High ciliary neuronotrophic specific activity in rat peripheral nerve

Ciliary neuronotrophic factors (CNTFs) are proteins defined by their ability to promote the survival in tissue culture of chick embryo ciliary ganglionic neurons. CNTF activity has been reported in several source materials. Among these sources, the highest specific activity, i.e. 16,000 TU/mg, has been found in the intraocular tissue innervated by the ciliary ganglionic neurons. We now report that extracts of adult rat peripheral nerve and spinal nerve roots contain CNTF at a specific activity equal to or greater than the intraocular tissue. Equally high concentrations of CNTF activity are found in both motor and sensory nerves. The possible cellular source(s) of this mammalian CNTF is discussed.     

Expression of Ciliary Neurotrophic Factor and the Neurotrophins-Nerve Growth Factor,Brain-Derived Neurotrophic Factor and Neurotrophin 3-in Cultured Rat Hippocampal Astrocytes

Cultured astrocytes are known to possess a range of neurotrophic activities in culture. In order to examine which factors may be responsible for these activities, we have examined the expression of the genes for four known neurotrophic factors – ciliary neurotrophic factor (CNTF), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin 3 (NT3) – in purified astrocyte cultures derived from neonatal rat hippocampus. Hippocampal astrocytes were found to express mRNA for three neurotrophic factors – CNTF, NGF and NT3 – at significantly higher levels than other cultured cell types or cell lines examined. BDNF messenger RNA (mRNA), however, was undetectable in these astrocytes. The levels of CNTF, NGF and NT3 mRNA in astrocytes were largely unaffected by their degree of confluency, while serum removal caused only a transient decrease in mRNA levels, which returned to basal levels within 48 h. Astrocyte-derived CNTF was found to comigrate with recombinant rat CNTF at 23 kD on a Western blot. Immunocytochemical analysis revealed strong CNTF immunoreactivity in the cytoplasm of astrocytes, weak staining in the nucleus, but no CNTF at the cell surface. NGF and NT3 were undetectable immunocytochemically. CNTF-like activity, as assessed by bioassay on ciliary ganglion neurons, was found in the extract of cultured astrocytes but not in conditioned medium, whereas astrocyte-conditioned medium supported survival of dorsal root ganglion neurons but not ciliary or nodose ganglion neurons. This conditioned medium activity was neutralized with antibodies to NGF. Astrocyte extract also supported survival of dorsal root ganglion and nodose ganglion neurons, but these activities were not blocked by anti-NGF. Part, but not all, of the activity in astrocyte extracts which sustained nodose ganglion neurons could be attributed to CNTF.     

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).     

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, beta-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 x 10(-5) M leu- and met-enkephalin as well as somatostatin supported sympathetic neurons to the same extent as NGF. At the same concentrations, leu-enkephalin, 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 x 10(-5) M had a small effect on ciliary neurons.(ABSTRACT TRUNCATED AT 250 WORDS)     

Polyornithine-attached neurite-promoting factors (PNPFs). Culture sources and responsive neurons

We have recently reported the existence within chick embryo heart cell conditioned medium (HCM) of two distinct and independently assayable factors. One agent, ciliary neuronotrophic factor (CNTF), supports the in vitro survival of 8-day chick embryo ciliary ganglionic (CG) neurons. The other factor, polyornithine-attachable neurite promoting factor (PNPF) is required for extensive neuritic growth from these same CNTF-supported CG neurons.In the present study we have examined the occurrence of PNPF activity within nearly 100 different conditioned media using our previously described chick CG bioassay system. From this screening we conclude that: (1) PNPF production is a rather widespread property of cultured neural as well as non-neural cells; and (2) the chick bioassay is sensitive to PNPF activity from all the species examined, including mouse, rat, human and chick cells.We next examined the effects of 3 representative PNPF-containing conditioned media (from chick heart, mouse Schwann and rat Schwannoma) on neurite production from 3 other peripheral ganglionic neuronal cultures (8-day chick dorsal root, 11-day chick sympathetic, and neonatal mouse dorsal root ganglia) as well as 4 central neuronal cultures (8-day chick embryo telencephalon, optic lobe and spinal cord and neonatal mouse cerebellum). The results of these studies indicate: (1) that the peripheral neurons exhibit a dramatic increase in neurite production in response to PNPF which can be easily recognized both qualitatively and quantitatively; whereas (2) the CNS neurons showed essentially no PNPF-induced increase in neurite production. The sole exception to the latter was the appearance within the chick spinal cord cultures of a neuronal population which extended very long neurites in response to PNPF.     

