Development of cholinergic pedunculopontine neurons in vitro: comparison with cholinergic septal cells and response to nerve growth factor, ciliary neuronotrophic factor, and retinoic acid

The well-documented role of nerve growth factor (NGF) in the function of cholinergic neurons in the mammalian basal forebrain can be regarded as a paradigm for the action of trophic substances on CNS neurons. Although several growth factors have been identified in recent years, the specificities and importance of such factors for the development of the nervous system are still unknown. In the present study it has been tested whether NGF affects the group of pedunculopontine cholinergic neurons. This population, which has been described in detail only recently, is located more caudally than but resembles, in some aspects, the basal forebrain cholinergic neurons. The cell bodies are located in the metencephalic pedunculopontine and dorsolateral tegmental nuclei. Similar to the forebrain cholinergic neurons, they are medium to large in size and ascend centrally with long axons. Projection areas are widespread throughout the mesencephalon and diencephalon. Dissociated pontine and septal cells of fetal rat brain (embryo ages E14 to E17) were grown in culture for 7 to 14 days in the presence or absence of NGF. Furthermore, a possible action of retinoic acid and ciliary neuronotrophic factor (CNTF) on cholinergic neurons of both the basal forebrain and the pontine area were tested. Differentiation of cultured cholinergic neurons was assessed by biochemical determination of choline acetyltransferase (ChAT) activity and by immunocytochemical staining for ChAT. NGF in concentrations of 1 to 1,000 ng/ml medium increased the number of immunostained cells and the staining intensity in ChAT immunocytochemistry and enhanced ChAT activity by at least 100% above control levels in septal cultures, thus confirming earlier results. In marked contrast, the same concentrations of NGF failed to influence ChAT activity or immunocytochemical staining in cultures of the pontine area. Retinoic acid (10(-8) M to 10(-5) M) and CNTF (0.2 and 2.0 ng/ml, corresponding to 1 and 10 trophic units, as defined in the ciliary ganglion cell assay) failed to enhance ChAT activity in either culture system and did not potentiate the NGF-mediated increase of ChAT activity in septal cultures. Our results, which indicate that pedunculopontine cholinergic neurons do not respond to NGF during development, are in line with those of NGF-receptor visualization studies that failed to demonstrate such receptors on cholinergic pontine cells in postnatal and adult rats. The findings further underline the specificity of NGF action in the central nervous system and, in particular, do not support the idea of transmitter-specific neurotrophic factors.(ABSTRACT TRUNCATED AT 400 WORDS)     

Synergistic effects of brain-derived neurotrophic factor and ciliary neurotrophic factor on cultured basal forebrain cholinergic neurons from postnatal 2-week-old rats.

Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, and ciliary neurotrophic factor (CNTF), a member of the neurocytokine family, are known to have synergistic effects on motoneurons, but such synergistic effect has not been studied in detail especially in the brain. In the present study, we examined the synergistic effects of BDNF and CNTF on the survival of basal forebrain cholinergic neurons cultured from postnatal 2-week-old (P2w) rats. Although BDNF is well-known to promote the survival of basal forebrain cholinergic neurons in P2w culture, CNTF had little effect on the survival of choline acetyltransferase (ChAT)-positive neurons and did not increase ChAT activity in the culture. However, CNTF enhanced BDNF-mediated promotion of cell survival of cholinergic neurons when added concomitantly. BDNF alone induced only a three-fold increase in ChAT activity in control cultures, but the concomitant addition of CNTF resulted in an eight-fold increase. CNTF did not enhance BDNF-mediated cell survival of total neurons from the basal forebrain, hippocampus or cerebellum, suggesting that the synergistic effects of CNTF on the BDNF-mediated increase of viability might be strong in basal forebrain cholinergic neurons. CNTF also enhanced the neurotrophin-4/5-mediated increase of ChAT activity, but not the nerve growth factor (NGF)-mediated one. Furthermore, the BDNF-mediated increase was also enhanced by leukemia inhibitory factor but not by interleukin-6. Similar synergistic pattern between neurotrophins and cytokines were also observed in the induction of ChAT activity in embryonic basal forebrain culture. These results suggest that TrkB, a functional high-affinity receptor of BDNF and NT-4/5, and LIFR beta, a receptor component contained in CNTF and LIF receptor complex, might be involved in the observed synergistic effects.     

