Altered NGF protein levels in different brain areas after immunolesion

Nerve growth factor (NGF) provides critical trophic support to the cholinergic basal forebrain neurons that express high levels of the low-affinity NGF receptor (p75^NGFR) in the adult rat brain. Intraventricular injection of 192 IgG-saporin, made by coupling the monoclonal antibody to p75^NGFR 192 IgG to the cytotoxin saporin, selectively destroys the p75^NGFR-bearing neurons in the basal forebrain and was used here to examine the effects of selective cholinergic lesions on brain NGF protein levels. We showed that 192 IgG-saporin produced significant long-lasting elevation of NGF protein levels in the hippocampus, cortex, and olfactory bulb, with profound reductions of ChAT activities representing complete cholinergic deafferentations of these areas. NGF level was maintained in the basal forebrain, even though there was almost complete loss of p75^NGFR-immunoreactive cells and significant decrease of ChAT activity. In addition, a mild glial response was observed in the basal forebrain, and most of the activated astroglia expressed NGF-like immunoreactivity there. The increases in NGF protein levels in the target areas of the basal forebrain were most likely due to loss of cholinergic basal forebrain neurons and retrograde transport of NGF from these areas. Glial-derived NGF is partially responsible for the maintained level of NGF in the basal forebrain after the loss of cholinergic neurons. The accumulation of NGF protein in the target areas may have some effects on synaptic rearrangement in denervated tissues. © 1996 Wiley-Liss, Inc.     

Specificity of 192 IgG-saporin for NGF receptor-positive cholinergic basal forebrain neurons in the rat

A monoclonal antibody to the rat nerve growth factor (NGF) receptor, 192 IgG, accumulates bilaterally and specifically in cholinergic basal forebrain (CBF) cells following intraventricular injection. An immunotoxin composed of 192 IgG linked to saporin (192 IgG-saporin) has been shown to destroy cholinergic neurons in the basal forebrain. We sought to determine if intraventricular 192 IgG-saporin affected choline acetyltransferase (ChAT) enzyme activity in the CBF terminal projection fields. ChAT assays from 192 IgG-saporin-treated animals showed significant time-dependent decreases in ChAT activity in the neocortex, olfactory bulb and hippocampus, compared to PBS- or OKT1-saporin-injected controls. ChAT and tyrosine hydroxylase activity in the striatum was always unchanged by 192 IgG-saporin. ChAT immunohistochemistry was confirmative of major cell loss in the CBF, while other cholinergic nuclei appeared unremarkable. The data provide further evidence of the selectivity of 192 IgG-saporin in abolishing cholinergic, NGF receptor-positive CNS neurons.     

Nerve growth factor levels and choline acetyltransferase activity in the brain of aged rats with spatial memory impairments

Nerve growth factor (NGF) and choline acetyltransferase (ChAT) activity levels were measured in 7 different brain regions in young (3-month-old) and aged (2-years-old) female Sprague-Dawley rats. Prior to analysis the spatial learning ability of the aged rats was assessed in the Morris' water maze test. In the aged rats a significant, 15–30%, increase in NGF levels was observed in 4 regions (septum, cortex, olfactory bulb and cerebellum), whereas the levels in hippocampus, striatum and the brainstem were similar to those of the young rats. The NGF changes did not correlate with the behavioral performance within the aged group. Minor 15–30%, changes in ChAT activity were observed in striatum, brainstem and cerebellum, but these changes did not correlate with the changes in NGF levels in any region. The results indicate that brain NGF levels are maintained at a normal or supranormal levels in rats with severe learning and memory impairments. Ther results, therefore, do not support the view that the marked atrophy and cell loss in the forebrain cholinergic system that is known to occur in the behaviorally impaired aged rats is caused by a reduced availability of NGF in the cholinergic target areas. The results also indicate that the slightly increased levels of NGF are not sufficient to prevent the age-dependent atrophy of cholinergic neurons, although they might be important for the stimulation of compensatory functional changes in a situation where the system is undergoing progressive degeneration.     

