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1.
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.  相似文献   

2.
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).  相似文献   

3.
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.  相似文献   

4.
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.  相似文献   

5.
Villa AE  Tetko IV  Dutoit P  Vantini G 《Bio Systems》2000,58(1-3):219-228
In the adult rat most of basal forebrain cholinergic neurons (BFCN) express the low-affinity p75 nerve growth factor recceptor (NGFr). The immunotoxin 192 IgG-saporin (SAP) provokes a selective loss of NGFr-positive BFCN, somewhat similar to the loss of integrity of BFCN associated with human senile dementia of Alzheimer's type, whereas NGF exerts a trophic action on BFCN. Cortico-cortical interactions are modulated by cholinergic projections of BFCN and it is proposed that alterations of these projections by SAP and by NGF produce opposite effects. This hypothesis was tested by recording multiple local field potentials (LFPs) in the rat temporal cortex and applying bispectral analysis to measure phase-coupled frequencies, somewhat analogous to frequencies of resonance. Choline acetyltransferase (ChAT) activity was measured in the septal area in order to assess the effects of the treatments. NGF-treatment increased ChAT activity by 45% and frequencies of non-linear coupling were shifted towards frequencies higher than 70 Hz, thus suggesting the presence of increased functional interactions in the short range. By contrast, SAP provoked a decrease of nearly 40% in ChAT activity and an increase of phase-coupling in the low frequencies (< 50 Hz), being interpreted as a decreased functional cortico-cortical interaction. Bispectral analysis revealed features of the effect of BFCN on cortical activity that could not be observed by other means and offers as a valuable tool of study that could be extended to the EEG of Alzheimer's patients.  相似文献   

6.
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.  相似文献   

7.
本文用免疫组化双标法观察了神经生长因子受体(NGF-R)及胆碱乙酰转移酶(ChAT)免疫反应阳性神经元在成鼠基底前脑内的分布,结果发现嗅结节、隔内侧核、斜角带核、腹侧苍白球及基底大细胞核均有NGF-R及ChAT免疫反应阳性神经元.免疫组化双标染色发现,大部分免疫反应阳性神经元的NGF-R与ChAT共存,部分神经元呈单纯NGF-R或ChAT阳性,但这种NGF-R和ChAT的共存情况在不同区域不完全相同.在隔内侧核和斜角带核,大多数的NGF-R阳性神经元和ChAT阳性神经元共存,但在腹侧仓白球和基底大细胞核,两者共存的神经元较前两区为少.此外ChAT阳性神经元在尾壳核中分布较均匀,而NGF-R阳性神经元较少见.研究结果表明,大多数胆碱能神经元有NGF-R,提示NGF对胆碱能神经元的保护和激活作用,部分可能是通过直接与NGF受体的结合而发生作用.  相似文献   

8.
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.  相似文献   

9.
Polyclonal antibodies against recombinant human nerve growth factor (rhNGF) potently inhibited PC12 neurite outgrowth, blocked high-affinity 125I-rhNGF binding but not its receptor, and cross-reacted with rat, mouse, and human nerve growth factor (NGF) but not with brain-derived neurotrophic factor, neurotrophin-3, ciliary neurotrophic factor, insulin-like growth factor, epidermal growth factor, or activin A. Immunocytochemistry revealed many NGF-positive neurons in the rat neostriatum. The NGF-positive neurons disappeared by 3 days after mechanical injury to the neostriatum and were replaced by intensely NGF- and glial fibrillary acidic protein-positive astrocytes. Enzyme-linked immunosorbent assay measurements revealed that the NGF content of the injured striatum was elevated by eightfold 3 days postinjury and by twofold 2 weeks later. The high-affinity choline uptake (HACU) into cholinergic nerve terminals was decreased by 23% at 2 and 4 weeks postinjury, yet choline acetyltransferase (ChAT) activity in these neurons was unchanged at 2 weeks and decreased by 14% at 4 weeks. Daily infusion of 1 microgram of rhNGF into the injury area did not alter the loss of HACU. However, this treatment elevated ChAT activity by 23-29% above intact neostriatal levels and by 53-65% relative to HACU at both survival times. Thus, lesion-induced increases in NGF levels within astrocytes are associated with maintenance of striatal ChAT activity at normal levels following cholinergic injury, even with decreases in HACU. Pharmacologic doses of rhNGF can further augment ChAT activity in damaged cholinergic neurons, showing the usefulness of exogenous NGF even when endogenous NGF is elevated in response to injury.  相似文献   

