p75 mediates death of cholinergic neurons during postnatal development of the neostriatum in mice

We have previously shown that p75 nerve growth factor receptor (p75^NGFR) mediates apoptosis of 25% of the cholinergic basal forebrain neurons in normal control mice between postnatal day 6 and 15, but only of cholinergic neurons that lacked the nerve growth factor receptor TrkA. Here, we investigated whether and when the cholinergic neurons of the neostriatum, which express TrkA and p75^NGFR during early postnatal times, undergo p75^NGFR-mediated death. The cholinergic neurons in the lateral neostriatal regions expressed choline acetyltransferase (ChAT) earlier (postnatal day 3–6) than those of the medial regions and TrkA appeared before ChAT in all regions. Between postnatal day 6 and 10, 40% of the ChAT-positive neurons in the most lateral regions disappeared in control mice but not in p75^NGFR-deficient mice. During this time, the neostriatum of control, but not p75^NGFR-deficient, mice contained many apoptotic cells. This suggests that, similar to the cholinergic neurons of the basal forebrain, the neostriatal cholinergic neurons of control mice die and that this process is mediated by p75^NGFR. However, the roles of p75^NGFR and TrkA appear to be more complicated in the neostriatum where relatively few neurons express p75^NGFR during the death phase (and predominantly in the lateral neostriatum where the neuronal loss is greatest), and TrkA-positive as well as TrkA-negative neurons may be lost.     

Nerve growth factor survival signaling in cultured hippocampal neurons is mediated through TrkA and requires the common neurotrophin receptor P75

The role of the common neurotrophin receptor p75 (p75NTR) in neuronal survival and cell death remains controversial. On the one hand, p75NTR provides a positive modulatory influence on nerve growth factor (NGF) signaling through the high affinity neurotrophin receptor TrkA, and hence increases NGF survival signaling. However, p75NTR may also signal independently of TrkA, causing cell death or cell survival, depending on the cell type and stage of development. Here we demonstrate that TrkA is expressed in primary cultures of hippocampal neurons and is activated by NGF within 10 min of exposure. In primary hippocampal cultures neuroprotection by NGF against glutamate toxicity was mediated by NF-kappaB and accompanied by an increased expression of neuroprotective NF-kappaB target genes Bcl-2 and Bcl-xl. In mouse hippocampal cells lacking p75NTR (p75NTR-/-) activation of TrkA by NGF was not detectable. Moreover, neuroprotection by NGF against glutamate toxicity was abolished in p75NTR-/- neurons, and the expression of bcl-2 and bcl-xl was markedly reduced as compared to wildtype cells. NGF increased TrkA phosphorylation in hippocampal neurons and provided protection that required phosphoinositol-3-phosphate (PI3)-kinase activity and Akt phosphorylation, whereas the mitogen-activated protein kinases (MAPK), extracellular-regulated kinases (Erk) 1/2, were not involved. P75NTR signaling independent of TrkA, such as increased neutral sphingomyelinase (NSMase) activity causing enhanced levels of ceramide, were not detected after exposure of hippocampal neurons to NGF. Interestingly, inhibition of sphingosine-kinase blocked the neuroprotective effect of NGF, suggesting that sphingosine-1-phosphate was also involved in NGF-mediated survival in our cultured hippocampal neurons. Overall, our results indicate an essential role for p75NTR in supporting NGF-triggered TrkA signaling pathways mediating neuronal survival in hippocampal neurons.     