Chronic alcohol intoxication in rats leads to a strong but transient increase in NGF levels in distinct brain regions

Summary. Nerve growth factor (NGF), a member of the neurotrophin family, is an essential mediator of neuronal activity and synaptic plasticity of basal forebrain cholinergic neurons. In this study NGF-protein levels were determined in areas of the basal forebrain cholinergic system, its projection areas as well as the striatum and the cerebellum after long-term exposure (6 and 9 months) to ethanol and a phase of withdrawal in male Sprague-Dawley rats. 6-month alcohol treatment led to an increase of NGF to 650–850% of controls in the basal forebrain and the septum and to a 210–485% increase in the cholinergic projection areas (anterior cortex, hippocampus and olfactory bulb). After 9 months exposure to ethanol, a decrease of NGF by 16% in the frontal cortex was observed compared to controls. In the other brain regions no differences in NGF expression were detectable at this time-point. These results support the idea of an endogenous neuroprotective mechanism acting through a transient NGF induction followed by a decrease in NGF-levels during the course of further neuronal degeneration. Authors contributed equally to this study     

Nerve growth factor and choline acetyltransferase activity levels in the rat brain following experimental impairment of cerebral glucose and energy metabolism.

Intracerebroventricular (ICV) injection of streptozotocin (STZ) has been reported to impair cerebral glucose utilization and energy metabolism (Nitsch and Hoyer: Neurosci Lett, 128:199-202, 1991) and also to prejudice passive avoidance learning in adult rats (Mayer et al.: Brain Res 532:95-100, 1990). It is well established that the forebrain cholinergic system, whose integrity is essential for learning and memory functions, depends on the target-derived retrograde messenger nerve growth factor (NGF). Therefore, we measured NGF and choline acetyltransferase (ChAT) activity levels in the forebrain cholinergic system in adult rats that had received a single injection of either STZ or artificial cerebrospinal fluid into the left ventricle 1 or 3 weeks prior to sacrifice. One week after ICV STZ treatment, NGF content was significantly decreased (-32%) in the septal region, where NGF-responsive cell bodies are located and NGF exerts its neurotrophic action after retrograde transport from NGF-producing targets. In contrast, NGF levels in the cortex and hippocampus, which are target regions for the basal forebrain cholinergic neurons, and in the brainstem and cerebellum were increased (+12% to +47%) within 3 weeks after ICV STZ treatment. The alterations in NGF levels were not related to changes in ChAT activity that decreased in the hippocampus by only 15%. This might be due to masking effects exerted by compensatory NGF-mediated stimulation of ChAT activity in remaining functional neurons. It is suggested that impaired behavior which has been observed after STZ-induced impairment of cerebral glucose and energy metabolism may be at least partially related to a diminished capacity of central NGF-responsive neurons to bind and/or transport NGF.(ABSTRACT TRUNCATED AT 250 WORDS)     

Time course of the elevation of nerve growth factor (NGF) content in the hippocampus and septum following lesions of the septohippocampal pathway in rats

Axotomy of cholinergic neurons in the medial septum-diagonal band and degeneration of their terminals in the hippocampus resulting from fornix-fimbria lesions induce elevation of NGF content in these two brain regions. Postlesion levels of cholinergic neuron-specific ChAT activity in the septum suggest that endogenously produced NGF may, at least partly, promote survival of axotomized cholinergic neurons or induce ChAT activity in undamaged cells or both. These findings thus support the proposed trophic role for NGF in central cholinergic neurons.     

Thyroid hormone regulation of NGF, NT-3 and BDNF RNA in the adult rat brain.

The effects of peripherally administered thyroid hormone (TH; 500 micrograms/kg; i.p.; q.d.) on the relative abundances of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) RNA were determined by rtPCR in the cortex and hippocampus of young adult rats. Corresponding changes in choline acetyltransferase (ChAT) activity were measured since NGF and BDNF have been shown to enhance the expression of this marker enzyme of central cholinergic pathways. Abundance levels of NGF and NT-3, relative to cyclophilin (cycl), were increased significantly (+50%, P < 0.05) in the hippocampus following TH treatment. Despite enhanced abundance of NGF in the hippocampus, ChAT activity was unchanged, whereas ChAT activity was modestly increased by 28% in the cortex without corresponding changes in NGF, NT-3 or BDNF. These results demonstrate that TH administration is capable of inducing the accumulation of NT-3, in addition to NGF but that the induction levels of RNA cannot be directly correlated with responsivity of the cholinergic system as measured by ChAT activity.     

