Hormonal modulation of neuronal cells behaviour in vitro

In this study we have investigated the effect of insulin and/or of nerve growth factor (NGF) on enzyme activities of cholinergic neurotransmission, in cultured embryonic rat mesencephali. Our data show that choline-O-acetyltransferase (ChAT) and acetylcholinesterase (AChE) activity display a prominent change in the embryonic brain tissues as a function of time in vitro. The change depends on the age of embryos from which the brain cell cultures have been set up. Namely, ChAT activity increases in the cultures taken from 13-17-day-old embryos as a function of time in vitro. AChE activity shows a striking decrease if the cultures have been set up from the older embryos (17-day-old), while AChE activity increases in the cultures prepared from 13-day-old embryos continuously. Insulin (amount ranging 10-27 micrograms/ml) causes a significant inhibition in the ChAT activity in comparison with the increased enzyme activity measured in control cultures (insulin ranging from 1 to 100 ng). AChE activity of 13-day-old embryos was not influenced by insulin (20-27 micrograms/ml) but the same amount of insulin prevents the decrease of AChE activity in cultured brain cells originating from 17-day-old-embryos. Biochemical studies of NGF treated cultures (30 ng/ml) revealed that nerve growth factor resulted in 5-12-fold increase in specific activity of the cholinergic enzyme, choline acetyltransferase (ChAT). NGF did not influence the AChE activity in cultured brain cells (13-17-day-old).     

Role of Thyroid Hormone and Nerve Growth Factor in the Development of Choline Acetyltransferase and Other Cell-Specific Marker Enzymes in the Basal Forebrain of the Rat

The effects of treatment with L-thyroxine (subcutaneously 0.3 microgram/g body weight daily from birth, i.e., day 1) and 2.5S nerve growth factor (NGF; intraventricularly 2 micrograms on 1, 3, 5, 7, and 9 postnatal days), separately and together, were studied on the biochemical development of different cell types in the basal forebrain of 10-day-old rats. The development of cholinergic, gamma-aminobutyric acid-ergic (GABAergic), and glutamatergic neurons was monitored respectively in terms of choline acetyltransferase (ChAT), glutamate decarboxylase (GAD), and glutaminase activities, whereas glutamine synthetase (GS) and 2',3'-cyclic nucleotide-3'-phosphohydrolase (CNPase) activities were used to judge the maturation of astroglial and oligodendroglial cells. Treatment with either thyroid hormone or NGF from birth significantly increased the expression of ChAT activity in the basal forebrain of neonatal rats. When both agents were administered to the same animal, in agreement with our earlier in vitro findings, the stimulation in ChAT activity was much greater than the sum of the individual effects. In hypothyroid rats, significant effects of NGF at the low doses used were not detectable, although the increase of ChAT activity induced by thyroxine was potentiated by NGF in these animals. Under the present experimental conditions neither thyroxine nor NGF treatment had an appreciable effect on the activities of glutaminase, GS, and lactate dehydrogenase. However, the administration of thyroxine markedly increased CNPase activity in normal rats, whereas in hypothyroid rats the effect on both CNPase and GAD was also significant. Similar elevations in CNPase and GAD activities were not observed after NGF treatment, suggesting that the effect of NGF was specific to the cholinergic cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intracellular Storage and Evoked Release of Acetylcholine from Postnatal Rat Basal Forebrain Cholinergic Neurons in Culture with Nerve Growth Factor

Cholinergic neurons from the septum area, the vertical limb of the diagonal band of Broca, and the nucleus basalis of Meynert of postnatal 13-day-old rats were cultured with or without nerve growth factor (NGF) conditions. Total choline acetyltransferase (ChAT) activities, acetylcholine (ACh) contents, and survival numbers of cholinergic neurons in culture from each of three distinct regions were increased by NGF treatment, but little difference was found in cellular ChAT activities and ACh contents obtained in cultures with or without NGF. The result shows that NGF promotes the survival of cholinergic neurons from 13-day-old rats. Furthermore, the release of ACh from cultured neurons was investigated. The cells cultured with NGF showed a larger increase of the high K+-evoked ACh release than those cultured without NGF. However, NGF had no effect on spontaneous release. This suggests that NGF could regenerate and sustain the stimulation-evoked release mechanisms of ACh in cultured cholinergic neurons from postnatal rats.     

