Increased Choline Acetyltransferase Research Articles

Development of CNS Cholinergic Neurons in Vitro: Selective Effects of CNTF and LIF on Neurons from Mesencephalic Cranial Motor Nuclei

Ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF) have previously been shown to increase the survival and/or differentiation of cholinergic spinal motoneurons in culture. We report here that CNTF and LIF increase choline acetyltransferase (ChAT) activity three-to fourfold in cultures from the ventral mesencephalon (VM) containing cholinergic neurons from cranial motor nuclei and the pedunculopontine nucleus, but decrease tyrosine hydroxylase activity to 50-60% of control values. In contrast, they do not increase cholinergic properties in cultured septal and striatal cholinergic neurons. In order to identify the subpopulations of cholinergic neurons in the mesencephalic cultures responding to CNTF and LIF, embryonic VM was cut into rostral, caudal, central, and laterocaudal portions and cultured separately. CNTF and LIF increased ChAT activity only in the rostral and central VM, which contain cranial motor nuclei, and not in the caudal VM, which contains the rostroventral part of the pedunculopontine nucleus. Together with the previous observations on spinal motoneurons, the present results indicate that only cholinergic motoneurons projecting to peripheral targets, but not cholinergic neurons projecting centrally, may respond to CNTF and LIF by an increase in ChAT activity or by increased neuronal survival.

Interferon-γ promotes cholinergic differentiation of embryonic septal nuclei and adjacent basal forebrain

In cultured rat embryonic septal nuclei with adjacent basal forebrain, murine interferon-γ (IFNγ) produces a striking increase in choline acetyltransferase (ChAT) activity and mRNA. The effect of IFNγ on cholinergic differentiation is more potent in E14 cultures than in older cultures. IFNγ does not cause a change in the affinity of ChAT for choline, nor does it affect cell proliferation. Whereas IFNγ doubles neuronal cell number, the cholinergic cell number increases more than 7-fold. Ameboid microglia respond to IFNγ with the translocation of p91 to the nucleus. The action of IFNγ is not mediated by NGF or bFGF. The enhancement of cholinergic expression that occurs with increased cell density may be partly attributable to an endogenous IFNy-like molecule, since antibodies to IFNy offset the effects of increased cell density.

Encapsulated genetically engineered fibroblasts: Release of nerve growth factor and effects in vivo on recovery of cholinergic markers after devascularizing cortical lesions

Genetically engineered rat fibroblasts producing nerve growth factor (NGF) were encapsulated in alginate-polylysine-alginate gels with the objective to produce viable “minifactories” continuously producing and secreting NGF into the rat brain. Microencapsulated fibroblasts (NGF secretors and NGF non-secretors) were placed onto the surface of the lesioned rat cortex (unilateral devascularizing lesion) and animals were sacrificed 30 days after surgery. Fibroblasts NGF-non secreters normally produce tumors after implantation, therefore, they were irradiated prior to encapsulation. Three other experimental groups were studied in parallel: non-lesioned (controls), lesioned rats receiving “empty” alginate spheres and lesioned animals without treatment and microspheres. Biochemical analysis of microdissected brain tissues of lesioned animals treated with encapsulated NGF-secretor fibroblasts showed a significant increase in choline acetyltransferase (ChAT) activity in cortices adjacent to the lesion but not far from it (entorhinal cortex). This may indicate a gradient of concentration of the released NGF and/or differential responsivity of lesioned vs nonlesioned target tissue. ChAT enzymatic activity in the microdissected nucleus basalis magnocellularis (NBM) was significantly decreased ( P < 0.05) in all lesioned animals treated with spheres without fibroblasts and those with fibroblasts not secreting NGF. Morphometric analysis of ChAT-IR and low affinity NGF-receptor IR cholinergic neurons in the middle portion of the NBM shows a prevention of neuronal shrinkage and extensive neuropil in animals treated with microencapsulated NGF-secretor fibroblasts. The results of this study demonstrate that NGF from encapsulated genetically engineered fibroblasts can be secreted for at least long enough to prevent degenerative changes of cholinergic neurons in the NBM.

IL-6 increases choline acetyltransferase but not neuropeptide transcripts in sympathetic neurons.