Cholinergic medial septum neurons do not degenerate in aged 129/Sv control or p75(NGFR)-/-mice

Cholinergic medial septum neurons express TrkA and p75 nerve growth factor receptor (p75(NGFR)) and interactions between TrkA and p75(NGFR) are necessary for high-affinity binding and signaling of nerve growth factor (NGF) through TrkA. In adult p75(NGFR)-deficient (-/-) mice, retrograde transport of NGF and other neurotrophins by these neurons is greatly reduced, however, these neurons maintain their cholinergic phenotype and size. Reduced transport of NGF has been proposed to play a role in Alzheimer's disease. Here, we investigated whether chronic and long-term absence of p75(NGFR) (and possibly reduced NGF transport and TrkA binding) would affect the cholinergic septohippocampal system during aging in mice. In young (6-8 months), middle aged (12-18 months), and aged (19-23 months) 129/Sv control mice the total number of choline acetyltransferase-positive medial septum neurons and the mean diameter and cross sectional area of the cholinergic cell bodies were similar. The cholinergic hippocampal innervation, as measured by the density of acetylcholinesterase-positive fibers in the outer molecular layer of the dentate gyrus was also similar across all ages. These parameters also did not change during aging in p75(NGFR) -/- mice and the number and size of the choline acetyltransferase-positive neurons and the cholinergic innervation density were largely similar as in control mice at all ages. These results suggest that p75(NGFR) does not play a major role in the maintenance of the number or morphology of the cholinergic basal forebrain neurons during aging of these mice. Alternatively, p75(NGFR) -/- mice may have developed compensatory mechanisms in response to the absence of p75(NGFR).     

Delayed NGF infusion fails to reverse axotomy-induced degeneration of basal forebrain cholinergic neurons in adult p75(LNTR)-deficient mice

The p75 low-affinity neurotrophin receptor (p75^LNTR) appears to have various functions that include enhancing nerve growth factor (NGF)-mediated survival by increasing TrkA (high-affinity NGF receptor) efficiency, and mediating apoptosis by acting as a ligand-regulated pro-apoptotic receptor. Here, we investigated the role of p75^LNTR for adult cholinergic basal forebrain neurons by comparing neuronal responses to injury in control and p75^LNTR-deficient mice. In both types of mice, 70% of the cholinergic neurons in the ipsilateral medial septum had lost their markers choline acetyltransferase and tyrosine kinase A by 28 days following unilateral transection of the dorsal septohippocampal pathway (fimbria fornix). A 7-day delayed infusion of NGF that started 28 days after the injury resulted in reversal of choline acetyltransferase expression and cell atrophy in control, but not in p75^LNTR-deficient, mice. This lack of response to delayed NGF treatment in p75^LNTR-deficient mice was most likely not due to cell death, as all of the septohippocampal neurons, labeled with Fluorogold before the lesion, were present at 28 days post-lesion, similar to control mice. p75^LNTR-deficient cholinergic neurons can respond to NGF as they were protected by NGF infusions that started immediately after the injury. These observations, the fact that lesioned p75^LNTR-deficient neurons atrophy faster, and that non-lesioned neurons hypertrophy in response to NGF in control but not in p75^LNTR-deficient mice, suggest that p75^LNTR is needed for tyrosine kinase A and NGF signaling efficiency.

In conclusion, during adulthood p75^LNTR appears to play a beneficial role in the response of cholinergic neurons to injury, consistent with the proposed role of p75^LNTR in the enhancement of TrkA signaling and the transport of neurotrophins by these neurons.     

Amelioration of cholinergic neurons dysfunction in aged rats depends on the continuous supply of NGF