Clinical trials of bestatin for leukemia and solid tumors

Dissociated cells from 13- and 17-day-old embryonic rat mesencephali have grown in primary cultures in order to compare the early and late influences of different agents - insulin, dexamethasone and nerve growth factor (NGF) - on the expression of cholinergic maturation process. We have studied cholin acetyltransferase (ChAT) activity, which is regarded as a specific marker for cholinergic function of the brain, and a widely used differentiation marker, the acetyl-cholinesterase (AchE) enzyme. Biochemical maturation of increasing specific activity of ChAT in both younger and older cells was taken into consideration. During cultivation the AchE activity was slightly increased in younger cells, but a dramatic decrease could be noted in older ones. Insulin in concentration from 10 to 27 µg mL^–1 causes a significant inhibition in ChAT activity in comparison with the enzyme activity measured in control cultures (insulin ranging from 1 to 100 ng), independently of embryos age. This polypeptide hormone is able to enhance AchE activity in the cultured cells, especially in older ones. With continuous treatment of the culture with dexamethasone, a synthetic glucocorticoid, the ChAT activity in younger cells reaches a maximum curve by day 9 (nine). At this time the AchE activity shows a slighter, no significant increase than at any other time during cultivation. In cell cultures taken from 17-day-old embryos however dexamethasone treatment evoked a significant decrease in ChAT activity with a concomitant increase of AchE activity which was compared to insulin treatment. In spite of the fact that the NGF is able to enhance the ChAT activity, no significant alteration in AchE activity can be measured in younger cell cultures. These results suggest an uneven expression of the enzymes in embryonic rat mesencephali in the presence of above agents depending on the age of cells.     

Effect of Nerve Growth Factor in Ethylcholine Mustard Aziridinium (AF64A)-Treated Rats: Sensitization of Cholinergic Enzyme Activity in the Septohippocampal Pathway

Abstract: In this study, we examined the effects of nerve growth factor (NGF) administration on cholinergic enzyme activity in both normal and ethylcholine mustard aziridinium (AF64A)-treated rats. Choline acetyltransferase (ChAT) and acetylcholinesterase activity were measured in the hippocampus and septum of rats chronically administered NGF (0.36–2.85 µg/day) into the lateral ventricle for 14 days. In both normal and AF64A-treated rats, NGF increased cholinergic enzyme activity in a dose-dependent manner. Furthermore, although NGF increased ChAT activity in normal rats by 147%, it had a greater effect in AF64A-treated rats, increasing ChAT activity as much as 273%. NGF increased acetylcholinesterase activity in normal rats by only 125% but produced a 221% increase in this activity in AF64A-treated rats. These data indicate that AF64A produces an increased sensitivity to NGF in cholinergic neurons.     

PACAP and NGF cooperatively enhance choline acetyltransferase activity in postnatal basal forebrain neurons by complementary induction of its different mRNA species

Both nerve growth factor (NGF) and pituitary adenylate cyclase activating polypeptide (PACAP) have neurotrophic effects on basal forebrain cholinergic neurons. They promote differentiation, maturation, and survival of these cholinergic neurons in vivo and in vitro. Here we report on the cooperative effects of NGF and PACAP on postnatal, but not embryonic, cholinergic neurons cultured from rat basal forebrain. Combined treatment with NGF, brain-derived neurotrophic factor (BDNF), neurotrophin-4 (NT-4), and PACAP induced an additive increase in choline acetyltransferase (ChAT) activity. There were no cooperative effects on the number of cholinergic neurons, suggesting that ChAT mRNA expression had been induced in each cholinergic neuron. Further analysis revealed that NGF and PACAP led to complementary induction of different ChAT mRNA species, thus enhancing total ChAT mRNA expression. These results explain the cooperative neurotrophic action of NGF and PACAP on postnatal cholinergic neurons.     