Protective Role of Nerve Growth Factor against Excitatory Amino Acid Injury during Neostriatal Cholinergic Neurons Postnatal Development

The interaction between excitatory amino acids (EAAs) and nerve growth factor (NGF) levels were studied on neostriatal cholinergic neurons during postnatal development. Striatal choline acetyltransferase (ChAT) activity and NGF levels were determined 7 days following EAA injection in 7-, 15-, 21-, 30-, and 50-day-old rats. ChAT activity was decreased 7 days after kainate (KA), quinolinate (QUIN), or quisqualate (QUIS) lesion. The reduction was most pronounced in 30-day-old rats. KA injection produced the greatest decrease in ChAT activity. Conversely, KA did not change NGF levels. QUIN and QUIS increased NGF protein and these effects were maximal with lesions in 21-day-old rats. In order to further characterize the effect of EAAs on NGF levels and ChAT activity, the time-course of the lesion was studied. We used 30-day-old rats as the maximal sensitivity of cholinergic neurons to EAAs was observed at this age. ChAT activity decreased 2 days following QUIN or QUIS injection and 1 day after KA. The EAA agonists also changed NGF levels. QUIN induced an increase in NGF levels 1 day after lesion. This effect was maintained to the last time point examined. In contrast, KA and QUIS induced transient increases in NGF levels that were only detected 2 and 4 days after injection, respectively. To study whether NGF is able to regulate EAA excitotoxicity on striatal cholinergic neurons, we studied ChAT activity 7 days after simultaneous injection of NGF plus QUIN, KA, or QUIS. Intrastriatal injection of exogenous NGF was able to block the decrease in ChAT activity observed following EAA injection alone. In conclusion, our results show that striatal cholinergic neurons have different vulnerabilities to excitotoxicity induced by EAAs during development. ChAT activity was decreased and NGF was increased by EAAs. However, those EAAs (QUIN and QUIS) that increased NGF had less effect on ChAT activity than KA which had little effect on NGF levels, suggesting that an increase in endogenous NGF levels by these agents may decrease their toxicity. This was confirmed by our finding that exogenous NGF protects cholinergic neurons against excitotoxic lesion. The combined results suggest that sensitivity to EAAs and the regulation of NGF may be crucial to the development of striatal cholinergic neurons.     

Nerve growth factor response to excitotoxic lesion of the cholinergic basal forebrain is slightly impaired in aged rats

Nerve growth factor (NGF) promotes survival and function of basal forebrain cholinergic neurons. We studied NGF and choline acetyltransferase (ChAT) activity after partial quisqualic acid induced lesions of the basal forebrain in 3 and 27 months-old rats, in order to investigate whether NGF-related regeneration is disturbed in old age. 2 weeks post lesion, ChAT activity decreased by 25 to 32% in adult and old rats. 3 months post lesion, the ChAT deficit receded in adult rats, but remained unchanged in old rats. 2 weeks post lesion, NGF levels were reduced by 36 to 44%, but there was no significant difference between adult and old rats. 3 months post lesion, we found increased NGF levels by 44% in the posterior cortex of adult rats. These results indicate that the compensatory NGF increase in the posterior cortex after partial cholinergic lesion of the basal forebrain is slightly impaired in old age.     

Chronic treatment with scopolamine and physostigmine changes nerve growth factor (NGF) receptor density and NGF content in rat brain

Nerve growth factor (NGF) and NGF receptors were measured in cortex and hippocampus of rats treated with drugs affecting cholinergic neurotransmission. High (K_d= 0.045nM) and low (K_d= 21nM) affinity¹²⁵I-NGF binding sites were present in both cortical and hippocampal membranes with hippocampus containing higher numbers of both sites than cortex. Chronic treatment of rats with the muscarinic receptor antagonist scopolamine (5 mg/kg, twice daily) decreased the density of high- and low-affinity sites by 50–90% in cortical and hippocampal membranes. These changes were seen after 7 days, but not 3 days, of scopolamine treatment. Chronic infusion of physostigmine (1 mg/kg/day) using minipumps increased the number of high- and low-affinity sites in cortex 3- and 6-fold, respectively. The changes in receptor-binding parameters induced by physostigmine were transient as they were evident after 3 days of treatment, but returned to control levels after 7 days. NGF content in cortex and hippocampus was reduced by about 50% following 7, but not 3, days of chronic physostigmine infusion. In contrast, scopolamine treatment failed to change NGF levels in the cholinergic neuronal target regions but it decreased NGF content in the septal area. The content of NGF mRNA in the cortex measured by Northern blot analysis failed to change following either scopolamine or physostigmine treatment. The results suggest that levels of NGF and NGF receptors in the target regions of cholinergic neurons are regulated by the extent of cholinergic neurotransmitter activity.     

