A transient role for ciliary neurotrophic factor in chick photoreceptor development

Previous studies suggest that ciliary neurotrophic factor (CNTF) may represent one of the extrinsic signals controlling the development of vertebrate retinal photoreceptors. In dissociated cultures from embryonic chick retina, exogenously applied CNTF has been shown to act on postmitotic rod precursor cells, resulting in an two- to fourfold increase in the number of cells acquiring an opsin-positive phenotype. We now demonstrate that the responsiveness of photoreceptor precursors to CNTF is confined to a brief phase between their final mitosis and their terminal differentiation owing to the temporally restricted expression of the CNTF receptor (CNTFR alpha). As shown immunocytochemically, CNTFR alpha expression in the presumptive photoreceptor layer of the chick retina starts at embryonic day 8 (E8) and is rapidly down-regulated a few days later prior to the differentiation of opsin-positive photoreceptors, both in vivo and in dissociated cultures from E8. We further show that the CNTF-dependent in vitro differentiation of rods is followed by a phase of photoreceptor-specific apoptotic cell death. The loss of differentiated rods during this apoptotic phase can be prevented by micromolar concentrations of retinol. Our results provide evidence that photoreceptor development depends on the sequential action of different extrinsic signals. The time course of CNTFR alpha expression and the in vitro effects suggest that CNTF or a related molecule is required during early stages of rod differentiation, while differentiated rods depend on additional protective factors for survival.     

Neuronal acetylcholine receptors with alpha7 subunits are concentrated on somatic spines for synaptic signaling in embryonic chick ciliary ganglia

Nicotinic acetylcholine receptors containing alpha7 subunits are widely distributed in the vertebrate nervous system. In the chick ciliary ganglion such receptors generate large synaptic currents but appear to be excluded from postsynaptic densities on the cells. We show here that alpha7-containing receptors are concentrated on somatic spines in close proximity to putative sites of presynaptic transmitter release. Intermediate voltage electron microscopy on thick sections, together with tomographic reconstruction, permitted three-dimensional analysis of finger-like projections emanating from cell bodies. The projections were identified as spines based on their morphology, cytoskeletal content, and proximity to presynaptic elements. Both in situ and after ganglionic dissociation, the spines were grouped on the cell surface and tightly folded into mats. Immunogold labeling of receptors containing alpha7 subunits showed them to be preferentially concentrated on the somatic spines. Postsynaptic densities were present in vivo both on the soma near spines and occasionally on the spines themselves. Synaptic vesicle-filled projections from the presynaptic calyx were interdigitated among the spines. Moreover, the synaptic vesicles often abutted the membrane and sometimes included Omega profiles as if caught in an exocytotic event, even when no postsynaptic densities were juxtaposed on the spine. The results suggest several mechanisms for delivering transmitter to alpha7-containing receptors, and they support new ideas about synaptic signaling via spines. They also indicate that neurons must have specific mechanisms for targeting alpha7-containing receptors to desired locations.     

Accumulation of acetylcholine receptors is a necessary condition for normal accumulation of acetylcholinesterase during in vitro neuromuscular synaptogenesis

To study a step of the very complex processes of the formation of the neuromuscular junction (NMJ), we have analysed the clustering of acetylcholine receptors (AChR) and acetylcholinesterase (AChE) in myotubes cultured in various conditions. On the surface of rat myotubes cultured in the presence of spinal cord cells from embryonic rat, numerous AChE clusters appeared. Such clusters are always co-localized with AChR clusters, but the reverse is not true: the number of AChR clusters largely exceeds that of AChE clusters. Very few AChE clusters formed when such co-cultures were treated with monoclonal antibodies (mAbs) against the main immunogenic region (MIR) of the AChR, which provoke internalization and degradation of the AChRs of the muscular membrane. The total levels of AChE and proportions of molecular forms were unaffected. We also used non-innervated myotubes in which addition of agrin, a protein normally synthesized by motoneurons, transported to nerve terminals and inserted into the synaptic basal lamina, induces the formation of small clusters of AChE. When added to rat myotubes devoid of membrane AChR, agrin-induced AChE clusters did not form. Finally, we analysed the capacity of the variant of the C2 mouse muscle cell line deficient in AChR (1R-) to form clusters of AChE in co-cultures with spinal cord cells from rat: no formation of AChE clusters could be observed. In all these different systems of cultures, the conditions which prevented clustering of AChR (anti-AChR antibodies, deficiency of the variant C2 cell line) also suppressed AChE clustering. We concluded that clustering of AChR is a prerequisite for clustering of AChE, so that NMJ formation implies the sequential accumulation of these two components.     