Hippocampal Membranes Contain a Neurotrophic Activity That Stimulates Cholinergic Properties of Fetal Rat Septal Neurons Cultured Under Serum-Free Conditions

Primary cultures of fetal rat septal neurons were used to identify a membrane-associated cholinergic neurotrophic activity. Under serum-free culture conditions, approximately 98% of the septal cells are neurons, and approximately 6% of the neurons are cholinergic as determined immunocytochemically. Crude membranes prepared from rat hippocampal homogenates stimulate choline acetyltransferase (ChAT) activity in treated septal neurons. The membrane-associated trophic activity is apparent at lower protein concentrations than activity present in the soluble fraction and is unevenly distributed in various brain regions; it is highest in hippocampus and striatum and negligible in cerebellum. Membrane trophic activity is developmentally regulated, is heat and trypsin sensitive, and increases the rate of expression of ChAT in septal neurons. Upon gel filtration chromatography of a high-salt membrane extract, trophic activity elutes as a broad peak in the 500 kilodalton (kD) molecular mass range. Stimulation of septal neuronal ChAT activity by either crude membranes or partially purified preparations is not inhibited by antibodies against nerve growth factor (NGF), and its maximal activity is additive to maximally active doses of NGF. The results indicate that hippocampal membranes contain cholinergic neurotrophic activity which may be important for the development of septal cholinergic neurons.     

NGF effects on developing forebrain cholinergic neurons are regionally specific

Nerve growth factor (NGF) has been shown to have an effect on neurons in the central nervous system (CNS). A number of observations suggest that NGF acts as a trophic factor for cholinergic neurons of the basal forebrain and the caudate-putamen. We sought to further characterize the CNS actions of NGF by examining its effect on choline acetyltransferase (ChAT) activity in the cell bodies and fibers of developing neurons of the septum and caudate-putamen. ChAT activity was increased after even a single NGF injection. Interestingly, the magnitude of the effect of multiple NGF injections suggested that repeated treatments may augment NGF actions on these neurons. The time-course of the response to NGF was followed after a single injection on postnatal day (PD) 2. NGF treatment produced long-lasting increases in ChAT activity in septum, hippocampus and caudate-putamen. The response in cell body regions (septum, caudate-putamen) was characterized by an initial lag period of approximately 24 hr, a rapid rise to maximum values, a plateau phase and a return to baseline. The response in hippocampus was delayed by 48 hr relative to that in septum, indicating that NGF actions on ChAT were first registered in septal cell bodies. Finally, developmental events were shown to have a regionally specific influence on the response of neurons to NGF. For though the septal response to a single NGF injection was undiminished well into the third postnatal week, little or no response was detected in caudate-putamen at that time. In highlighting the potency and regional specificity of NGF effects, these observations provide additional, support for the hypothesis that NGF is a trophic factor for CNS cholinergic neurons.Dedicated to Dr. E. M. Shooter and Dr. S. Varon as part of a special issue (Neurochemical Research, Vol. 12, No. 10, 1987).     

K-252a Promotes Survival and Choline Acetyltransferase Activity in Striatal and Basal Forebrain Neuronal Cultures

Abstract: The organic molecule K-252a promoted cell survival, neurite outgrowth, and increased choline acetyltransferase (ChAT) activity in rat embryonic striatal and basal forebrain cultures in a concentration-dependent manner. A two- to threefold increase in survival was observed at 75 n M K-252a in both systems. A single application of K-252a at culture initiation prevented substantial (>60%) cell death that otherwise occurred after 4 days in striatal or basal forebrain cultures. A 5-h exposure of striatal or basal forebrain cells to K-252a, followed by its removal, resulted in survival equivalent to that observed in cultures continually maintained in its presence. This is in contrast to results found with a 5-h exposure of basal forebrain cultures to nerve growth factor (NGF). Acute exposure of basal forebrain cultures to K-252a, but not to NGF, increased ChAT activity, indicating that NGF was required the entire culture period for maximum activity. Striatal cholinergic and GABAergic neurons were among the neurons rescued by K-252a. Of the protein growth factors tested in striatal cultures (ciliary neurotrophic factor, neurotrophin-3, NGF, brain-derived neurotrophic factor, interleukin-2, basic fibroblast growth factor), only brain-derived neurotrophic factor promoted survival. The enhancement of survival and ChAT activity of basal forebrain and striatal neurons by K-252a defines additional populations of neurons in which survival and/or differentiation is regulated by a K-252a-responsive mechanism. The above results expand the potential therapeutic targets for these molecules for the treatment of neurodegenerative diseases.     

Cholinergic fiber growth in co-cultures of CNS tissue.