The present study was designed to examine whether NGF-induced improvement in morphology of senile basal forebrain cholinergic neurons persist after discontinuation of NGF treatment. Trophic effect of continuous intraventricular infusion of NGF was tested in the 4- and 28 months old male Wistar rats immediately after cessation of NGF and 3 or 6 weeks after termination of treatment. Immunohistochemical procedure for ChAT, TrkA, and p75(NTR) receptor has been applied to identify cholinergic cells in the basal forebrain structures. Using the quantitative image analyzer, morphometric and densitometric parameters of cholinergic cells were measured. In untreated 28-month-old rats a reduction in the number, size and intensity of staining of cholinergic neurons was observed in all basal forebrain structures. NGF significantly improved morphological parameters of ChAT- and TrkA-positive cells in aged rats. In 28-month-old rats tested within 3 and 6 weeks after discontinuation of infusion a renewed progressive deterioration of cholinergic phenotype of basal forebrain neurons was observed when compared with the NGF-treated immediately tested group. The parallel staining for p75(NTR) revealed normal morphology of the basal forebrain neurons, despite of the age of rats or the NGF treatment. Analysis of Nissl stained sections also showed that 28-month-old rats did not display significant losses of neurons in the basal forebrain when compared with the young animals. These findings demonstrate that senile impairment of cholinergic neurons is induced by a loss of cholinergic phenotype rather than an acute degeneration of cell bodies. NGF may be beneficial in enhancing cholinergic neurochemical parameters, but the protective effects seem to be dependent on the continuous supply of NGF.     

Nerve growth factor activates aorta endothelial cells causing PI3K/Akt- and ERK-dependent migration

Nerve growth factor (NGF) is a well-known neurotrophin. We determined whether NGF can activate endothelial cell migration and signalling that underlie angiogenic processes. We showed that aorta endothelial cells express mRNA for both the receptor tyrosine kinase TrkA and the p75 neurotrophin receptor (p75NTR) that associates with TrkA when signalling occurs. Pig aortic endothelial cells migrated when exposed to an NGF gradient, due to the simultaneous activation of the phosphatidylinositol 3-kinase and extracellular signal-regulated kinase signalling pathways. Furthermore, morphological changes were found in migrating cells: they appear with elongated structures with a smaller cell volume than control cells. Our data show that NGF is an activator of endothelial cells and suggest that NGF plays a role in mediating angiogenesis.     

Nicotine increases the expression of neurotrophin receptor tyrosine kinase receptor A in basal forebrain cholinergic neurons

In this study, we investigated whether the potential positive effects of nicotine in Alzheimer's disease (AD) may involve neurotrophic factors, such as nerve growth factor (NGF), closely associated with basal forebrain (BF) cholinergic function and survival. To this aim, we studied the effects of prolonged nicotine treatment on neurotrophin receptors expression and on NGF protein levels in the rat BF cholinergic circuitry. Both in vivo and in vitro experiments were conducted. We found that s.c. nicotine infusion (1.2 mg free base/kg/d delivered by mini-pumps for 7 days) induced in vivo an increase in tyrosine kinase receptor A (TrkA)—but not TrkB, TrkC or low affinity neurotrophin receptor p75 (p75)—expression in BF cholinergic neurons targeting the cerebral cortex. Nicotine did not produce statistically significant long-lasting effects on NGF levels in the cerebral cortex, or in the BF. In vitro experiments performed on primary BF neuronal cultures, showed that 72 h exposure to nicotine increased both TrkA expression, and NGF release in culture medium. Neutralization experiments with an anti-NGF antibody showed that NGF presence was not necessary for nicotine-induced increase of TrkA levels in cultured cholinergic neurons, suggesting that nicotine may act through NGF-independent mechanisms. This study shows that nicotine, independently of its action on NGF levels, may contribute to the restoration of the trophic support to BF cholinergic neurons by increasing TrkA levels.     

Co-expression of the P75 neurotrophin receptor and neurotrophin receptor-interacting melanoma antigen homolog in the mature rat brain