Hepatocyte growth factor promotes motor neuron survival and synergizes with ciliary neurotrophic factor

Hepatocyte growth factor (HGF) has been shown to function as a potent mitogen for a variety of cells, transducing its signal through the c-met tyrosine kinase receptor. Ciliary neurotrophic factor (CNTF) is a cytokine that has been shown to promote survival of motor neurons. We show here that c-met mRNA is present in the embryonic rat spinal cord. Peak expression of c-met (at E14) coincides with the period of naturally occurring cell death in motor neurons, suggesting a possible role of HGF in the regulation of this process. Utilizing a neuron-enriched culture system, we established that HGF, like CNTF, stimulates choline acetyltransferase (CAT) activity in motor neurons. When co-administered to motor neuron cultures, saturating concentrations of HGF and CNTF produced a synergistic increase in CAT levels. We show that this synergy reflects enhanced motor neuron survival. Exposure of motor neuron cultures to the cytostatic agent vincristine markedly decreased CAT levels; co-treatment with HGF and CNTF (but not either factor alone) restored CAT activity to control levels. Our findings indicate that HGF is a survival factor for motor neurons, that it acts synergistically with CNTF, and that HGF and CNTF can together be neuroprotective in the face of vincristine toxicity.     

Adenoviral gene transfer of ciliary neurotrophic factor and brain-derived neurotrophic factor leads to long-term survival of axotomized motor neurons

OBJECTIVE: This study was undertaken to investigate the influence of oral supplementation with creatine monohydrate on muscular performance during repeated sets of high-intensity resistance exercise. SUBJECTS/DESIGN: Fourteen active men were randomly assigned in a double-blind fashion to either a creatine group (n = 7) or a placebo group (n = 7). Both groups performed a bench press exercise protocol (5 sets to failure using each subject's predetermined 10-repetition maximum) and a jump squat exercise protocol (5 sets of 10 repetitions using 30% of each subject's 1-repetition maximum squat) on three different occasions (T1, T2, and T3) separated by 6 days. INTERVENTION: Before T1, both groups received no supplementation. From T1 to T2, both groups ingested placebo capsules. From T2 to T3, the creatine group ingested 25 g creatine monohydrate per day, and the placebo group ingested an equivalent amount of placebo. MAIN OUTCOME MEASURES: Total repetitions for each set of bench presses and peak power output for each set of jump squats were determined. Other measures included assessment of diet, body mass, skinfold thickness, and preexercise and 5-minute postexercise lactate concentrations. RESULTS: Lifting performance was not altered for either exercise protocol after ingestion of the placebos. Creatine supplementation resulted in a significant improvement in peak power output during all 5 sets of jump squats and a significant improvement in repetitions during all 5 sets of bench presses. After creatine supplementation, postexercise lactate concentrations were significantly higher after the bench press but not the jump squat. A significant increase in body mass of 1.4 kg (range = 0.0 to 2.7 kg) was observed after creatine ingestion. CONCLUSION: One week of creatine supplementation (25 g/day) enhances muscular performance during repeated sets of bench press and jump squat exercise.     

IGF-I enhances survival of embryonic chick ciliary ganglion neurons in a calcium-dependent way

We have shown that neurons from embryonic chick ciliary ganglia in primary culture possess receptors for insulin-like growth factor I (IGF-I). When added to serum- and insulin-free culture medium, the factor potently enhanced neuronal survival as observed after 24 and 48 h of culture. The effect saturated at 5 ng/ml. Laminin was not necessary for the trophic effects of IGF-I; in the absence of the factor, it had no effect on neuronal survival. Insulin exerted a trophic effect similar to that observed with IGF-I, but at higher doses. The trophic effect of IGF-I was sharply and specifically reduced when either a membrane-permeable calcium chelating agent or blockers of voltage-dependent calcium channels were added to the medium.     

Cholinesterases precede "ON-OFF" channel dichotomy in the embryonic chick retina before onset of synaptogenesis

Retinal physiology is balanced by the interplay between an ON and an OFF channel. The formation of this wiring dichotomy is not understood. Two neuropil sublayers of the inner plexiform layer (IPL) represent levels of cholinergic synaptic circuitry. These two sublayers are better segregated in avians than in most vertebrates; in the chick they are called a and d and participate in the OFF and ON channel, respectively. Both levels can be detected easily in the mature retina by choline acetyltransferase (ChAT) or by acetylcholinesterase (AChE); however, the usefulness of these enzymes as developmental markers is restricted, since ChAT is detected too late, while AChE labels not only cholinergic neuropil. To establish that individual AChE+ cells indeed project into cholinergic subbands a or d (and thus are involved in either channel), in the first part of this study we used the AChE-specific monoclonal antibody 1A2 plus confocal microscopy. We show that at embryonic stage E17 two AChE+ cell types of the inner nuclear layer project into the cholinergic subband a, and another one projects into d. Moreover, two others project into noncholinergic subbands, b and c, which develop only from E14 onwards. In contrast to immunocytochemistry, sensitive AChE histochemistry allows us to trace back the establishment of subbands a and d before E10, with the first AChE subband appearing along with IPL differentiation at E7. The establishment of AChE subbands is preceded by butyrylcholinesterase subbands; with differentiation following a central-peripheral gradient. Since synapses do not develop before E12 in the chick, we conclude that retinal ON-OFF dichotomy is laid down long before the formation of chemical synapses.     