Transplantation of fetal and early postnatal rat septal cholinergic neurons cultured in serum-free and serum-containing medium with nerve growth factor

Fetal rat (E17-E19) septal neurons were cultured in a defined, serum-free medium for 6-8 days with or without nerve growth factor (NGF) and transplanted into the hippocampus or the surrounding ventricle of 28 adult rats denervated of its septal input by a fimbria-fornix transection. The cholinergic septal neurons, which were visualized by acetylcholinesterase (AChE) histochemistry, always survived in transplantation to the adult brains from nearly pure neuronal cultures. Although choline acetyltransferase (ChAT) activity of septal neurons in culture was greatly increased (5.59-fold) by the addition of NGF to the defined medium, this ChAT induction appeared to have little effect on the subsequent survival or growth of the septal neurons after transplantation. These results demonstrate that survival of cultured fetal septal cholinergic neurons following transplantation is not dependent upon the presence of NGF or serum- or glia-derived factors during the preliminary culture. Postnatal rat (P4) septal neurons cultured for 5 days in serum-containing medium with NGF were also successfully transplanted in one of 3 cases.     

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)     

Effects of nerve growth factor and basic fibroblast growth factor on survival of cultured septal cholinergic neurons from adult rats

We have established a primary culture technique for neuronal cells from rat basal forebrain from postnatal day 58 (P58) to study the effects of neurotrophic factors on the neurons. The survival of acetylcholinesterase (AChE)-positive neurons of 2-week-old rat septum has already been reported to be strongly supported by nerve growth factor (NGF) in culture. In this culture study of neurons from adult rat brains, the survival of AChE-positive neurons from P58 rat septum was slightly improved by NGF, although low affinity NGF receptor expression was also observed on cultured P58 rat septum neurons as well as on those from 2-week-old rats. The addition of basic fibroblast growth factor (bFGF) improved the survival of AChE-positive neurons cultured from P58 rat septum, but did not promote the survival of neurons from P12 rat septum. These results suggest that NGF changes to a maintenance factor in adult rat brain from a survival factor in postnatal 2-week-old rats. The survival of cholinergic neurons in culture of adult rat septum might be supported by factor(s) other than NGF, such as bFGF.     

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.     

High oxygen atmosphere for neuronal cell culture with nerve growth factor. I. Primary culture of basal forebrain cholinergic neurons from fetal and postnatal rats

Cholinergic neurons cultured from postnatal days 11-13 (P11-P13) rat basal forebrain showed better survival in the culture condition using a 50% O2 atmosphere with and without nerve growth factor (NGF) than in a low (10 or 20%) O2 atmosphere. Except for the culture at a low cell density, the beneficial effect of the highly oxidized culture condition was found in the culture from P3 neurons, but not from embryonic day 18 neurons. The survival of microtubule-associated protein 2 (MAP2)-positive neurons in culture from P3 basal forebrain regions was more enhanced in a 50% O2 atmosphere than in 20% and also 10% O2 atmosphere. The viable number of the MAP2-positive neurons in a 10% O2 condition was about half of that in a 20% condition. These results suggest that the response of the cultured neurons to an incubator O2 concentration changes during the neuronal development in CNS from fetal to postnatal stages.     

Reversibility of Nerve Growth Factor's Enhancement of Choline Acetyltransferase Activity in Cultured Embryonic Rat Septum

Choline acetyltransferase (ChAT) activity and survival of acetylcholinesterase (AChE)-positive neurons were measured in low-density cultures of embryonic (Day 14-15) rat septum exposed to various sequences of nerve growth factor (NGF) exposure and deprivation for up to 7 weeks in vitro. Most septal cultures grown 4-5 weeks with no exogenous NGF (including exposure to monoclonal or polyclonal antibodies against NGF) retained both a basal ChAT activity and the ability to increase ChAT activity in response to subsequently added NGF. When cultures were exposed to NGF (7S, 0.75 nM) for 2-3 weeks and then deprived of NGF for 2 weeks, ChAT activity fell gradually, but the number of AChE-positive neurons remained unchanged, and in many cases ChAT activity could be restored by subsequent re-exposure to NGF. Thus NGF's enhancement of ChAT activity in embryonic septal neurons in vitro is largely reversible and is not mediated by differential survival of cholinergic neurons.     