The effect of interleukin (IL)-6 on cell survival, and the expression of choline acetyltransferase (ChAT) and neuropeptide mRNAs was examined in cultured rat sympathetic neurons. Reverse transcription-polymerase chain reaction analysis indicated that IL-6 treatment led to six-fold increase in ChAT mRNA without a concomitant increase in ChAT immunoreactivity. In contrast, treatment with ciliary neurotrophic factor or leukemia inhibitory factor/cholinergic differentiation factor increased ChAT mRNA levels 25-fold and resulted in intense ChAT immunoreactivity. Moreover, the latter factors increased somatostatin and preprotachykinin mRNA levels 10-fold whereas IL-6 had no effect. Maximal doses of IL-6 had no effect on cell survival. These findings provide a basis for the overlapping and yet divergent actions of these three factors which share some components of their signal transduction machinery.

Acetyl- l-carnitine treatment increases choline acetyltransferase activity and NGF levels in the CNS of adult rats following total fimbria-fornix transection

Transection of the fimbria-fornix in adult rats is a useful model for producing impairments of cholinergic activity in the hippocampus (HIPP) and atrophy of the medial septum cholinergic perikarya, similar to those observed during senescence, that are possibly due to the lack of nerve growth factor (NGF) retrogradely transported from the hippocampus. In our investigation we used choline acetyltransferase (ChAT) as an index of cholinergic activity in HIPP, frontal cortex (FCX), septum and nucleus basalis magnocellularis (NBM) along with measurements of NGF levels in the HIPP. Three-month-old rats with unilateral total fimbria transection received acetyl- l-carnitine (ALCAR) (150 mg/kg/day in drinking water for 1 week before and 4 weeks after the lesion). ALCAR is a substance known to ameliorate some morphological and functional disturbances in the aging central nervous system (CNS). ChAT activity in septum and FCX, and NGF levels in HIPP were significantly increased in the treated group, compared with untreated control groups, while no changes were found in the NBM. On the other hand, a similar ALCAR treatment in unoperated animals induced an increase in ChAT activity in FCX but not in septum nor in NBM. These data are suggestive of a neurotrophic property of ALCAR exerted on those central cholinergic pathways typically damaged by aging.

New chlorine-containing prenylated C6-C3 compounds increasing choline acetyltransferase (ChAT) activity in culture of postnatal rat septal neurons from Illicium tashiroi.

The structures of two new chlorine-containing C6-C3 compounds isolated from the woods of Illicium tashiroi have been established as 2(R)-12-chloro-2,3-dihydroillicinone E (1) and 12-chloroillicinone E (2) by X-ray crystallographic analysis and spectroscopic data, respectively. Compound 1 has been found to significantly increase ChAT activity in culture of P10 rat septal neurons.

Early in ovo exposure of chick embryos to ethanol prevents the neuronotrophic effects of intracerebral NGF administration on cholinergic phenotypic expression

We have reported that ethanol administration during early neuroembryogenesis significantly alters neuronal phenotypic expression. In addition, previous findings have indicated that ethanol may interfere with the neurotrophic effects of NGF. In this study, we examined the cholinergic neuronal response to NGF given intracerebrally to embryos at embryonic day 8 (E8) which were exposed to ethanol in ovo via the air sac at E1–3. We found that doses of NGF ranging from 0.01 to 1 ng/2 μl/embryo given intracerebrally to untreated embryos at E8, and sacrificed at E10, significantly increased choline acetyltransferase (ChAT) activity, the marker for cholinergic neuronal expression. This response was most marked in spinal cord as compared with the low response observed in cerebral hemispheres. In control embryos treated with saline at E1–3 and then receiving NGF intracerebrally at E8, ChAT activity in the spinal cord increased with increasing NGF doses; the highest value was obtained with 0.1 ng NGF. In contrast, in ethanol-treated embryos, ChAT activity was not affected by intracerebral administration of NGF and, in fact, the highest dose (0.1 ng) produced a decrease in ChAT activity. We conclude that: (1) intracerebral administration of NGF produces differential cholinotrophic effects in the embryonic chick CNS; and (2) exposure to ethanol during early neuroembryogenesis interferes with the cholinotrophic effects of NGF.