Age-dependent control of dorsal root ganglion neuron survival by macromolecular and low-molecular-weight trophic agents and substratum-bound laminins

Chick embryo dorsal root ganglion (DRG) neurons can be supported in vitro by nerve growth factor (NGF) and ciliary neuronotrophic factor (CNTF). Pyruvate is also required for survival of neurons from embryonic day 8 (E8) chick ciliary ganglia and from several chick and rat embryonic central nervous system sources. Here we have examined the survival requirements of chick DRG neurons between E6.5 and E15. These DRG neurons, initially dependent only on NGF, become dependent also on CNTF and later on increasingly independent from both factors. Pyruvate nearly doubles neuronal survival at all ages under all conditions. The pyruvate concentration permitting this additional survival was reduced two-fold with serine present. In the presence of polyornithine-bound laminins, nearly all seeded neurons were rescued by pyruvate plus NGF (E8 on), or pyruvate plus CNTF (E10 on), or pyruvate without trophic factors (E15). The same maximal survival was achieved without pyruvate by supplying E10 or older neurons with both NGF and CNTF. Unmodified polyornithine substrata yielded about one-half this number of surviving neurons.     

Survival of purified embryonic chick retinal ganglion cells in the presence of neurotrophic factors

In a search for neurotrophic factors (NTFs) regulating retinal ganglion cell (RGC) death in the chick embryo we have used purified and cultured RGCs. Purification of RGCs from embryonic day 10 was achieved by employing the "panning" method (Silverstein and Chun: Soc Neurosci Abstr 13:1054, 1987). The obtained neuron population consisted of 97% RGCs as demonstrated by retrograde labeling with a fluorescence dye. RGCs were cultured at low density in a chemically defined medium and short-term survival (24 hr) was determined. In the absence of NTFs, less than 3% of the RGCs survived. In the presence of various crude or purified NTFs (eye, brain, and tectum extracts; glial-conditioned medium; ciliary neurotrophic factor [CNTF]; nerve growth factor [NGF]) 31% to 52% of the RGCs were maintained. The effects of NGF and CNTF were not additive. Neither acidic nor basic fibroblast growth factor was able to maintain RGCs in culture. Our results, obtained with a culture system which allowed the analysis of direct trophic actions, suggest that NGF and CNTF may be NTFs for overlapping subpopulations of chick RGCs.     

Cholinergic neuronotrophic factors: I. Survival, neurite outgrowth and choline acetyltransferase activity in monolayer cultures from chick embryo ciliary ganglia

Two key components of neural development and regeneration, survival of the involved neurons and elongation of neuritic elements, are likely to depend on the availability of an appropriate trophic drive to these neurons. At present, only one trophic factor, Nerve Growth Factor, is known to ensure both survival and neuritic growth for its target neurons. A search for a second such agent, a putative cholinergic neuronotrophic factor (CNTF), has been undertaken using as indicators neuronal survival, neurite outgrowth and choline acetyltransferase (CAT) activity in monolayer cell cultures. Eight-day chick embryo ciliary ganglia yielded two monolayer culture systems which appear to be well suited for a CNTF assay. Ciliary ganglionic dissociates, seeded on a highly adhesive collagen substratum, show no neuronal survival by 24 h if the medium is supplemented only with serum or chick embryo extract. However serum and embryo extract combined support survival of, and extensive neuritic outgrowth from, nearly the theoretical number of ganglionic neurons seeded. Alternatively, ciliary ganglionic neurons can be made to survive and produce a profuse neuritic outgrowth on polyornithine-coated dishes if supplied with medium conditioned over chick embryo heart muscle cultures, as already described by other laboratories. The two trophic sources differ markedly in their effects on the ganglionic neurons when tested on collagen or polyornithine substrata, and in some cases when different serum supplements are used. Neuronal survival, neurite production and, possibly, CAT activity appear to be subject to independent regulation. The culture systems used in this study can be developed into quantitative bioassays for the isolation of the different agents responsible for neuronal survival and neurite promotion, and for the investigation of their activities.     