Axotomy-dependent stimulation of choline acetyltransferase activity by exogenous mouse nerve growth factor in adult rat basal forebrain

Transection of the adult rat dorsal fornix and fimbria (F-F) induced a sensitivity of the cholinergic neurons in the medial septum and diagonal band (MS/DB) to exogenous mouse nerve growth factor (mNGF). Continuous infusion of mNGF for two weeks after complete unilateral F-F aspiration resulted in a stimulation of choline acetyltransferase (ChAT)-specific activity in precise micro-dissections of the MS/DB ipsilateral to the transection to a level that was 200% higher than that measured in normal adult animals. This supranormal stimulation of ChAT activity reached plateau levels after 10 days of NGF infusion and was dose-dependent with an E.D.50 equal to 120 ng/day. Administration of mNGF had no effect on the ChAT activity in the MS/DB of normal animals or animals with a unilateral transection of only the supracallosal dorsal septo-hippocampal pathway. Partial transection experiments indicated that a predominent pathway for cholinergic neurons potentially sensitive to exogenous mNGF runs in the paramedian F-F. Administration of mNGF also induced a stimulation of ChAT activity in dissections of the caudate-putamen both ipsi- and contralateral to the infusion cannula. This indicates that unlike the cholinergic projection neurons of the MS/DB, adult cholinergic striatal interneurons are sensitive to exogenous NGF without prior axotomy.     

Differential expression of mRNAs for the NGF family of neurotrophic factors in the adult rat central olfactory system.

The cellular localization of mRNAs for nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT3), in the rat central olfactory system was evaluated with in situ hybridization of 35S-labeled cRNA probes. In the main olfactory bulb, low levels of NGF and BDNF mRNA expression were detected. NGF mRNA was restricted to the glomerular region while BDNF mRNA was predominantly localized to the granule cell layer. No cellular hybridization to NT3 cRNA was seen. The accessory olfactory bulb did not express detectable levels of mRNA for any of the three related neurotrophic factors. Areas which receive olfactory bulb afferents expressed comparatively high levels of both NGF and BDNF mRNA. Cell labeling with cRNAs for NGF and BDNF occurred throughout the cellular layers of the anterior olfactory nucleus and in layers 2 and 3 of rostral piriform cortex. BDNF mRNA expression in these areas appeared more robust than that of NGF mRNA, while NT3 mRNA was not detectable. In contrast, tenia tecta exhibited dense labeling with the cRNAs for all three neurotrophic factors. The localization of NGF mRNA to primary target neurons of the olfactory nerve in the periglomerular region of the main olfactory bulb suggests that bulb cells may influence the ingrowth and continual turnover of olfactory sensory afferents. However, as there is a strong correlation between the distribution of neurotrophic factor mRNAs within rostral olfactory structures and the distribution of centrifugal cholinergic afferents, it is more likely that bulb-derived NGF, and possibly BDNF, act on the cholinergic neurons of the basal forebrain.     

Effect of nerve growth factor on the lesioned septohippocampal cholinergic system of aged rats

Nerve growth factor (NGF) was injected intraventricularly into aged (24 months) rats with unilateral lesions of the lateral fimbria. The activity of choline acetyltransferase (ChAT) was determined in the septum and hippocampus from the normal unlesioned rats, lesioned and cytochrome c-treated rats (controls), and lesioned and NGF-treated rats at different times after the lesion. NGF-injection for 15 days after the lesion resulted in an increase of the ChAT activity in both the contralateral hippocampus and the entire septum, to about 130% of that in the normal animals, but resulted in a slight increase in the ipsilateral lesioned hippocampus, when compared to the activity in the ipsilateral side of the cytochrome c-treated controls. NGF-injection for 30 days after the lesion resulted in a 48% increase of the ChAT activity in the ipsilateral hippocampus as compared to cytochrome c-treated controls, but failed to result in a significant increase in the contralateral hippocampus. These findings indicate that atrophic cholinergic neurons in aged animals are similarly responsive to NGF treatment, like these in the young animals. Moreover, these findings suggest that the responses of basal forebrain cholinergic neurons to NGF treatment varies with time after the lesion and imply that the NGF administration can promote the collateral sprouting from spared cholinergic fibers after the lesion in the aged forebrain.     