10.
We studied the effects of insulin, nerve growth factor (NGF), and tetrodotoxin (TTX) on cellular metabolism and the activity of glutamic acid decarboxylase (GAD) and choline acetyltransferase (ChAT) in neuron-rich cultures prepared from embryonic day 15 rat striatum. Insulin (5 micrograms/ml) increased glucose utilization, protein synthesis, and GAD activity in cultures plated over a range of cell densities (2,800-8,400 cells/mm2). TTX reduced GAD activity; NGF had no effect on GAD activity. Insulin treatment reversibly reduced ChAT activity in cultures plated at densities of greater than 4,000 cells/mm2, and the extent of this reduction increased with increasing cell density. The number of acetylcholinesterase-positive neurons was not reduced by insulin, suggesting that insulin acts by down-regulating ChAT rather than by killing cholinergic neurons. Insulin-like growth factor-1 (IGF-1) reduced ChAT activity at concentrations 10-fold lower than insulin, suggesting that insulin's effect on ChAT may involve the IGF-1 receptor. NGF increased ChAT activity; TTX had no effect on ChAT activity. These results suggest that striatal cholinergic and GABAergic neurons are subject to differential trophic control.  相似文献   

11.
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)  相似文献   

12.
Abstract: Effects of thyroxine (T4) on nerve growth factor (NGF) level and choline acetyltransferase (ChAT) activity of rat brains were investigated. Repetitive intraperitoneal administration of T4 caused increases in both NGF level and ChAT activity in the frontal cortex, septum, hippocampus, and striatum and decreases in the cerebellum in 2-day-old rats. Only ChAT activity was elevated in the olfactory bulb, and the NGF level remained unchanged there. No changes were observed in the midbrain and pons/medulla. Furthermore, T4 was effective on the post-natal rats only up to day 11. These results suggest that T4 plays a role in the developmental regulation of NGF level and ChAT activity in rat brain in a region- and/or stage-specific manner. That (1) changes in NGF level and ChAT activity occurred in regions nearly identical to those that contained NGF-responding neurons, and (2) the change in NGF level in the hippocampus and frontal cortex was followed by the change of ChAT activity after a single injection of T4 suggest that the effects of T4 on cholinergic differentiation are, at least in part, mediated via NGF, which itself is quantitatively regulated by T4.  相似文献   

13.
Nerve growth factor (NGF) acts through the receptor tyrosine kinase trkA to serve as a trophic factor for cholinergic neurons in the medial septal nucleus and vertical limb of the diagonal band. We have previously shown that the neuronal isoform of nitric oxide synthase (NOS) is selectively expressed in a large fraction of trkA-expressing cholinergic neurons in these brain regions in the adult rat, and that NGF induces the expression of neuronal-NOS in these cells. Herein, we show that: 1) neuronal-NOS is also localized to these neurons in the developing septum; 2) the expression of neuronal-NOS is regulated in the developing medial septal nucleus and vertical limb of the diagonal band; 3) neuronal-NOS regulation parallels that for other markers of basal forebrain cholinergic neuron differentiation, such as cholineacetyltransferase; and 4) NGF infusion in the postnatal period induces robust increases in neuronal-NOS mRNA and in NOS activity in the basal forebrain. Taken together with earlier findings, our results suggest that neuronal-NOS has a role in the differentiation and mature function of septal cholinergic neurons. Through enhancing neuronal-NOS synthesis, endogenous NGF is likely to regulate NO functions in vivo. Special issue dedicated to Dr. Hans Thoenen.  相似文献   