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.     

Neuron-enriched cultures of adult rat dorsal root ganglia: establishment, characterization, survival, and neuropeptide expression in response to trophic factors

It is unknown whether adult dorsal root ganglion (DRG) neurons require trophic factors for their survival and maintenance of neuropeptide phenotypes. We have established and characterized neuron-enriched cultures of adult rat DRGs and investigated their responses to nerve growth factor (NGF), ciliary neuronotrophic factor (CNTF), pig brain extract (PBE, crude fraction of brain-derived neuronotrophic factor, BDNF), and laminin (LN). DRGs were dissected from levels C1 through L6 and dissociated and freed from myelin fragments and most satellite (S-100-immunoreactive) cells by centrifugation on Percoll and preplating. The enriched neurons, characterized by their morphology and immunoreactivity for neuron-specific enolase, constituted a population representative of the in vivo situation with regard to expression of substance P (SP), somatostatin (SOM), and cholecystokinin-8 (CCK) immunoreactivities. In the absence of trophic factors and using polyornithine (PORN) as a substratum, 60-70% of the neurons present initially (0.5 days) had died after 7 days. LN as a substratum did not prevent a 30% loss of neurons up to day 4.5, but it subsequently maintained DRG neurons at a plateau. This behavior might reflect a cotrophic effect of LN and factors provided by non-neuronal cells, whose proliferation between 4.5 and 7 days could not be prevented by addition of mitotic inhibitors of gamma-irradiation. CNTF, but not NGF, slightly enhanced survival at 7 days on either PORN or LN. No neuronal losses were found in non-enriched cultures or when enriched neurons were supplemented with PBE, indicating that non-neuronal cells and PBE provide factor(s) essential for adult DRG neuron survival. Proportions of SP-, SOM-, and CCK-immunoreactive cells were unaltered under any experimental condition, with the exception of a numerical decline in SP cells in 7-day cultures with LN, but not PORN, as the substratum. Our data, considered in the context of recent in vivo and vitro studies, suggest that a combination of trophic factors or an unidentified factor, rather than the established molecules NGF, CNTF, and BDNF, which address embryonic and neonatal DRG neurons, are required for the in vitro maintenance of adult DRG neurons.     

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.     

Choroid coat extract and ciliary neurotrophic factor strongly promote neurite outgrowth in the embryonic chick retina

Previous studies have shown that extracts from the target optic tectum stimulate neurite outgrowth from retinal explants. The present study indicates that the choroid coat is an even richer source of retinotrophic activity. We thus studied the effects of recombinant rat ciliary neurotrophic factor (CNTF) on primary cultures of dissociated chick ciliary ganglion neurons and retinal explants for a comparison with choroid coat extract from the E 18 chick. For our assays, E9 ciliary neurons were incubated in collagen gels and retinal explants were cultured on collagen gels with the addition of the trophic factors and maintained for two or four days. Survival of ciliary neurons per area as well as maximal neurite length in retinal cultures were determined. Growth responses occurred in a dose-dependent manner both to CNTF and choroid extract. Immunofluorescence examination of cells and developing processes showed 200 kdal neurofilament positivity demonstrating that the cells studied were neurons with neurites. It is concluded that a trophic activity of the choroid as well as the recombinant CNTF stimulate retinal neuron survival and neurite extension. The results suggest that CNTF may have developmental functions in the establishment of the visual pathways.     

Site-directed mutagenesis of human CNTF: Functional analysis of recombinant variants