Transient up-regulation of ciliary neurotrophic factor receptor-alpha mRNA in axotomized rat septal neurons

PURPOSE: To determine prospectively the feasibility, complications, and mid- and long-term advantages of peripheral insertion of central catheters in infants and children. MATERIALS AND METHODS: During a 15-month period between March 1995 and June 1996, a total of 285 catheter placement attempts were made to peripherally insert central catheters in 183 pediatric patients (89 boys, 94 girls). Phlebographic guidance was used, and the catheters were inserted below the elbow in 99% of cases. Catheter insertion was indicated for prolonged antibiotic therapy in 108 patients (158 catheter placement attempts), hematologic or oncologic care in 24 patients (40 attempts), total parenteral nutrition in 16 patients (46 attempts), and venous access for fluid or blood in 35 patients (41 attempts). The success rate and complications were recorded along with the indication, patient age, and duration of catheter placement. RESULTS: One hundred fifty-two of 158 (96%) catheter placement attempts were successful in outpatients (n = 108), 124 of 127 (98%) in hospitalized patients (n = 75), and 70 of 73 (96%) in patients aged less than 1 year. Infection and pericatheter venous thrombosis were the main complications and were seen in 17 of 276 (6%) and one of 276 (0.3%) catheter placement attempts, respectively. Catheter occlusion occurred in 23 of 276 (8%) catheter placement attempts. CONCLUSION: Peripheral insertion of central catheters was highly feasible in infants and children with this protocol. Such catheters were well tolerated in the pediatric population with a low frequency of complications.     

Release of ciliary neurotrophic factor from cultured astrocytes and its modulation by cytokines

CNTF rescues various types of lesioned neurons in vivo, and it needs to be released from astrocytes into the extracellular space to have the effect. However, direct evidence for CNTF release has not been unequivocally demonstrated. We hypothesized that the rapid sequestration by CNTF receptor present on cultured astrocytes might be the cause of the inability to detect CNTF released into astrocyte-conditioned medium (ACM). Therefore, we measured CNTF immunoreactivity in medium conditioned by astrocytes treated with phosphatidylinositol-specific phospholipase C (PI-PLC) which was used to prevent released CNTF from binding to the CNTF receptor, since PI-PLC cleaves glycosyl-phosphatidylinositol anchor of CNTFR alpha, the unique component involved in CNTF binding. CNTF was not detectable in untreated ACM, but was detectable in PI-PLC-treated ACM. These results together with the evidence that PI-PLC treatment did not have a toxic effect on astrocytes prove the fact that CNTF can be released from astrocytes without cell lysis. Subsequently, the effect of cytokines such as IL-1 beta, TNF-alpha, and EGF on CNTF release was examined. These cytokines increased CNTF protein levels in ACMs without increasing CNTF protein levels in astrocyte-extracts, indicating that they enhanced CNTF release from astrocytes.     

Systemic injections of ciliary neurotrophic factor induce sprouting by adult motor neurons

The ability of ciliary neurotrophic factor (CNTF) to induce sprouting by undamaged adult motor neurons was studied in gluteal muscles of adult ICR mice. Low doses of CNTF (0.02 mg kg-1 day-1) only induced sprouting in gluteus muscles that were beneath the site of injection, whereas high doses of CNTF (0.4-1.2 mg kg-1 day-1) acted systemically to induce motor neuron sprouting. We found little difference between the type or quality of sprouting induced by CNTF compared with muscle paralysis. Both stimuli induced sprouts of the same length, although muscle paralysis tended to induce more sprouts per end-plate. Paralysis also induced more nodal sprouting than did CNTF, but both were weaker stimuli for nodal sprouting than was partial denervation. In addition to its effects on motor neuron sprouting, high doses of CNTF induced loss of up to 36% of the body weight of treated mice. The substantial wasting caused by CNTF indicates that the factor has potent cachectic activity.     

Toxicity and tolerability of recombinant human ciliary neurotrophic factor in patients with amyotrophic lateral sclerosis

We examined the toxicity of both single and multiple subcutaneous injections of recombinant human ciliary neurotrophic factor (rhCNTF) in 72 patients with ALS, in doses ranging from 2 to 100 micrograms/kg. Adverse events were generally dose related and ranged from mild to severe. The tolerability of daily subcutaneous rhCNTF was equivalent to placebo at doses < or = 5 micrograms/kg/day. At higher doses, anorexia, weight loss, reactivation of herpes simplex virus (HSV1) labialis/stomatitis, cough, and increased oral secretions occurred.     