14.
Brain-derived neurotrophic factor (BDNF) was found to promote the survival of E17 rat embryo septal cholinergic neurons in culture, as assessed by a histochemical stain for acetylcholinesterase (AChE). A 2.4-fold increase in neuronal survival was achieved with 10 ng/ml BDNF. After initial deprivation of growth factor for 7 days, BDNF failed to bring about this increase, strongly suggesting that BDNF promotes cell survival and not just induction of AChE. BDNF was also found to increase the levels of cholinergic enzymes; choline acetyltransferase (ChAT) and AChE activities were increased by approximately 2-fold in the presence of 50 ng/ml BDNF. BDNF produced a 3-fold increase in the number of cells bearing the NGF receptor, as detected by the monoclonal antibody IgG-192. Although NGF had no additive effect with BDNF in terms of neuronal survival, suggesting that both act on a similar neuronal population, the combination of both produced an additive response, approximately a 6-fold increase, in ChAT activity.  相似文献   

15.
The potential of cell therapy is promising in nerve regeneration, but is limited by ethical considerations about the proper and technically safe source of stem cells. We report the successful differentiation of human EnSCs (endometrial stem cells) as a rich source of renewable and safe progenitors into high-efficiency cholinergic neurons. The extracellular signals of NGF (nerve growth factor) and bFGF (basic fibroblast growth factor) could induce cholinergic neuron differentiation. ChAT (choline acetyltransferase), MAP2 (microtubule associated protein 2) and NF-l (neurofilament L) increased after administration of bFGF and NGF to the EnSC cultures. trkC and FGFR2 (fibroblast growth factor receptor 2), which belong to the NGF and bFGF receptors respectively, were determined in populations of EnSCs. NGF, bFGF and their combination differentially influenced human EnSCs high efficiency differentiation. By inducing cholinergic neurons from EnSCs in a chemically defined medium, we could produce human neural cells without resorting to primary culture of neurons. This in vitro method provides an unlimited source of human neural cells and facilitates clinical applications of EnSCs for neurological diseases.  相似文献   

16.
Most mammalian sympathetic neurons are noradrenergic, and their dependence upon nerve growth factor (NGF) for survival during development is well established. A minor population of sympathetic neurons, including those that innervate sweat glands, is cholinergic. To determine whether cholinergic sympathetic neurons, like their noradrenergic counterparts, require NGF during development, neonatal rats were treated with NGF-antiserum and 3 weeks later their sweat glands were examined for the presence of innervation. Acetylcholinesterase (AChE) staining and vasoactive intestinal polypeptide-like immunoreactivity (VIP-IR) which mark the mature sweat gland innervation were absent from the sweat glands of the anti-NGF treated animals. Further, when the glands were examined with the electron microscope, no axons or nerve terminals were evident. These observations indicate that the elaboration of the sweat gland plexus is NGF-dependent and suggest that at least one population of cholinergic sympathetic neurons in the rat requires NGF for survival. Our findings are consistent with the idea that during development NGF is a required trophic factor not only for noradrenergic sympathetic but also for cholinergic sympathetic neurons.  相似文献   

17.
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.  相似文献   

18.
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.  相似文献   

19.
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.  相似文献   

20.
T Hagg  D Quon  J Higaki  S Varon 《Neuron》1992,8(1):145-158
Recombinant human ciliary neurotrophic factor (CNTF) was infused for 2 weeks into the lateral ventricle of fimbria-fornix transected adult rats, and its effects were compared with those of purified mouse nerve growth factor (NGF). We provide evidence that CNTF can prevent degeneration and atrophy of almost all injured medial septum neurons (whereas NGF protects only the cholinergic ones). CNTF is also involved in up-regulation of immunostainable low affinity NGF receptor (LNGFR) in cholinergic medial septum and neostriatal neurons and in a population of lateral septum neurons. In contrast to NGF, CNTF did not stimulate choline acetyltransferase in the lesioned septum and normal neostriatum (pointing to different mechanisms for the regulation of choline acetyltransferase and LNGFR), cause hypertrophy of septal or neostriatal cholinergic neurons, or cause sprouting of LNGFR-positive (cholinergic) septal fibers.  相似文献   

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