Effects of acidic fibroblast growth factor on cholinergic neurons of nucleus basalis magnocellularis and in a spatial memory task following cortical devascularization

The ability of acidic fibroblast growth factor to elicit a trophic response in the nervous system of the rat was tested in vitro and in vivo. Treatment of cultured septal cells with acidic fibroblast growth factor resulted in an elongation of glial processes as assessed by immunostaining for glial fibrillary acidic protein. Increased choline acetyltransferase was also observed. The responses to acidic fibroblast growth factor in vivo were studied in rats trained in a spatial memory task, using the Morris water maze. Randomly selected animals were subjected to unilateral cortical devascularization. This lesion results in partial unilateral infarction of the neocortex, and in retrograde degeneration of the nucleus basalis magnocellularis. Animals were tested post-lesion for memory retention and were then killed for morphological studies. Intracerebroventricular administration of acidic fibroblast growth factor (0.6μg/h for seven days starting at surgery) prevented the lesion-induced impairment in this test, and reduced the nucleus basalis magnocellularis cholinergic degeneration, as assessed by morphometric choline acetyltransferase-like immunoreactivity and radioenzymatic assay for choline acetyltransferase activity. The preservation of the phenotype of injured cholinergic neurons of the nucleus basalis magnocellularis by acidic fibroblast growth factor was indicated by the maintenance of the cross-sectional area of cell bodies and mean length of neuritic processes one month after surgery. The effect of acidic fibroblast growth factor in non-cholinergic cells remains to be investigated. It is suggested that acidic fibroblast growth factor may alleviate the lesion-induced deficit in the memory retention task by preventing disruption of functional connections between nucleus basalis magnocellularis and intact cortical areas. Acidic fibroblast growth factor may act together with other trophic factors such as nerve growth factor to modulate the development and/or plasticity of cholinergic central nervous system neurons. These observations are consistent with the idea that acidic fibroblast growth factor may ameliorate nucleus basalis magnocellularis-dependent memory impairments that occur in degenerative diseases.

Early nerve growth factor-induced events in developing rat septal neurons

A culture system enriched for nerve growth factor (NGF) receptor bearing cells was developed to investigate signal transduction events activated by NGF in postmitotic central nervous system neurons. Cells from the septal region of embryonic rats at 16 days of gestation were grown on glass coverslips above a glial cell layer established from postnatal rat cortex. The separation of glial and neuronal planes in this “bilaminar” system permits the diffusion of glial-derived factors required by septal neurons for survival yet allows the investigation of NGF responses in a pure neuronal population. Approximately 15% of the neurons in this culture system were immunoreactive for the low affinity NGF receptor. NGF rapidly increased MAP kinase activity (2–5 min) and transiently induced expression of c- fos in septal neurons. NGF treatment also increased choline acetyltransferase activity, while the number of cholinergic neurons remained constant. Septal neuron survival depended on the presence of glial cells, but neuronal viability in the bilaminar system was unaffected by anti-NGF antiserum, indicating that glial-derived neurotrophic support is not mediated by NGF alone. These data suggest that the bilaminar culture system is a useful system for the study of early events in NGF-activated signal transduction and the nature of glial-derived trophic support of developing basal forebrain neurons.

The neurotrophins BDNF, NT-3 and NT-4/5, but not NGF, up-regulate the cholinergic phenotype of developing motor neurons.

Although developing motor neurons express low-affinity nerve growth factor (NGF) receptors, there is no known biological effect of NGF on developing or adult motor neurons. In this study, we found that, unlike NGF, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4/5 (NT-4/5) stimulated cholinergic phenotype by increasing choline acetyltransferase (CAT) activity in cultures enriched with embryonic rat motor neurons. Ciliary neurotrophic factor (CNTF) also stimulated CAT activity. The effects of BDNF and NT-4/5 on CAT activity appeared to be synergistic with that of CNTF. Cotreatment with BDNF and NT-3 resulted in an additive effect, suggesting that signal transduction was mediated through different high-affinity receptors tyrosine kinases B and C (Trk B and Trk C). However, cotreatment with BDNF and NT-4/5 did not result in an increase in CAT activity greater than that of either BDNF or NT-4/5 alone, suggesting that their effects were mediated via the same receptor Trk B. Supporting our findings that spinal cholinergic neurons are responsive to trophic actions of members of the neurotrophin family, motor neuron-enriched cultures were found to express mRNA for Trk B and Trk C, which have been identified as high-affinity receptors for BDNF and NT-4/5, and NT-3, respectively.