In co-cultures prepared from the septum and the hippocampus, cholinergic fibers originating in the septal slices grew into the neighboring hippocampal tissue and established functional cholinergic connections with pyramidal cells. To get further insight into the mechanisms governing cholinergic fiber growth, we have added TTX to the growth medium (2 x 10(-7) M) to block propagated electrical activity. Under these conditions, considerably fewer cholinergic cells appeared to survive. A few cholinergic fibers still invaded hippocampal target tissue, but their number was markedly reduced compared with control cultures. Simultaneous application of NGF together with TTX, however, not only increased enzyme levels and enhanced survival of cholinergic neurons, but also led to hippocampal ingrowth in virtually all septo-hippocampal co-cultures. These data, therefore, suggest, that in the absence of spiking activity, cholinergic fibers are capable of growing into a co-cultured target tissue. To test the specificity of growth of septal cholinergic fibers, we have co-cultured septal slices with slices of various brain areas which in situ lack a major cholinergic innervation, in particular the cerebellum. In the vast majority of such co-cultures, cholinergic fibers remained restricted within the septal slices, without innervating cerebellar tissue. This failure might in part be related to the lack of trophic factors released by the target tissue. We have, therefore, grown septo-cerebellar cultures in the presence and absence of NGF. Following application of 100 ng/ml NGF during the entire growth of the cultures, numerous AChE-positive fibers originating in the septal slices invaded the co-cultured cerebellar slices.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ciliary neurotrophic factor induces cholinergic differentiation of rat sympathetic neurons in culture

Ciliary neurotrophic factor (CNTF) influences the levels of choline acetyltransferase (ChAT) and tyrosine hydroxylase (TH) in cultures of dissociated sympathetic neurons from newborn rats. In the presence of CNTF both the total and specific activity of ChAT was increased 7 d after culture by 15- and 18-fold, respectively, as compared to cultures kept in the absence of CNTF. Between 3 and 21 d in culture in the presence of CNTF the total ChAT activity increased by a factor of greater than 100. Immunotitration demonstrated that the elevated ChAT levels were due to an increased number of enzyme molecules. In contrast to the increase in ChAT levels, the total and specific activity levels of TH were decreased by 42 and 36%, respectively, after 7 d in culture. Half-maximal effects for both ChAT increase and TH decrease were obtained at CNTF concentrations of approximately 0.6 ng and maximal levels were reached at 1 ng of CNTF per milliliter of medium. The effect of CNTF on TH and ChAT levels were seen in serum-containing medium as well as in serum-free medium. CNTF was shown to have only a small effect on the long-term survival of rat sympathetic neurons. We therefore concluded that the effects of CNTF on ChAT and TH are not due to selective survival of cells that acquire cholinergic traits in vitro, but are rather due to the induction of cholinergic differentiation of noradrenergic sympathetic neurons.     

Evidence for a physiological role of nerve growth factor in the central nervous system of neonatal rats

Forebrain cholinergic neurons have been shown to respond in vivo to administration of nerve growth factor (NGF) with a prominent and selective increase of choline acetyltransferase (ChAT) activity. This has suggested that NGF can act as a trophic factor for these neurons. To test this hypothesis directly, anti-NGF antibodies (and their Fab fragments) were intracerebroventricularly injected into neonatal rats to neutralize endogenously occurring NGF. The anti-NGF antibody administration produced a decrease of ChAT activity in the hippocampus, septal area, cortex, and striatum of rat pups. This finding was substantiated by a concomitant decrease of immunopositive staining for ChAT in the septal area. These effects indicate that the occurrence of endogenous NGF in the CNS is physiologically relevant for regulating the function of forebrain cholinergic neurons.     

Differential Effects of Nerve Growth Factor on Expression of Choline Acetyltransferase and Sodium-Coupled Choline Transport in Basal Forebrain Cholinergic Neurons in Culture

Abstract: Nerve growth factor (NGF) treatment of primary cultures of embryonic day 17 rat basal forebrain differentially altered activity of choline acetyltransferase (ChAT) and high-affinity choline transport; ChAT specific activity was increased by threefold in neurons grown in the presence of NGF for between 4 and 8 days, whereas high-affinity choline transport activity was not changed relative to control. Dose-response studies revealed that enhancement of neuronal ChAT activity occurred at low concentrations of NGF with an EC₅₀ of 7 ng/ml, with no enhancement of high-affinity choline transport observed at NGF concentrations up to 100 ng/ml. In addition, synthesis of acetylcholine (ACh) and ACh content in neurons grown in the presence of NGF for up to 6 days was increased significantly compared with controls. These results suggest that regulation of ACh synthesis in primary cultures of basal forebrain neurons is not limited by provision of choline by the high-affinity choline transport system and that increased ChAT activity in the presence of NGF without a concomitant increase in high-affinity choline transport is sufficient to increase ACh synthesis. This further suggests that intracellular pools of choline, which do not normally serve as substrate for ACh synthesis, may be made available for ACh synthesis in the presence of NGF.     