Ciliary neurotrophic factor (CNTF), interleukin-6 (IL-6), leukemia inhibitory factor (LIF), and oncostatin M (OSM) share functional properties, a predicted common helical framework, and partially identical receptor components. CNTF is a survival promoting factor for various types of neurons in vitro and in vivo. In the present study, structural features essential for the biological function of human CNTF were investigated. Several recombinant CNTF variants were constructed by PCR and expressed in E.coli. Their survival promoting activities were determined using cultures of embryonic chick and newborn rat dorsal root ganglion cells. Deletion of 14 N-terminal and 18 C-terminal amino acids significantly increased bioactivity compared to wild-type (wt) CNTF. Further truncation of the CNTF molecule at the N- or C-terminus resulted in a significant reduction or complete loss of activity. Substitution of two amino acids (Lys154Glu and Trp157Pro) abolished the survival promoting effect. Recently described analogous substitutions in IL-6 had resulted in a partial IL-6 receptor antagonist. However, the double substitution variant had no significant inhibitory effect on wtCNTF activity in assays with both wt and mutant factor. The CNTF variants constructed had almost identical effects on both chick and rat neurons indicating a close similarity of the avian and the mammalian CNTF receptor complex. The present results also demonstrate that a core segment of the CNTF molecule is indispensable for biological function. Analogous segments important for activity have already been identified in the related molecules IL-6, LIF, and OSM. Thus, our data confirm the close structural relationship of CNTF to these “neuropoietic” cytokines. In addition, they demonstrate that site-directed mutagenesis of recombinant human CNTF can yield molecules which show increased survival promoting activity on mammalian neurons. © 1995 Wiley-Liss, Inc.     

An examination of ciliary neuronotrophic factors from avian and rodent tissue extracts using a blot and culture technique

We have previously reported a technique for determining the apparent molecular weight (Mr) of ciliary neuronotrophic factors (CNTFs) in crude extracts. This method involves SDS-polyacrylamide gel electrophoresis of the extract. Western blotting and culture of purified ciliary ganglion neurons on the paper containing the blotted lane. Neurons will survive only if in direct contact with the trophic factor band and the surviving neurons, when stained with a vital dye, will outline the CNTF band thereby indicating the Mr of the active polypeptide. Here we have modified this 'blot and culture' technique by including Mr standard proteins in the same electrophoretic lane with the samples, identifying the proteins by staining the nitrocellulose blot with Amido black, marking the standard bands with pinholes and destaining the blot prior to seeding neurons onto it. The active CNTF polypeptides can then be identified by their ability to support the 24-h survival of cultured ciliary neurons. This modified technique was used to determine the Mr of CNTF activities in several chick and rat tissue extracts of selected developmental ages and to ascertain if the two forms of CNTF are exclusive to chick and rat, embryonic and adult, or eye and nerve tissues. We report that the above modifications permitted a more accurate method for Mr determination than the previous method, only two apparent forms of CNTF were recognized, the Mr found for each form is 25 kDa and 28 kDa, both forms can be present in chick and rat tissues and from embryonic and adult sources and the 28 kDa form is predominant in rat while the 25-kDa form is predominant in chicken tissues.     

Survival effect of ciliary neurotrophic factor (CNTF) on chick embryonic motoneurons in culture: comparison with other neurotrophic factors and cytokines

In previous studies, it has been demonstrated that ciliary neurotrophic factor (CNTF) has a potent survival effect on various populations of neurons in culture, in particular, neurons isolated from chick ciliary, dorsal root sensory, and sympathetic ganglia (Barbin et al., 1984). After recent investigations demonstrated that CNTF prevents the degeneration of motoneurons in newborn rats after axonal lesion (Sendtner et al., 1990), the question arose as to whether CNTF also has a survival effect on embryonic chick motoneurons at the developmental stage where physiological cell death occurs. To study this, it was essential to develop an isolation and culture procedure for the survival of chick E6 spinal motoneurons in which non-neuronal cells were eliminated and the motoneurons were highly enriched. In these cultures, virtually all of the initially plated motoneurons survived for at least 3 d in the presence of muscle extract, which was chosen as a positive control. Retrograde labeling of the motoneurons prior to their isolation showed that there is more than an 80% enrichment for motoneurons by the method used. The retrogradely labeled neurons also fulfilled the morphological criteria (diameter of neurons, appearance of processes) to identify motoneurons independent of retrograde labeling. Under these conditions, CNTF at a concentration of 1.5 ng/ml (EC50, 0.023 ng/ml) supported maximally 64% of the initially plated spinal motoneurons after 3 d and 53% after 6 d (the longest time period investigated). Other neurotrophic factors, such as NGF, brain-derived neurotrophic factor (BDNF), and neurotrophin-3, had no survival effect at all, even at concentrations up to 10 micrograms/ml for NGF and BDNF.(ABSTRACT TRUNCATED AT 250 WORDS)     