Effects of repeated administration of rolipram, a cAMP-specific phosphodiesterase inhibitor, on acetylcholinergic indices in the aged rat brain

The effects of repeated treatment of rolipram, a cAMP specific phosphodiesterase inhibitor (0.1 mg/kg/day i.p., 14 days), on several neuronal cholinergic indices, especially on those in aged rats were examined. In young-adult rats, rolipram treatment increased choline acetyltransferase (ChAT) activity ( V maxvalue) in the striatum as well as in thalamus + midbrain, whereas it decreased choline esterase activity in the hippocampus. The ChAT activity ( V maxvalue) and the M1-R binding ( B maxvalue) in the aged control rats were significantly reduced in all the brain regions examined, compared with the young-adult rats, but consecutive rolipram treatment ameliorated the reductions of both indices in the frontal cortex and the hippocampus to approximately the young-adult control levels. Since high membrane binding site concentrations for rolipram itself were revealed in the frontal cortex and the hippocampus, where the rolipram treatment showed ameliorating effects on the ChAT activity and the M1-R binding, the present findings indicate that repeated rolipram administration easily affects these two brain regions. Thus, repeated rolipram administration could restore both the presynaptic ChAT activity and the postsynaptic muscarinic cholinergic M1-R binding which are decreased with aging.

Detailed characterization of the biological activities of recombinant human nerve growth factor expressed in Chinese hamster ovary cells.

The biological activities of recombinant human nerve growth factor (rhNGF) produced by Chinese hamster ovary (CHO) cells that were transfected with human NGF gene were investigated in vitro and in vivo. rhNGF showed the same immunoreactivity as mouse NGF (mNGF) in a highly sensitive two-site enzyme immunoassay system employing mouse monoclonal antibody against mouse beta-NGF (MAb 27/21) for both the primary and the secondary antibodies. In PC12 cells, rhNGF promoted neurite extension and induced acetylcholinesterase (AChE) with the same potency as mNGF, showing an ED50 of 10-20 ng/mL. In fetal rat septal neurons cultured on a feeder layer of astroglial cells, rhNGF promoted survival and neurite extension as well as an increase in choline acetyltransferase (ChAT) activity and acetylcholine (ACh) content. At a maximal effective concentration of 30 ng/mL, rhNGF promoted a 1.4-, 2.8-, and 4-fold increase in surviving cell number, ACh content, and ChAT activity, respectively. rhNGF was five times more potent than mNGF for the increase in ChAT activity and ACh content showing an ED50 of 0.5 ng/mL, although the maximal response was the same for the two NGFs. Transection of the fimbria-fornix resulted in a loss of AChE-positive cells in the medial septum (MS) and vertical limb of the diagonal band of Broca (VDB). The administration of rhNGF or mNGF (3 or 30 micrograms in gel form) attenuated the loss of AChE-positive cells; rhNGF was as potent as or even more potent than mNGF. Radio frequency lesion of the basal forebrain (BF) including the nucleus basalis magnocellularis (NBM) resulted in severe impairment of memory and/or learning in passive avoidance and Morris' water maze tasks. Repeated injection of rhNGF (5 micrograms x 5 over 2 wk) into the lateral ventricle ameliorated the behavioral impairment in the water maze task but not in passive avoidance. rhNGF treatment increased ChAT activity in the frontal cortex and even in other subregions of the cerebral cortex where ChAT activity was not decreased by BF lesion. These results indicate that human NGF can be measured in an enzyme immunoassay system using monoclonal antibody against mNGF (MAb 27/21) and that rhNGF has potent biological activity, comparable to or greater than mNGF, both in vitro and in vivo.