Development of septal cholinergic neurons in culture: plating density and glial cells modulate effects of NGF on survival, fiber growth, and expression of transmitter-specific enzymes

To characterize the role of NGF in the development of forebrain cholinergic neurons, we established primary cell culture systems to grow these cells under controlled in vitro conditions. Cultures of dissociated cells were prepared from the septal area of fetal (E17) rats, which contained part of the group of basal forebrain cholinergic neurons. Cultures were treated either with NGF (100 ng/ml) or with an antiserum against NGF (1:500 dilution). To assess the influence of non-neuronal cells, 2 types of high-density cultures were prepared: mixed neuronal-glial cultures and pure neuronal cultures. Cholinergic neurons were identified using choline acetyltransferase (ChAT) immunocytochemistry and AChE cytochemistry. Receptors for NGF (NGF-R) were located immunocytochemically using monoclonal antibodies against rat NGF-R. We report that, first, NGF-R are exclusively localized on cholinergic neurons in septal cultures. All neurons labeled with antibodies against NGF-R also contained AChE. Twenty-one percent of all AChE-positive neurons were not stained in NGF-R immunocytochemistry (AChE has earlier been shown to be colocalized with ChAT in septal cultures). Second, NGF treatment increases and anti-NGF treatment reduces the number of AChE-positive neurons in cultures of low plating density, suggesting that NGF promotes survival of septal cholinergic neurons in these cultures. In cultures of high plating density, NGF increased the number of NGF-R and ChAT-positive neurons without affecting the number of AChE-positive neurons in these cultures. These results suggest that exogenous NGF is not required for survival of cholinergic neurons in high-density cultures but stimulates the expression of ChAT and NGF-R. Third, NGF stimulates fiber growth of septal cholinergic neurons, as assessed by computerized image analysis of AChE-positive neurons. Fourth, NGF specifically increases ChAT and AChE activities in septal cultures. These NGF-mediated increases in enzyme activities are more pronounced when neurons are grown together with glial cells. In pure neuronal cultures, NGF increased ChAT and AChE activities by 101 and 16%, and in mixed neuronal-glial cultures by 318 and 87%, respectively. Anti-NGF blocked the effects of NGF but failed to reduce ChAT and AChE activities below control levels in cultures of high plating density. Fifth, astrocytes attenuate the expression of ChAT and AChE by septal neurons in the absence of NGF.(ABSTRACT TRUNCATED AT 400 WORDS)     

Nerve growth factor increases choline acetyltransferase activity in developing basal forebrain neurons

Nerve growth factor (NGF) is a neuronotrophic protein. Its effects on developing peripheral sensory and sympathetic neurons have been extensively characterized, but it is not clear whether NGF plays a role during the development of central nervous system neurons. To address this point, we examined the effect of NGF on the activity of neurotransmitter enzymes in several brain regions. Intracerebroventricular injections of highly purified mouse NGF had a marked effect on the activity of choline acetyltransferase (ChAT), a selective marker of cholinergic neurons. NGF elicited prominent increases in ChAT activity in the basal forebrain of neonatal rats, including the septum and a region which contains neurons of the nucleus basalis and substantia innominata. NGF also increased ChAT activity in the hippocampus and neocortex, terminal regions for the fibers of basal forebrain cholinergic neurons. In analogy with the response of developing peripheral neurons, the NGF effect was shown to be selective for basal forebrain cholinergic cells and to be dose-dependent. Furthermore, septal neurons closely resembled sympathetic neurons in the time course of their response to NGF. These observations suggest that endogenous NGF does play a role in the development of basal forebrain cholinergic neurons.     

Comparison of nerve growth factor's effects on development of septum, striatum, and nucleus basalis cholinergic neurons in vitro

In the central nervous system, nerve growth factor (NGF) affects basal forebrain cholinergic neurons during early development and in the adult mammalian brain. These neurons are located in medial septum, diagonal band of Broca, and nucleus basalis of Meynert. While the effects of NGF on the development of septal cholinergic neurons are well documented, only little is known about the influence of NGF on development of cholinergic neurons in the nucleus basalis. In addition to the basal forebrain cholinergic neurons, there are cholinergic interneurons in the corpus striatum, which form an anatomically and functionally distinct population of cholinergic neurons. These striatal interneurons have been reported to respond to NGF during early development; however, it is not known whether the effects of NGF on their development are similar to those on septal cholinergic neurons. We prepared cultures of dissociated cells from fetal rat septum, striatum, and nucleus basalis and investigated the development of cholinergic neurons localized in these three different areas in the presence or absence of NGF. We now report that, first, cholinergic neurons of striatum and nucleus basalis develop a more extensive fiber network and contain more acetylcholinesterase (AChE) per neuron than do cholinergic neurons of septum. The amount of choline acetyltransferase (ChAT) per cholinergic neuron is approximately the same in all three culture types when grown in the absence of NGF. Second, NGF treatment increases and anti-NGF treatment decreases the number of AChE-positive neurons in cultures of low plating density, suggesting that NGF is able to promote survival of cholinergic neurons of all three areas studied. Third, NGF increases the total length of fibers and the number of branching points of cholinergic neurons in septal cultures but not in cultures of striatum and nucleus basalis. Fourth, NGF treatment increases AChE activity in septal but not in nucleus basalis or striatal cultures, suggesting that AChE activity reflects the extent of the fiber network of cholinergic neurons of all areas. Fifth, NGF treatment produces severalfold elevations in ChAT activity in septal cultures and more modest increases in cultures of nucleus basalis and striatum, suggesting that NGF is able to stimulate ChAT activity also in the absence of a stimulatory effect on survival and fiber growth. Our results demonstrate that, during early development, NGF is able to affect survival and differentiation of all three populations of forebrain cholinergic neurons.(ABSTRACT TRUNCATED AT 400 WORDS)     