Developmental stage-dependent protective effect of NGF against lead cholinotoxicity in the rat septum

The ability of nerve growth factor (NGF) to ameliorate developmental cholinotoxicity of inorganic lead (Pb) for the septal neurons was investigated by making intracerebroventricular injections of single doses of 30 microg 2.5S NGF into maternally lead-exposed suckling rats on postnatal days P2, P4, P11, or P18. Administration of NGF on P4 or later induced septal choline acetyltransferase (ChAT) activity to the same relative extent in both Pb-exposed as in control rats but failed to reverse the net reductions of ChAT activity induced by Pb. In contrast, injection of NGF at P2 completely restored ChAT activity in Pb-exposed pups to control levels by preventing the loss of ChAT-immunoreactive cells in the septum. It is concluded that although NGF retains the capacity to upregulate ChAT throughout the period of Pb exposure, it protects against the Pb-induced loss of septal cholinergic neurons only when applied within the critical period of Pb-vulnerability between postnatal days 2 and 4.     

Nerve growth factor receptor and choline acetyltransferase colocalization in neurons within the rat forebrain: response to fimbria-fornix transection

Although it is well known that magnocellular cholinergic basal forebrain neurons are trophically responsive to nerve growth factor (NGF) and contain NGF receptors (NGFr), the exact distribution of forebrain NGFr-immunoreactive neurons and the degree to which cholinergic neurons are colocalized with them have remained in question. In this study we employed a very sensitive double-labelling method and examined in the same tissue section the distribution and cellular features of NGFr-positive and choline acetyltransferase (ChAT)-immunolabelled neurons within the rat basal forebrain. Throughout this region the majority of magnocellular basal forebrain neurons were immunoreactive for both NGFr and ChAT. However, a small percentage of neurons in the ventral portion of the vertical limb of the diagonal band of Broca were immunoreactive only for NGFr, whereas a larger population of magnocellular neurons in the substantia innominata exhibited only ChAT immunoreactivity. No NGFr-immunoreactive cells were found associated with ChAT-positive neurons in the striatum, neocortex, or hippocampus, and no single-labelled NGFr-immunoreactive neurons were found outside the basal forebrain area, except for a large number of positive-labelled cells along the ventricular walls of the third ventricle. In addition to its function in maintaining the normal integrity of the basal forebrain and cholinergic, peripheral sympathetic, and neural-crest-derived sensory neurons, NGF may also have a role in the growth of these neurons after damage to the nervous system. To examine this postulate the hippocampus was denervated of its septal input and examined 8 weeks later. Two populations of neurons were found to have undergone collateral sprouting--namely, the midline magnocellular cholinergic neurons of the dorsal hippocampus and the sympathetic noradrenergic neurons of the superior cervical ganglion. Both of these neuronal populations also stained strongly for NGFr. In contrast, the small intrinsic cholinergic neurons of the hippocampus exhibited neither sprouting response nor staining for NGFr. In view of these results, we suggest that the differing sprouting responses demonstrated by these three neuronal populations may be due to their responsiveness to NGF, as indicated by the presence or absence of NGF receptors.     

Enhancement of choline acetyltransferase activity in coculture of rat septal and hippocampal neurons

We report that choline acetyltransferase (ChAT) activity and neuronal survival were enhanced in rat septal neurons cocultured with hippocampal neurons. The enhancement of ChAT activity also occurred as a result of the addition of hippocampal conditioned medium (HpCM). When septal neurons from embryonic day 17 (E17) rats were cocultured with hippocampal neurons, ChAT activity was increased 2-fold compared with homogeneous culture of septal neurons. By contrast, no increase in ChAT activity was observed in coculture of septal and neocortical neurons. Treatment with HpCM obtained from cultured E19 rat hippocampal neurons enhanced the ChAT activity of E17 rat septal neurons. The enhancement of ChAT activity caused by coculture with hippocampal neurons and that caused by the addition of HpCM were not blocked by the addition of anti-nerve growth factor (NGF) antibody, suggesting that NGF, which is known to increase the ChAT activity of septal neurons both in vivo and in vitro, did not participate in the increase of ChAT activity. These findings indicate that possible target-derived neurotrophic factor(s), other than NGF, from hippocampal neurons enhance(s) the ChAT activity of septal neurons.     

