinic agents. Autoradiography of [125I]alpha-bungarotoxin binding to 4-week-old cultures revealed a uniform labeling pattern over cell somas and processes. Although the relation of toxin binding to functional acetylcholine receptors is not known, the appearance of toxin binding sites may have some developmental significance for the maturation of cholinergic transmission or the maintenance of synaptic connections.     

Specific innervation of neurons in the paravertebral sympathetic ganglia of the chick

I have explored the chick sympathetic system as a model for the study of specific synapse formation by examining the pattern of ganglion cell innervation by preganglionic axons in 2 different ganglia. Using intracellular recording and HRP labeling techniques, the innervation of the 12th and 15th cervical ganglia (C12 and C15) was examined. Sympathetic ganglion cells of the chick are innervated in a stereotyped manner by preganglionic axons arising from different levels of the spinal cord. While each ganglion is innervated by preganglionic axons arising from several spinal cord segments, individual ganglion cells are innervated by only some of the spinal segments that supply each ganglion as a whole. The subset of spinal segments is always contiguous, with 1 segment providing the dominant innervation to the cell. Spinal segments adjacent to the dominant segment provide synaptic inputs that diminish as a function of distance from the dominant segment. This pattern of ganglion cell innervation in the chick is similar to that of the mammal, where re-innervation studies have suggested that ganglion cell innervation is selective. The similarity in the innervation of avian and mammalian sympathetic neurons suggests that the rules that underlie the specificity of synapse formation in the sympathetic system of the 2 species are the same. Because of the accessibility of the chick embryo for experimental manipulations during development, it is now possible to study the cellular basis that underlies the specificity of synapse formation in this relatively simple nervous system.     

Differential effects of nerve transection on the ACh and GABA receptors of chick ciliary ganglion neurons

Chick ciliary ganglion neurons have nicotinic acetylcholine receptors (AChRs) that mediate chemical transmission through the ganglion, and GABAA receptors of unknown significance. Previous experiments examining the role of cell-cell interactions in regulating neuronal AChRs have shown that postganglionic axotomy of ciliary ganglia in newly hatched chicks causes a 10-fold decline in total AChRs within 5 d compared with unoperated contralateral ganglia and that preganglionic denervation causes a 3-fold decline within 10 d. Many of the AChRs are known to be intracellular; of those present on the cell surface, only a small fraction appears to be functionally available normally. In the present experiments, the effects of the operations on functional AChRs and GABAA receptors in the plasma membrane of the neurons were examined by removing the ganglia 5 d after axotomy or 10 d after denervation, dissociating them into single cells, and immediately measuring their ACh and GABA sensitivities with intracellular recording techniques. The ACh sensitivity of axotomized ciliary ganglion neurons was reduced 10-fold compared with neurons from unoperated contralateral ganglia of the same chicks. The reduction could be largely accounted for by a decrease in the maximum response and did not arise from a change either in the dose-response curve or the acetylcholinesterase activity of the neurons. Autoradiographic studies using a radiolabeled anti-AChR monoclonal antibody also demonstrated a substantial decrease in the total number of surface AChRs associated with axotomized neurons. In contrast, axotomy had no unilateral effect on the GABA response.(ABSTRACT TRUNCATED AT 250 WORDS)