Selective Expression of Factors Preventing Cholinergic Dedifferentiation

Chicken retina neurons from 8-9-day-old embryos developed prominent cholinergic properties after several days in stationary dispersed cell (monolayer) culture. These cells accumulated [3H]choline by a high-affinity, hemicholinium-sensitive transport system, converted [3H]choline to [3H]-acetylcholine [( 3H]ACh), released [3H]ACh in response to depolarization stimuli, and developed choline acetyltransferase (ChAT) activity to levels comparable to those of the intact retina. The cholinergic state, however, was not permanent. After 7 days in culture, the capacity for [3H]ACh release decreased drastically and continued to diminish with longer culture periods. Loss of this capacity seemed not to be due to loss of cholinergic neurons, because high-affinity choline uptake was unchanged. However, a substantial decrease of ChAT activity was observed as a function of culture age, and probably accounted for the low level of ACh synthesis in long-lasting cultures. The loss of ChAT activity could be prevented in at least two different ways: (a) Maintaining the neurons in rotary (aggregate) rather than stationary culture completely blocked the loss of enzyme activity and gave a developmental profile identical to the known "in situ" pattern of differentiation; and (b) Conditioned medium from aggregate cultures significantly reduced the drop in ChAT activity of neurons maintained in stationary, dispersed cell cultures. Activity that stabilized cholinergic differentiation was nondialyzable, heat-sensitive, and not mimicked by functional nerve growth factor. Production of activity by aggregates was developmentally regulated; medium obtained from aggregates after 3 days in culture had no effect on cholinergic differentiation, whereas medium obtained from aggregates between 6 and 10 days in culture produced a fivefold increase of ChAT in monolayers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Exogenous Nerve Growth Factor Increases the Activity of High-Affinity Choline Uptake and Choline Acetyltransferase in Brain of Fisher 344 Male Rats

The objective of this study was to determine the effect of age and chronic intracerebral administration of nerve growth factor (NGF) on the activity of the presynaptic cholinergic neuronal markers hemicholinium-sensitive high-affinity choline uptake (HACU) and choline acetyltransferase (ChAT) in the brain of Fisher 344 male rats. In 24-month-old rats, a substantial decrease in ChAT activity (30%) was measured in striatum, and decreases in HACU were found in frontal cortex (28%) and hippocampus (23%) compared with 4-month-old controls. Cholinergic neurons in brain of both young adult and aged rats responded to administration of exogenous NGF by increased expression of both phenotypes. In 4-month-old animals, NGF treatment at 1.2 micron/day resulted in increased activities of both ChAT and HACU in striatum (175 and 170%, respectively), frontal cortex (133 and 125%), and hippocampus (137 and 125%) compared with untreated and vehicle-treated 4-month-old animals; vehicle treatment had no effect on the activity of either marker. In 24-month-old animals treated with NGF for 2 weeks, ChAT activity was increased in striatum (179%), frontal cortex (134%), and hippocampus (119%) compared with 24-month-old control animals. Synaptosomal HACU in 24-month-old rats was increased in striatum (151%) and frontal cortex (128%) after 2 weeks of NGF treatment, but hippocampal HACU was not significantly different from control values.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of Chronic Basic Fibroblast Growth Factor Administration to Rats with Partial Flmbrial Transections on Presynaptic Cholinergic Parameters and Muscarinic Receptors in the Hippocampus: Comparison with Nerve Growth Factor