Nerve Growth Factor and Basic Fibroblast Growth Factor Protect Cholinergic Neurons Against Quinolinic Acid Excitotoxicity in Rat Neostriatum

In the present work we have characterized a possible mechanism leading to the early survival of neostriatal cholinergic neurons after quinolinic acid injection. Different doses of quinolinic acid were injected in rat neostriatum and two different parameters were analysed 7 days after the lesion: choline acetyltransferase (ChAT) activity and nerve growth factor (NGF) levels. We have observed that ChAT activity decreased (until 68 nmol quinolinic acid) and NGF levels increased (until 34 nmol quinolinic acid) in a dose-dependent manner. In order to characterize the time-course of the lesion on NGF levels and ChAT activity, and the possible protective effect of NGF and basic fibroblast growth factor (bFGF) on cholinergic neurons, we have used the quinolinic acid dose (68 nmol) at which the first decrease of ChAT activity was observed. ChAT activity and NGF levels showed different patterns of response to quinolinic acid injection, since the maximal effect was reached at 1 day for ChAT activity and at 2 days for NGF levels. NGF or bFGF simultaneously injected with quinolinic acid (68 nmol) completely prevented the decrease in ChAT activity in a dose-dependent manner but NGF was more effective than bFGF. Furthermore, differences observed in ChAT activity after NGF but not bFGF treatment were correlated with changes in the number of ChAT immunoreactive cells. Finally, we have also observed that, although bFGF alone was not able to modify NGF levels, bFGF simultaneously injected with quinolinic acid produced an increase of NGF levels higher than that observed after quinolinic acid injection alone. Our results show that NGF and bFGF protect striatal cholinergic neurons against quinolinic acid injury, and bFGF is able to potentiate the increase of NGF after the lesion, suggesting a cooperative action between different trophic factors in neuronal protection after excitotoxic injury. Thus, administration of trophic factors may be relevant in the prevention and treatment of neurodegenerative disorders, such as Huntington's disease.     

Characterization of a neurotrophic factor produced by cultured astrocytes involved in the regulation of subcortical cholinergic neurons.

When dissociated subcortical cells were cultured in the presence of conditioned medium of relatively differentiated astrocytes (ACM), a marked increase was observed in the expression of choline acetyltransferase (ChAT), an enzyme required for the synthesis of the neurotransmitter acetylcholine. Astrocytes from the target regions of subcortical neurons, the hippocampus and the cerebral cortex, produced neurotrophic factor consistently more than those derived from the nontarget region, the cerebellum. The production of cholinergic trophic activity was increased with the maturation of astrocytes. Even though, nerve growth factor (NGF) and ciliary neurotrophic factor (CNTF) are known cholinergic trophic compounds produced by astrocytes in vitro, a large part of the neurotrophic activity in our ACM was not related to either of these 2 factors. This is because (i) ACM and NGF produced an additive effect on ChAT activity, (ii) only a small proportion of the cholinergic trophic activity in ACM was abolished by anti-NGF antibody, and (iii) treatment with CNTF had no effect on ChAT activity of basal forebrain cholinergic neurons. On the other hand, when cholinergic neurons are cultured on a preformed layer of astrocytes, addition of basal fibroblast growth factor (bFGF) failed to increase further the ChAT activity. Similarly the effects of ACM and bFGF were not additive. A large proportion of the cholinergic trophic activity in ACM was neutralized by anti-bFGF antibody. These findings would suggest that the trophic activity on septal cholinergic neurons in our ACM was due to bFGF or a bFGF-like compound.