Differential Actions of Neurotrophins in the Locus Coeruleus and Basal Forebrain

The neurotrophin gene family, including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and NT-4/NT-5, supports the survival of distinct peripheral neurons, however, actions upon central neurons are relatively undefined. In this study we have compared different neurotrophins in the regulation of neuronal survival and function using dissociated embryonic cell cultures from two brain regions, the basal forebrain (BF) and locus coeruleus (LC). In the BF, NGF increased choline acetyl transferase (ChAT) activity, but did not influence cholinergic cell survival. In contrast to NGF, BDNF, NT-3, and the novel neurotrophin, NT-4, all increased ChAT activity and cholinergic cell survival. We also examined embryonic LC neurons in culture. LC neurons are unresponsive to NGF. In contrast, NT-3 and NT-4 elicited significant increases in survival of noradrenergic LC neurons, the first demonstration of trophic effects in this critical brain region. Identification of factors supporting coerulcal and basal forebrain neuronal survival may provide insight into mechanisms mediating degeneration of these disparate structures in clinical disorders.

Effects of fetal forebrain transplants in ibotenic-injured nucleus basalis: an anatomical investigation.

Survival of fetal basal forebrain transplant (TP) into ibotenic-injured nucleus basalis of rats was examined after a delay lesion and TP (1 or 2 weeks) and a delay between harvest and TP (1-4.5 hours). Optimal TP survival occurred for TP made 2 weeks postlesion and less than 2 hours after harvesting. In these cases large, healthy TP-neurons displayed robust cytochrome oxidase (CO) activity and sent cholinergic processes throughout the TP and occasionally into host tissue. A mild astrocytic reaction was observed within the TP and at the host-TP interface. Surviving TPs increased choline acetyltransferase innervation and CO activity within the ipsilateral frontoparietal cortex. Therefore data suggest that fetal cholinergic TPs into the damaged NBM reduced neuronal degeneration within the NBM and stimulated remaining neurons spared by the lesion.

Effects of Nerve Growth Factor (NGF) in Rats with Basal Forebrain Lesions

Effects of nerve growth factor (NGF) on the basal forebrain (BF) lesion-induced amnesia in rats were investigated. When NGF infusion was begun immediately after the formation of BF lesions, NGF ameliorated amnesia in a water maze task and showed a tendency to increase choline acetyltransferase (CAT) activity in the fronto-parietal cortex. The amnesia and the decrease of CAT activity were not ameliorated when NGF infusion was begun 4 weeks after BF lesion formation. These observations suggest that NGF may act as a trophic and/or a protective factor on partially damaged cholinergic neurons and that the efficacy of NGF was influenced by the phase of neuronal damage.

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.

Role of the cell recognition molecule, cognin, in GABAergic differentiation in chick retina

Previous work showed that GABAergic differentiation in developing chick retina depends on insulin and cell interactions. Here, we investigated whether it depended on cell signaling mediated by retina cognin, a 50 kDa cell recognition molecule. Cognin mediates cell adhesion in vitro and occurs on retinal neurons that become both GABAergic and cholinergic. We investigated two markers of GABAergic differentiation: glutamate decarboxylase (GAD) activity and high-affinity GABA uptake. Both increase during differentiation of retinal neurons in culture and can be easily measured. We blocked cognin-mediated cell signaling with cognin antibody and found a reduction of the developmental increase in GAD activity in cultures of retinal neurons from 7 and 11 day chick embryos. There was no reduction of high-affinity GABA uptake. This suggested that cognin-mediated signaling was necessary for the normal developmental increase in GAD but not for high-affinity GABA uptake. These results contrasted with our previous observations on cholinergic differentiation in cultured retinal neurons. We found that cognin antibody blocked the normal developmental increase in choline acetyltransferase (ChAT) only if the cells were exposed before embryonic day 7. Thus, while both GAD and ChAT activity appear to be controlled by cell signaling involving cognin, the periods of developmental sensitivity for the two differentiation markers are different. Antibodies to other adhesion molecules, Ng-CAM, and N-cadherin, did not similarly affect GAD activity. Antibodies to laminin at a 10-fold higher concentration inhibited GAD activity only in early embryonic retina. Tests for protein synthesis and “housekeeping” enzyme activity demonstrated that the cognin antibody effect was selective for neuronal differentiation pathways. Thus, GABAergic differentiation in developing retina is sensitive to cell signaling mediated in part by cognin.