Abstract: The present study compares the effects of chronic administration of basic fibroblast growth factor (bFGF) and nerve growth factor (NGF) on various hippocampal cholinergic parameters in rats with partial unilateral fimbrial transections. Lesions resulted in marked reductions of several presynaptic cholinergic parameters: choline acetyltransferase (ChAT) activity (by 50%), [³H]-acetylcholine ([³H]ACh) synthesis (by 59%), basal and ve-ratridine (1 μM)-evoked [³H]ACh release (by 44 and 57%, respectively), and [³H]vesamicol binding site densities (by 35%). In addition, [³H]AF-DX 116/muscarinic M₂ binding site densities were also modestly decreased (by 23%). In contrast, [³H]pirenzepine/muscarinic M₁ and [³H]AF-DX 384/muscarinic M₂/M₄ binding site densities were not altered by the lesions, nor were they affected by any of the treatments. Intracerebroventricular administration of bFGF (10 ng, every other day, for 21 days) partially prevented the lesion-induced deficit in hippocampal ChAT activity, an effect that was not markedly different from that measured in the NGF-treated (1 μg intracerebroventricularly, every other day, for 21 days) rats. In rats treated with a combination of bFGF and NGF, ChAT activity was not different from that in rats treated with the individual factors alone. In contrast, the lesion-induced deficits in the other cholinergic parameters were not attenuated by bFGF treatment, although they were at least partially prevented by NGF administration. To determine whether higher concentrations of bFGF are necessary to affect cholinergic parameters other than hippocampal ChAT activity, rats were treated with 1 μg (every other day, 21 days) of the growth factor. In this group of rats, detrimental effects of bFGF, manifested by an increased death rate (46%), and marked reductions in body weight of the survivors, were observed. In addition, this concentration of bFGF appeared to exacerbate the lesion-induced reduction in [³H]ACh synthesis by hippocampal slices; [³H]ACh synthesis in lesioned hippocampi represented 36 and 52% of that in contralateral unlesioned hippocampi for the bFGF-treated and control groups, respectively. In conclusion, although bFGF administration attenuates the deficit in hippocampal ChAT activity induced by partial fimbrial transections, this does not appear to translate into enhanced functional capacity of the cholinergic terminals. This is clearly in contrast to NGF, which enhances not only hippocampal ChAT activity, but also other parameters indicative of increased function in the cholinergic terminals.     

Nerve Growth Factor Increases the Intracellular Content of Acetylcholine in Cultured Septal Neurons from Developing Rats

The effects of nerve growth factor (NGF) on the intracellular content of acetylcholine (ACh) in cultured septal neurons from developing rats have been examined. The content of ACh could be measured by using HPLC and electrochemical detection (HPLC-ECD), coupled with an immobilized enzyme column. This method of determination is very simple and rapid, and is highly sensitive. The content of ACh and the activity of choline acetyltransferase (ChAT) in cultured postnatal day 1 (P1) septal neurons grown on an astroglial "feeder" layer was increased during the period of cultivation by the addition of NGF. The activities of ChAT and the content of ACh increased in a dose-dependent manner in direct relationship to the different amounts of NGF employed. These effects of NGF, i.e., elevating the intracellular content of ACh, accompanied by an increase in activity of ChAT, also were confirmed in the P1 septal organotypic cultures. Additionally, embryonic day 17 (E17) septal neurons in a serum-free medium displayed a similar responsiveness to NGF with respect to the elevation in the content of ACh and the increase in activity of ChAT. These results suggest that intracellular levels of ACh are likely to be regulated by NGF in a fashion similar to that of the activity levels of the biosynthetic enzyme.     

Does senile impairment of cholinergic system in rats concern only disturbances in cholinergic phenotype or the progressive degeneration of neuronal cell bodies?

The trophic effect of continuous intraventricular infusion of nerve growth factor (NGF) on morphology of the basal forebrain (BF) cholinergic neurons was tested in 4- and 28-month-old male Wistar rats. All studies were conducted using behaviorally uncharacterized animals from the same breeding colony. Immunohistochemical procedure for choline acetyltransferase (ChAT) and p75NTR receptor has been applied to identify cholinergic cells in the structures of basal forebrain (BF). Using a quantitative image analyzer, morphometric and densitometric parameters of ChAT- and p75NTR-positive cells were measured immediately after cessation of NGF infusion. In 28-month-old non-treated rats the number of intensively ChAT-positive cells in all forebrain structures was reduced by 50-70% as compared with young animals. The remaining ChAT-positive cells appeared shrunken and the neuropil staining was NTR markedly reduced. In contrast, the same neurons when stained for p75 were numerous and distinctly visible with perfect morphology. Analysis of Nissl stained sections also showed that 28-month-old rats did not display significant losses of neuronal cell bodies. NGF restored the number of intensely stained ChAT-positive cells to about 90% of that for young controls and caused a significant increase in size of those cells in 28-month-old rats as compared with the control, age-matched group. NGF did not influence the morphology of p75NTR-positive neurons, which were well labeled, irrespective of treatment and age of the rats. In 4-month-old rats, NGF infusion decreased the intensity of both ChAT and p75NTR immunostaining. These data provide some evidence for preservation of BF cholinergic neurons from atrophy during aging and indicate that senile impairment of the cholinergic system in rats concerns decrease in ChAT-protein expression rather than an acute degeneration of neuronal cell bodies. Treatment with NGF resulted in restoration of cholinergic phenotype in the BF neurons of aged rats. However, the present study also rises issue of possible detrimental effects of NGF in young normal animals.     