Cooperative effects of Sonic Hedgehog and NGF on basal forebrain cholinergic neurons

In ventromedial cells of the developing CNS, Sonic hedgehog (Shh) has been shown to affect precursor proliferation, phenotype determination, and survival. Here we show that Shh and its receptor, Ptc-1, are expressed in the adult rat basal forebrain, and that Ptc-1 is expressed specifically by cholinergic neurons. In basal forebrain cultures, Shh was added alone and in combination with nerve growth factor (NGF), and the number of cholinergic neurons was determined by choline acetyltransferase (ChAT) immunocytochemistry. By 8 days in vitro, Shh and NGF show a synergistic effect: the number of ChAT-positive cells after treatment with both factors is increased over untreated cultures or cultures treated with either factor alone. While Shh increases the overall basal level of proliferation, double-labeling of dividing neuronal precursors with [(3)H]thymidine followed by ChAT immunocytochemistry after they mature, demonstrates that the specific increase in cholinergic neurons is not due to this proliferation enhancement. These experiments imply a role for Shh in the development of postmitotic cholinergic neurons and suggest a therapeutic value for Shh in neurodegenerative disease.     

Antioxidants N-acetylcysteine (NAC) and 2-mercaptoethanol (2-ME) affect the survival and differentiative potential of cholinergic precursors from the embryonic septal nuclei and basal forebrain: involvement of ras signaling.

We investigated the effects of antioxidants N-acetylcysteine (NAC) and 2-mercaptoethanol (2-ME) on the expression of choline acetyltransferase (ChAT) in cultured cholinergic precursors from the embryonic rat septal nuclei and basal forebrain. Carboxy-dichlorofluorescein fluorescence confirmed that 2-ME inhibited intracellular oxidation. Low micromolar concentrations of 2-ME produce as much as a 12-fold increase in ChAT; this is enhanced further by inclusion of nerve growth factor (NGF). NAC effects are biphasic: 0.15 mM produces profound increases in ChAT while 1.5 mM has no effect. Immature (E16) cultures respond with increases in ChAT while more highly differentiated cultures (E18) do not. Labeling of single precursors with a lacZ-expressing retrovirus reveals that the increase in ChAT is due primarily to an increased number and size of clones, not an increase in cholinergic neurons per clone, suggesting an effect on precursor survival. Inhibition of ras farnesylation inhibits both 2-ME and NAC induction of ChAT suggesting a ras-mediated pathway. Inclusion of the MEK inhibitor PD98059 does not affect low doses of NAC, but at doses of NAC that fail to increase ChAT activity, inhibition of the pathway actually raises ChAT. Immunocytochemical investigation of the cultures indicates that cells exposed to low doses of NAC develop healthy neuronal arbors in the apparent absence of glial support. At higher concentrations of NAC, neurons were found in association with astrocytes, making contact via elaborate varicose fibers. Treatment of the cultures with PD98059 to inhibit MEK returned cultures to a 'low-dose' phenotype. These data suggest that redox status of basal forebrain precursors affect both their survival and differentiative potential.     

Survival-promoting effect of NGF on in vitro septohippocampal neurons with cholinergic and GABAergic phenotypes.

Recently, we demonstrated a survival-promoting effect of nerve growth factor (NGF) on cultured hippocampus-projecting neurons from developing septum/diagonal band region using fluorescent latex microspheres as a retrograde neuronal marker (Arimatsu et al., 1989). In the present study, we characterized these projection neurons by combining the retrograde cell labeling and histochemical stainings for acetylcholinesterase (AChE) activity and NGF receptor-, choline acetyltransferase- (ChAT-) and gamma-aminobutyric acid- (GABA-) immunoreactivities. The surviving microsphere-labeled neurons were, for the most part, immunoreactive for NGF receptor in the culture. A great majority (about 90%) of the microsphere-labeled neurons showed strong AChE activity and ChAT-immunoreactivity. The number of strongly AChE-positive neurons and that of ChAT-immunoreactive neurons in the culture supplemented with NGF was much greater with than without exogenous NGF. In addition, a major part (about 70%) of the microsphere-labeled neurons exhibited GABA-immunoreactivity in the presence of NGF. The number was also much greater than that without NGF. A considerable portion of cultured septal cholinergic neurons were shown to express GABA-immunoreactivity by a two-color immunofluorescence labeling experiment for ChAT and GABA. These findings are consistent with the assumption that NGF plays an important role in the development and organization of the cholinergic and GABAergic septohippocampal systems by supporting the neuronal survival, and raise a possibility that cholinergic and GABAergic fractions of the septohippocampal neurons may be developmentally correlated.     