Effect of delayed treatment with nerve growth factor on choline acetyltransferase activity in the cortex of rats with lesions of the nucleus basalis magnocellularis: dose requirements

Rats received bilateral ibotenic acid-lesions of the nucleus basalis magnocellularis. Starting two weeks after the lesion, cytochrome c (0.3 μg/rat/day) or 0.01, 0.1, 1 or 10 μg/rat/day human recombinant nerve growth factor (NGF) was infused into the lateral ventricle. The highest dose of NGF reduced the weight gain of the animals. Six weeks, but not two weeks of treatment with 10 μg/rat/day NGF increased choline acetyltransferase (ChAT) activity in the frontal cortex, parietal cortex and hippocampus, predominantly on the side of the ventricular cannula. The 1 μg/rat/day dose only increased ChAT activity in the frontal cortex on the infused side. Six weeks of treatment with 10 μg/rat/day NGF increased the size, but not the number of NGF-receptor-immunoreactive neurons in the nucleus basalis. This treatment did not affect the levels of dopamine, norepinephrine and serotonin in any of the brain regions studied. These data suggest that prolonged treatment with relatively high doses of NGF is necessary to increase ChAT activity in cortical regions of nucleus basalis-lesioned rats. This treatment will also increase ChAT activity in the intact septohippocampal system, but does not affect the levels of several non-cholinergic neurotransmitters.

1,1,3 Tricyano-2-amino-1-propene (Triap) stimulates choline acetyltransferase activity in vitro and in vivo

1,1,3 Tricyano-2-amino-1-propene (Triap) is a small molecule that has neurotrophic properties similar to nerve growth factor (NGF). Studies have shown that NGF increases choline acetyltransferase (ChAT) activity, the enzyme responsible for the synthesis of acetylcholine, in several cell lines and in the CNS of adult animals. To investigate whether Triap can cause similar increases in ChAT enzyme activity, we used the PC12 cell line and primary cultures of rat fetal brain tissue to examine Triap's effects. Nanomolar concentrations of Triap produced a 4.2- and 2.1-fold increase in the ChAT activity of PC12 cells and cultured rat fetal brain cells, respectively. This stimulation reached a plateau within 4 days of treatment in the primary fetal brain cultures with the first increases evident within 24 h. In the PC12 cell line, Triap's stimulation of ChAT activity was significantly greater than increases produced by optimal concentrations of NGF. Triap also matched NGF's stimulation of ChAT activity in primary neuronal culture. Triap also potentiated NGF's actions on ChAT activity in the PC12 cell line and in primary fetal neuronal cultures. These increases in enzyme activity correlated with increases in cellular enzyme levels as assessed using immunochemical identification of the ChAT enzyme. We also conducted experiments to determine if Triap also induced these same increases in ChAT activity in adult animals. Ten-day chronic injections of Triap in mice resulted in significant increases in specific ChAT enzyme activity in the cortex and septal-hippocampal area. Similar increases in ChAT enzyme levels were also detected using western blotting techniques. Thus, it appears that Triap increases ChAT activity, similar to NGF, in both cultured cells and in adult animals. Triap's effects on ChAT enzyme activity seem to be mediated primarily through increases in enzyme concentration. These results show that Triap is a neurotrophic agent with potent effects on the cholinergic nervous system and may be useful as a potential therapeutic agent in diseases exhibiting cholinergic deficits.

Cholinergic differentiation factor (CDF/LIF) promotes survival of isolated rat embryonic motoneurons in vitro

We present evidence that the cholinergic differentiation factor (CDF), originally purified from cardiac and skeletal muscle cell-conditioned medium and found to be identical to leukemia inhibitory factor (LIF), promotes survival of embryonic day 14 rat motoneurons in vitro. These neurons were retrogradely labeled with the fluorescent tracer Dil and enriched on a density gradient or purified to homogeneity by fluorescence-activated cell sorting. Subnanomolar concentrations of CDF/LIF supported the survival of 85% of the motoneurons that would have died between days 1 and 4 of culture. The enhanced survival was accompanied by a 4-fold increase in choline acetyltransferase (ChAT) activity per culture. CDF/LIF also increased ChAT activity in dorsal spinal cord cultures, but had no detectable effect on ChAT levels in septal or striatal neuronal cultures. For comparison, other neurotrophic molecules were tested on motoneuron cultures. Ciliary neurotrophic factor had effects on motoneuron survival similar to those of CDF/LIF, whereas basic fibroblast growth factor was somewhat less effective. Nerve growth factor had no effect on the survival of rat motoneurons.