The p75 neurotrophin receptor (p75(NTR)) is involved in the regulation of neuronal survival and phenotype, but its signal transduction mechanisms are poorly understood. Recent evidence has implicated the cytoplasmic protein NRAGE (neurotrophin receptor-interacting MAGE (from Melanoma AntiGEn) homolog) in p75(NTR) signaling. To gain further insight into the role of NRAGE, we investigated the co-expression of NRAGE and p75(NTR) in mature rat brain. In all areas examined, NRAGE appeared to be confined to neurons. In the basal forebrain cholinergic complex, NRAGE immunoreactivity was evident in all p75(NTR)-positive neurons. There were many more NRAGE-positive than p75(NTR)-positive neurons in these regions, however. NRAGE was also expressed in areas of the basal forebrain that did not express p75(NTR), including the lateral septal nucleus and the nucleus accumbens. A finding in marked contrast to previous studies was the presence of p75(NTR) immunoreactivity in neuronal cell bodies in the hippocampus. Hippocampal p75(NTR) immunoreactivity was apparent in rats 6 months and older, and was localized to the dentate gyrus and stratum oriens. All p75(NTR)-positive neurons in the dentate gyrus and hippocampal formation were positive for NRAGE. The majority of granular cells of the dentate gyrus and pyramidal cells in the hippocampal formation were positive for NRAGE and negative for p75(NTR). NRAGE was also present in some neuronal populations that express p75(NTR) after injury, including striatal cholinergic interneurons, and motor neurons. A region of marked disparity was the cerebral cortex, in which NRAGE immunoreactivity was widespread whereas p75(NTR) was absent. The results are consistent with an important role for NRAGE in p75(NTR) signaling, as all cells that expressed p75(NTR) also expressed NRAGE. The wider distribution of NRAGE expression suggests that NRAGE may also participate in other signaling processes.     

Leukaemia inhibitory factor prevents loss of p75-nerve growth factor receptor immunoreactivity in medial septal neurons following fimbria-fornix lesions.

Transection of the fimbria-fornix leads to retrograde degeneration of axotomized septal cholinergic neurons as manifested by loss of choline acetyltransferase and low-affinity nerve growth factor receptor (p75NGFR) immunoreactivity. Nerve growth factor administered into cerebral ventricles at the time of axotomy can prevent these changes, while ciliary neurotrophic factor can prevent the loss of p75NGFR immunostaining. Leukaemia inhibitory factor shares structural homologies with ciliary neurotrophic factor and has similar actions in the nervous system. Both proteins share the same signalling pathways, which involve the interleukin-6 transducing receptor components leukaemia inhibitory factor receptor beta and gp130. In this study, we compared the effects of leukaemia inhibitory factor, ciliary neurotrophic factor and nerve growth factor, administered into cerebral ventricles, on p75NGFR and choline acetyltransferase immunoreactivity in septal neurons after fimbria-fornix transection. We found that leukaemia inhibitory factor, like ciliary neurotrophic factor, prevents the loss of p75NGFR-stained medial septal neurons after fimbria-fornix axotomy, without maintaining choline acetyltransferase expression in these neurons. In addition, p75NGFR-immunostained neurons had significantly smaller mean diameter after axotomy in leukaemia inhibitory factor- and ciliary neurotrophic factor-treated animals as compared with either nerve growth factor-treated or unlesioned animals. These findings suggest that both leukaemia inhibitory factor and ciliary neurotrophic factor can prevent the axotomy-induced cell death of septal cholinergic neurons, but that, in contrast to nerve growth factor, these growth factors do not maintain the expression of choline acetyltransferase or the normal neuronal size of these injured neurons.     

Cholinergic neurons of mouse intrinsic cardiac ganglia contain noradrenergic enzymes, norepinephrine transporters, and the neurotrophin receptors tropomyosin-related kinase A and p75