Reduced levels of acetylcholine receptor expression in chick ciliary ganglion neurons developing in the absence of innervation

Chick ciliary ganglion neurons receive innervation from a single source, the accessory oculomotor nucleus (AON), and nicotinic ACh receptors (AChRs) mediate chemical synaptic transmission through the ganglion. Previous experiments examining the developmental expression of AChRs in embryonic chick ciliary ganglion neurons in situ have shown that AChR levels increase substantially in the neurons at the time of innervation. Prior to synapse formation, few AChRs are detected in the neurons. In the present experiments, the role of presynaptic inputs in inducing an increase in AChRs was established by examining AChR levels in ciliary ganglion neurons that have been deprived of innervation by surgical ablation of the AON prior to synapse formation. AChR levels were dramatically reduced in neurons of input-deprived ganglia as compared to control innervated neurons at all developmental stages examined from embryonic day (ED) 5 to ED 12 as determined by indirect immunocytochemical labeling of frozen ganglion sections with the anti-AChR monoclonal antibody mAb 35, and light microscopy. In contrast, neuronal somata of input-deprived and control ganglia had equivalent levels of immunolabeling for three other components, a transmembrane glycoprotein of synaptic vesicles, SV2, and two microtubule-associated proteins, MAP 1B and MAP 2, from ED 5 up to ED 10. The results demonstrate that presynaptic inputs specifically increase the levels of AChR expression in developing neurons. In addition, changes in the levels of immunolabeling for AChRs, SV2, MAP 1B, and MAP 2 in neuronal somata after ED 10 demonstrate that other major developmental events also influence the levels of these components in neurons. Declines in the intensity of AChR, SV2, MAP 1B, and MAP 2 immunolabeling within a subset of neuronal somata in both operated and control ganglia at ED 10 and 12 coincide with the period of neuronal cell death. Increases in AChR labeling in the rest of the neuronal population of input-deprived ganglia at ED 12 suggest that, in addition to innervation, synapse formation with the peripheral target tissue influences AChR levels in developing neurons in situ.     

Reconstitution of the response to leukemia inhibitory factor, oncostatin M, and ciliary neurotrophic factor in hepatoma cells

Ciliary neurotrophic factor (CNTF) has been described as a neuro-active cytokine that shares functional similarities with the leukemia inhibitory factor (LIF). We demonstrate here that, like LIF, CNTF stimulates expression of acute phase plasma proteins in rat H-35 hepatoma cells. Transfection of the LIF receptor into Hep3B hepatoma cells reconstituted LIF and oncostatin M regulation of acute phase plasma protein genes. Co-expression of the LIF receptor and the CNTF receptor, but not expression of either subunit alone, generated CNTF responsiveness in Hep3B cells, suggesting cooperativity of these receptor subunits. Evidence is presented for direct interaction of the LIF receptor with the intracellular signal transduction machinery.     

Magnocellular vasopressinergic neurons in explant cultures are rescued from cell death by ciliary neurotrophic factor and leukemia inhibiting factor

The genus Mycobacterium includes the major human pathogens Mycobacterium tuberculosis and Mycobacterium leprae. The development of rational drug treatments for the diseases caused by these and other mycobacteria requires the establishment of basic molecular techniques to determine the genetic basis of pathogenesis and drug resistance. To date, the ability to manipulate and move DNA between mycobacterial strains has relied on the processes of transformation and transduction. Here, we describe a naturally occurring conjugation system present in Mycobacterium smegmatis, which we anticipate will further facilitate the ability to manipulate the mycobacterial genome. Our data rule out transduction and transformation as possible mechanisms of gene transfer in this system and are most consistent with conjugal transfer. We show that recombinants are not the result of cell fusion and that transfer occurs from a distinct donor to a recipient. One of the donor strains is mc(2)155, a highly transformable derivative that is considered the prototype laboratory strain for mycobacterial genetics; the demonstration that it is conjugative should increase its genetic manipulability dramatically. During conjugation, extensive regions of chromosomal DNA are transferred into the recipient and then integrated into the recipient chromosome by multiple recombination events. We propose that DNA transfer is occurring by a mechanism similar to Hfr conjugation in Escherichia coli.     

Evidence for a tropic role of brain-derived neurotrophic factor in transplanted embryonic retinae

Developing retinal axons must follow a stereotypic course directing them to the subcortical visual centres which on arrival they must recognise. Transplantation studies suggest that local substrate cues close to the surface of the brainstem and diffusible factors emanating from the target region are important. To test a role for diffusible factors, we transplanted retinae to the cerebral cortex and have shown that outgrowth can be promoted by BDNF secreting fibroblasts.