Pituitary adenylate cyclase-activating polypeptide promotes the survival of basal forebrain cholinergic neurons in vitro and in vivo: comparison with effects of nerve growth factor

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a member of the vasointestinal polypeptide gene family for which neurotrophic activity has been postulated. PACAP mRNA is expressed in the developing and adult hippocampus, which is the principal target region of septal cholinergic neurons. We therefore studied the effects of PACAP on septal cholinergic neurons. In primary cultures from septum of embryonic and postnatal rats, PACAP increased the number of neurons immunohistochemically stained for the low-affinity nerve growth factor (NGF) receptor p75 and for the enzyme choline acetyltransferase (ChAT). PACAP also caused a corresponding increase in ChAT activity. In comparison, NGF had a greater effect than PACAP on the number of p75- and ChAT-positive neurons in these cultures. In vivo, following fimbria fornix transection, the number of immunohistochemically stained septal cholinergic neurons fell significantly to 18% in rats given continuous intracerebroventricular infusion of vehicle, whereas in rats given NGF the number of these neurons did not differ significantly from unoperated controls. In PACAP-treated rats the number was 48% of unoperated values, which represented a significant increase compared with vehicle-treated rats and a significant decrease compared with NGF-treated rats or unoperated controls. Double-staining experiments revealed that most ChAT-positive neurons in rat medial septum also express PACAP receptor 1. Together the results show that PACAP promotes the survival of septal cholinergic neurons in vitro, and after injury in vivo, suggesting that PACAP acts as a neurotrophic factor influencing the development and maintenance of these neurons.     

Trophic actions of recombinant human nerve growth factor on cultured rat embryonic CNS cells

NGF is a neurotrophic factor for basal forebrain cholinergic neurons and may serve to counteract the cholinergic deficits that are observed in Alzheimer's disease. Prior to the introduction of clinical trials, it is essential that recombinant human NGF (rhNGF) be produced and that its actions on target cells in the CNS be demonstrated. We prepared rhNGF and examined its actions on fetal rat brain neurons in culture including, in particular, the cholinergic neurons of the basal forebrain. rhNGF was more potent in increasing choline acetyltransferase (ChAT) activity in septal cultures than NGF purified from mouse salivary glands (mNGF). ED50s of the beta-NGF dimers were 4.9 pM for rhNGF and 12.4 pM for mNGF. The maximal ChAT activity response was achieved at approximately 35 pM with both NGFs and their efficacies were not significantly different. The two NGFs were not additive in effect. Identical to the results with mNGF, rhNGF strongly enhanced the intensity of ChAT immunostaining in septal cultures. Neither rhNGF nor mNGF affected the appearance of the cultures under phase-contrast illumination. Survival of cells at very low plating density on polyornithine/laminin-coated culture dishes was not affected by rhNGF or mNGF. Protein content and the uptake of GABA were also unaffected. At concentrations of up to 10 micrograms/ml, rhNGF did not significantly increase uptake of dopamine into cultures of ventral mesencephalon. We conclude that rhNGF produces potent and selective actions on cholinergic neurons of the basal forebrain as previously shown for mNGF.     

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.     

Nerve growth factor does not influence the expression of β amyloid precursor protein mRNA in rat brain: in vivo and in vitro studies

We investigated the effect of NGF on amyloid precursor protein (APP) mRNA levels in the rat septal/nucleus basalis system. Total APP mRNA and APP 695 mRNA were determined in basal forebrain primary cell cultures exposed acutely and chronically to NGF (150–300 ng/ml) and, in vivo, in the septal area and striatum of rat pups after multiple intracerebroventricular injections of NGF. The trophic factor was able to affect cholinergic neurons in both paradigms, as evidenced by the significant increase of choline acetyltransferase (ChAT) activity induced by NGF in cell cultures (+80%) and in the striatum (+240%) of rat pups. In spite of this effect, no significant change of APP mRNA expression was observed in neuronal cultures and brain tissues. These data indicate that the neurotrophic effect of NGF on forebrain cholinergic neurons is not always associated with an alteration of APP expression.