Half of the cholinergic neurons of human and primate intrinsic cardiac ganglia (ICG) have a dual cholinergic/noradrenergic phenotype. Likewise, a large subpopulation of cholinergic neurons of the mouse heart expresses enzymes needed for synthesis of norepinephrine (NE), but they lack the vesicular monoamine transporter type 2 (VMAT2) required for catecholamine storage. In the present study, we determined the full scope of noradrenergic properties (i.e. synthetic enzymes and transporters) expressed by cholinergic neurons of mouse ICG, estimated the relative abundance of neurons expressing different elements of the noradrenergic phenotype, and evaluated the colocalization of cholinergic and noradrenergic markers in atrial nerve fibers. Stellate ganglia were used as a positive control for noradrenergic markers. Using fluorescence immunohistochemistry and confocal microscopy, we found that about 30% of cholinergic cell bodies contained tyrosine hydroxylase (TH), including the activated form that is phosphorylated at Ser-40 (pSer40 TH). Dopamine beta-hydroxylase (DBH) and norepinephrine transporter (NET) were present in all cholinergic somata, indicating a wider capability for dopamine metabolism and catecholamine uptake. Yet, cholinergic somata lacked VMAT2, precluding the potential for NE storage and vesicular release. In contrast to cholinergic somata, cardiac nerve fibers rarely showed colocalization of cholinergic and noradrenergic markers. Instead, these labels were closely apposed but clearly distinct from each other. Since cholinergic somata expressed several noradrenergic proteins, we questioned whether these neurons might also contain trophic factor receptors typical of noradrenergic neurons. Indeed, we found that all cholinergic cell bodies of mouse ICG, like noradrenergic cell bodies of the stellate ganglia, contained both tropomyosin-related kinase A (TrkA) and p75 neurotrophin receptors. Collectively, these findings demonstrate that mouse intrinsic cardiac neurons (ICNs), like those of humans, have a complex neurochemical phenotype that goes beyond the classical view of cardiac parasympathetic neurons. They also suggest that neurotrophins and local NE synthesis might have important effects on neurons of the mouse ICG.     

Expression and p75 neurotrophin receptor dependence of cholesterol synthetic enzymes in adult mouse brain

Normal brain function depends critically on cholesterol. Although cholesterol is synthesized locally in the adult brain, the precise anatomical localization of cholesterogenic enzymes is not known. Here we show that 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoAred) and 7-dehydrocholesterol reductase (7dhcred), the first and last enzymes, respectively, in the cholesterol biosynthesis pathway, are co-expressed in neurons throughout adult murine brain. Co-localization is most prominent in cortical, hippocampal, and cholinergic neurons. Since adult hippocampal and cholinergic neurons express p75 neurotrophin receptors (p75NTR) we hypothesized that p75NTR regulates expression of cholesterogenic enzymes. Treatment of Neuro2a neuroblastoma cells or primary cerebellar cultures with siRNA downregulates p75NTR and decreases the expression level of HMG-CoAred and 7dhcred. Native neuroblastoma cell lines with differential expression of p75NTR differentially express 7dhcred; 7dhcred expression correlates with p75NTR expression. This suggests that, in p75NTR-expressing cells, p75NTR regulates cholesterol synthesis through regulation of HMG-CoAred and 7dhcred expression. The unexpected localization of cholesterogenic enzymes in adult neurons suggests that at least some adult neurons retain the ability to synthesize cholesterol.     

Loss of GABAergic neurons in medial septum after fimbria-fornix transection

Neurons in the medial septum of the rat brain undergo retrograde degeneration after transection of their projection to the hippocampal formation, the fimbria-fornix. This cell death has been characterized for both Nissl-stained neurons and acetylcholinesterase-stained neurons. The major cell type in the medial septum is GABAergic, and many of these GABAergic neurons project to the hippocampal formation. Because the fimbria-fornix transection causes more neuronal death than can be accounted for by the loss of cholinergic neurons, we have sought to determine if the GABAergic neurons undergo a cell death similar to that reported for the cholinergic neurons. We report here that GABAergic neurons are indeed lost after the transection but the time course is considerably slower than that for the cholinergic neurons.     