Regulation of cholinergic gene expression by nerve growth factor depends on the phosphatidylinositol-3'-kinase pathway

Nerve growth factor (NGF) exerts anti-apoptotic, trophic and differentiating actions on sympathetic neurons and cholinergic cells of the basal forebrain and activates the expression of genes regulating the synthesis and storage of the neurotransmitter acetylcholine (ACh). We have been studying the intracellular signaling pathways involved in this process. Although, in the rat pheochromocytoma cell line PC12, NGF strongly activates the mitogen-activated protein kinase (MAPK) pathway, prolonged inhibition of MAPK kinase (MEK) activity by PD98059 or U0126 did not affect the ability of NGF to up-regulate choline acetyltransferase (ChAT) or to increase intracellular ACh levels. In contrast, the treatment with the phosphatidylinositol 3'-kinase (PI3K) inhibitor LY294002, but not with its inactive analogue LY303511, completely abolished the NGF-induced production of ACh. Inhibition of PI3K also eliminated the NGF effect on the intracellular ACh level in primary cultures of septal neurons from E18 mouse embryos. Blocking the PI3K pathway prevented the activation of cholinergic gene expression, as demonstrated in RT/PCR assays and in transient transfections of PC12 cells with cholinergic locus promoter-luciferase reporter constructs. These results indicate that the PI3K pathway, but not the MEK/MAPK pathway, is the mediator of NGF-induced cholinergic differentiation.     

Cytokine Regulation of Nerve Growth Factor-Mediated Cholinergic Neurotrophic Activity Synthesized by Astrocytes and Fibroblasts

The neurotrophic activity of astrocytes and fibroblasts and its regulation by various cytokines were investigated. Astrocyte conditioned medium (ACM) enhanced the survival of neurons and the proliferation of astrocytes in embryonic cortical cultures grown in serum-free defined medium. However, these results were not affected by acidic fibroblast growth factor, interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF alpha), and transforming growth factor-beta 1. In contrast, ACM induced choline acetyltransferase expression in septal cholinergic neurons via nerve growth factor (NGF)-dependent and -independent mechanisms. However, neither acidic nor basic fibroblast growth factor is involved in this biological activity in ACM. The cytokines listed above mainly stimulate NGF-mediated cholinergic neurotrophic activity in ACM. A combination of IL-1 beta and TNF alpha significantly enhanced choline acetyltransferase activity in septal neurons co-cultured with astrocytes, and this effect was found to be mediated by NGF produced by activated astrocytes. Effects of astrocytes on GABAergic neurons were also examined. ACM was found to increase glutamate decarboxylase activity in neuronal cultures from septum in the presence of Ara-C. However, the cytokines did not enhance this activity in ACM. Moreover, a combination of IL-1 beta and TNF alpha had no effect on glutamate decarboxylase activity in septal neurons co-cultured with astrocytes. In a final set of experiments, cholinergic neurotrophic activity in skin-derived fibroblast conditioned medium (FCM) was examined. FCM was found to possess biological activity similar to that of ACM on septal neurons grown in serum-free defined medium with Ara-C. The cytokines also enhanced NGF-mediated cholinergic neurotrophic activity in FCM. Astrocytes and fibroblasts were found to possess NGF-type and non-NGF-type cholinergic neurotrophic activity, and various cytokines were found to regulate the NGF-type cholinergic neurotrophic activity in both types of cells. NGF produced by astrocytes and fibroblasts that are activated by cytokines is likely to be important for development and regeneration of NGF-sensitive neurons in the central and peripheral nervous systems.