An automated colorimetric microassay for neuronotrophic factors

A microassay is described for determining the number of neurons surviving after 24 h in response to added neuronotrophic factors. Neuronal cultures in 96-well microtiter plates are supplied with a yellow tetrazolium derivative, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide), which is taken up selectively by viable neurons and converted to a blue formazan product. The amount of blue color development can be rapidly quantified using an automatic microplate spectrophotometer. The resulting optical density is directly proportional to the number of viable neurons. The spectrophotometer has been interfaced with a computer allowing a print out of individual absorbance values and calculation of half-maximal (one trophic unit) neuronal survival. The assay has been used for the quantification of the trophic activities of nerve growth factor and ciliary neuronotrophic factor using, respectively, dorsal root and ciliary ganglionic neurons from 8-day chick embryos. Assay parameters were optimized so that about 2000 individual cultures of ganglionic neurons can be set up and analyzed each day, thus allowing the serial titration in duplicate of 80-120 separate samples. The determination of neuronal number and titer calculation steps now requires about 2 min per microplate (96 cultures), a 50-fold reduction in time over existing methods.     

Control of Na,K-pump activity in dorsal root ganglionic neurons by different neuronotrophic agents

Na⁺,K⁺-pump activity is indispensable for neuronal survival in vitro and a specific role in its regulation has been demonstrated for the NGF action on its target neurons. We have extended these earlier studies to include two other neuronotrophic agents: the chick eye-derived ciliary neuronotrophic factor (CNTF); and 12-O-tetradecanoyl-phorbol-13-acetate (TPA). CNTF and TPA individually supported the survival of an identical (and maximal) number of embryonic day 10 (E10) dorsal root ganglion (DRG) neurons as did NGF. E10 DRG neurons, seeded as monolayer cultures with⁸⁶Rb⁺ (as K⁺ tracer) but no trophic supplement in their medium, received NGF, CNTF, TPA, or no agent at 2, 4 or 6 h after seeding. The cultures were analyzed at 6 and 24 h for Na⁺,K⁺-pump performance and at 24 h for neuronal survival. Neurons receiving no agent lost their pump activity over the first 6 h and died over the 10–24 h incubation period. Both pump performance and survival were fully supported by any one of the 3 agents when provided at seeding time. Delayed presentation of NGF also led to full restoration of pump activity and survival support, as expected. In contrast, CNTF and TPA failed to correct the increasing pump deficits incurred with increasing times of trophic deprivation, and neuronal survival was proportionally reduced. Delayed addition of CNTF and TPA did, however, prevent further losses of both pump and viability. Close similarities were observed between pump failure and cell losses, demonstrating a linear correlation between pump performance and neuronal survival. Thus, neuronal survival is strongly correlated with the Na⁺,KK⁺-pump performance regardless of whether the DRG neurons are supported by NGF, CNTF or TPA. All 3 agents protect the neurons against pump losses (hence, against death), but only NGF appears to be able to restore pump function and cell viability.     

Ciliary neurotrophic factor enhances neuronal survival in embryonic rat hippocampal cultures

First described as a survival factor for chick ciliary ganglion neurons, ciliary neurotrophic factor (CNTF) has recently been shown to promote survival of chick embryo motor neurons. We now report neurotrophic effects of CNTF toward three populations of rat hippocampal neurons, the first demonstration of effects of CNTF upon rodent CNS neurons in culture. CNTF elicited an increase in the neurofilament content of hippocampal cultures prepared from embryonic day 18 (E18) rat brain. This was accompanied by increases of 2-, 28-, and 3-fold in the number of GABAergic, cholinergic, and calbindin-immunopositive cells, respectively. CNTF totally prevented the 67% loss of GABAergic neurons that occurred in control cultures over 8 d. CNTF also increased high-affinity GABA uptake and glutamic acid decarboxylase activity. Effects of CNTF were in all cases dose dependent, with maximal stimulation at approximately 100 pg/ml. When addition was delayed for 3 d, CNTF failed to elicit increases either in the number of cholinergic neurons or in GABA uptake.     

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.