Human cholinergic basal forebrain: chemoanatomy and neurologic dysfunction

The human cholinergic basal forebrain (CBF) is comprised of magnocellular hyperchromic neurons within the septal/diagonal band complex and nucleus basalis (NB) of Meynert. CBF neurons provide the major cholinergic innervation to the hippocampus, amygdala and neocortex. They play a role in cognition and attentional behaviors, and are dysfunctional in Alzheimer's disease (AD). The human CBF displays a continuum of large cells that contain various cholinergic markers, nerve growth factor (NGF) and its cognate receptors, calbindin, glutamate receptors, and the estrogen receptors, ER and ERβ. Admixed with these cholinergic neuronal phenotypes are smaller interneurons containing the m2 muscarinic acetylcholine receptor (mAChRs), NADPH-diaphorase, GABA, calcium binding proteins and several inhibitory neuropeptides including galanin (GAL), which is over expressed in AD. Studies using human autopsy material indicate an age-related dissociation of calbindin and the glutamate receptor GluR2 within CBF neurons, suggesting that these molecules act synergistically to induce excitotoxic cell death during aging, and possibly during AD. Choline acetyltrasnferease (ChAT) activity and CBF neuron number is preserved in the cholinergic basocortical system and up regulated in the septohippocampal system during prodromal as compared with end stage AD. In contrast, the number of CBF neurons containing NGF receptors is reduced early in the disease process suggesting a phenotypic silence and not a frank loss of neurons. In end stage AD, there is a selective reduction in trkA mRNA but not p75^NTR in single CBF cells suggesting a neurotrophic defect throughout the progression of AD. These observations indicate the complexity of the chemoanatomy of the human CBF and suggest that multiple factors play different roles in its dysfunction in aging and AD.     

The effects of nerve growth factor upon the neuropeptide content of the suprachiasmatic nucleus of rats withdrawn from ethanol are mediated by the nucleus basalis magnocellularis

It has been previously shown that withdrawal from alcohol decreases the synthesis and expression of vasopressin (VP) and vasoactive intestinal polypeptide (VIP) in the suprachiasmatic nucleus (SCN), and that the infusion of NGF over 1 month completely restores these changes. Because SCN neurons do not express TrkA, NGF might have exerted its effects either through direct signalling of the neurons via p75^NTR or by enhancing the activity of the cholinergic afferents to the SCN, which arise from the nucleus basalis magnocellularis (NBM). The observation that the infusion of NT-3 to withdrawn rats does not elicit any change in neuropeptide expression in the SCN suggests that ACh might be implicated in this process, a hypothesis that we have attempted to clarify in this study. For this purpose we destroyed, with quinolinic acid, the NBM of rats withdrawn from ethanol and later infused them with NGF over a period of 13 days. The total number and the somatic volume of SCN neurons immunoreactive for VP and VIP were stereologically estimated. No differences were found in the total number of neurons between quinolinic-injected NGF-treated withdrawn animals and intact withdrawn rats. However, the somatic volume of SCN neurons from quinolinic-injected animals was significantly reduced relative to control and withdrawn rats. The present results unequivocally demonstrate that the trophic effects exerted by NGF upon SCN neurons do not depend on direct neuronal signalling. Instead, they are indirect and, according to our results, NBM neurons, whose axons give rise to a cholinergic projection to the SCN, seem to be essential for eliciting those effects.     

P75 nerve growth factor receptor is important for retrograde transport of neurotrophins in adult cholinergic basal forebrain neurons

The role of the p75 nerve growth factor receptor in the retrograde transport of neurotrophins in the adult CNS was investigated by comparing the transport of 125I-labeled neurotrophins by normal and p75 nerve growth factor receptor-deficient cholinergic septohippocampal neurons. In control mice, nerve growth factor was selectively transported from the hippocampal formation to the cholinergic neurons in the septum. Nerve growth factor labeling was found in three to four times as many septal cholinergic neuronal cell bodies than labeling for neurotrophin-3 or neurotrophin-4/5, and transported brain-derived neurotrophic factor was barely detectable. Cells were considered as labeled when the number of grains per cell exceeded five times background. In p75 nerve growth factor receptor-deficient mice, the number of cholinergic neurons labeled with each of the neurotrophins was reduced by 85-95%. Retrograde labeling of septohippocampal neurons with Fluorogold was not obviously reduced in p75 nerve growth factor receptor-deficient mice, suggesting that general transport mechanisms were not impaired. Despite the reduced neurotrophin transport, cholinergic neurons of p75 nerve growth factor receptor-deficient mice were larger than controls and had an apparently normal density of immunostaining for choline acetyltransferase. Since nerve growth factor is reportedly involved in size regulation and choline acetyltransferase expression, this raises the possibility that the retrograde transport itself is not essential for these events. Thus, p75 nerve growth factor receptor plays an important, although not exclusive, role in the transport of neurotrophins by cholinergic basal forebrain neurons, and retrograde transport of nerve growth factor may not be needed for regulating certain cellular processes.     

Thymocyte depletion affects neurotrophin receptor expression in thymic stromal cells

Thymocytes and thymic stromal cells cross-talk in a bidirectional manner within the thymus, thus contributing to the generation of mature T-cells. The thymic stromal cells in the rat express the high- (TrkA, TrkB) and low-affinity (p75NTR) receptors for neurotrophins. In this study we analysed the regulation of TrkA, TrkB and p75NTR expression in the rat thymus by thymocytes. We induced thymocyte apoptosis by administration of corticoids in rats, and then analysed the expression and distribution of these receptors 1, 4 and 10 days later. Thymocyte death was assessed by the activation of caspase-3 in cells undergoing apoptosis. We observed massive thymocyte apoptosis 1 day after injection and, to a lesser extent, after 4 days, which was parallel with a reduction in the density of thymic epithelial cells normally expressing TrkA and p75NTR. Furthermore, TrkA expression was found in cortical thymic epithelial cells, which normally lack this receptor. The expression of TrkB was restricted to a subset of macrophage-dendritic cells, and remained unchanged with treatment. The normal pattern of neurotrophin receptor expression was almost completely restored by day 10. The results demonstrate that the expression of neurotrophin receptors by thymic epithelial cells, but not by macrophage-dendritic cells, is regulated by thymocytes.     

Nerve growth factor and glial cell line-derived neurotrophic factor restore the cholinergic neuronal phenotype in organotypic brain slices of the basal nucleus of Meynert

Loss of cholinergic neurons is found in the medial septum and nucleus basalis of Meynert in Alzheimer's disease. Recent observations suggest that cholinergic neurons down-regulate their phenotype and that growth factors may rescue cholinergic neurons. The aim of this study was to investigate whether cholinergic neurons of the basal nucleus of Meynert can be cultured in rat organotypic slices, and if nerve growth factor and glial cell line-derived neurotrophic factor can rescue the cholinergic phenotype. In the organotypic slices, glial cells, GABAergic and cholinergic neurons were visualized using immunohistochemistry. The number of cholinergic neurons was found to be very low in slices cultured without exogenous nerve growth factor. Analysis of nerve growth factor tissue levels by enzyme-linked immunosorbent assay revealed very low endogenous tissue levels. When slices were incubated with 100ng/ml nerve growth factor during the initial phase of culturing, a stable expression of choline acetyltransferase was found for up to several weeks. After eight weeks in culture with nerve growth factor or two to three weeks after nerve growth factor withdrawal, numbers of detected cholinergic neurons decreased. Neurons incubated with nerve growth factor displayed a significantly enlarged cell soma compared to neurons without growth factors. In cultures incubated for up to nine weeks, it was also found that glial cell line-derived neurotrophic factor was capable of restoring the cholinergic phenotype. The low-affinity p75 and high-affinity trkA receptors, as well as the glial cell line-derived neurotrophic factor receptor GFRalpha-1, could be visualized in slices using immunohistochemistry. In conclusion, it is shown that, in the axotomized organotypic slice model, the number of cholinergic neurons is decreased, but can be partly restored by nerve growth factor and glial cell line-derived neurotrophic factor.