Cultured basal forebrain cholinergic neurons in contact with cortical cells display synapses,enhanced morphological features,and decreased dependence on nerve growth factor

Prior studies examining the dependence of basal forebrain cholinergic neurons (BFCNs) on nerve growth factor (NGF) for survival have reached differing conclusions depending on the experimental paradigm employed, suggesting the importance of environmental and developmental variables. The present study examined the NGF dependence of BFCNs and modulatory effects of target (cortical) neurons under the controlled conditions of dissociated cell cultures. Initial experiments found BFCNs (identified by using choline acetyltransferase immunocytochemistry) in pure basal forebrain (BF) cultures to be dependent on NGF between the 2nd and 4th week in vitro. During that developmental period, NGF deprivation for 3 days, induced by application of anti-NGF antibody, resulted in degeneration of over 80% of BFCNs, whereas at earlier or later times, BFCNs were largely resistant to NGF deprivation. When BF neurons were plated together with cortical neurons (as dissociated co-cultures), the BFCNs grew neuritic processes (labeled with acetylcholinesterase histochemistry) that appeared to specifically target cortical neurons; electron microscopy revealed that synapses formed between these cells. BFCNs in co-cultures were more resistant to NGF deprivation, were larger, and had much more extensive neuritic growth than BFCNs in pure BF cultures. The resistance of BFCNs to NGF deprivation provided by cortical neurons could be largely reproduced by addition of other trophic factors (brain-derived neurotrophic factor, BDNF; neurotrophin 3, NT3; neurotrophin 4/5, NT4/5; or glial-derived neurotrophic factor, GDNF) during NGF deprivation in pure BF cultures. These results suggest that developing BFCNs undergo a critical period requiring trophic influences that may be provided by NGF or other trophic factors, as well as unknown factors derived from cortical neurons. © 1996 Wiley-Liss, Inc.     

Reduced neuronotrophic activity of fibroblasts from individuals with dysautonomia in cultures of newborn mouse sensory ganglion cells

The neuronotrophic activity of human skin fibroblast cell lines from two normal individuals and 3 individuals with Familial Dysautonomia (FD) was compared in terms of their ability to support neuron survival and neurite regeneration in cultures of newborn mouse sensory neurons. Neuronotrophic activity was assayed by culturing dissociated sensory neurons from newborn mice: (1) on fibroblast 'cell beds' consisting of monolayer cultures of living fibroblasts, (2) with medium that had been conditioned by monolayer fibroblast cultures and (3) with extracts of the cultured fibroblasts. Neurite regeneration was compared by determining the percentage of neurons that had regenerated neurites after 24 or 48 h in culture. Neuron survival was determined by counts at 48 h, 7 days and 14 days after culture initiation. Neurite regeneration and neuron survival was found to be significantly less on FD cell beds or with FD-conditioned medium than in replicate cultures with normal fibroblasts or their conditioned medium. The reduced neuronotrophic activity of FD fibroblasts or conditioned medium was still observed in the presence of antibody sufficient to block the neuronotrophic effects of purified nerve growth factor (NGF). Thus, fibroblasts from individuals with FD exhibit reduced neuronotrophic activity for newborn mouse sensory neurons that appears to be due to factor(s) other than NGF.     

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.     

Immunosympathectomy: Lack of evidence for a complement-mediated cytotoxic mechanism

To determine whether the destruction of peripheral sympathetic neurons by anti-NGF-antibodies results from a complement-mediated cytotoxic action or from the deprivation of endogenous NGF or immunologically NGF-like cross-reacting molecules we investigated the time-course of the reversibility of the effect of NGF-antibodies by neutralizing doses of NGF, together with the effect of NGF-antibodies in complement-deficient mice. After administration of a single dose of 50 mg/kg of purified antibodies to newborn rats the TH level was reduced to 75% of controls after 12 h, to 48% after 24 h, 39% after 36, and 28% after 48 h. After this time no further reduction occurred and levels remained constant up to 14 days. The effect of the NGF-antibodies was reversible on addition of NGF up to 48 h after antibody administration. Although the reversibility was not complete (85% of controls) the extent of the reversibility was the same whether NGF was given 12 or 48 h after the antibodies. The incompleteness of the reversibility is reflected by the small number of degenerating neurons apparent as early as 12 h after antibody administration. Since these early degenerative effects were also seen in complement-deficient mice it is concluded that they involve a small population of neurons, sensitive to short-term NGF deprivation whereas the majority of the neurons can withstand deprivation for up to 48 h without sustaining irreversible damage.     

Substance P content in cultured neonatal rat vagal sensory neurons: the effect of nerve growth factor

To begin to study the factors regulating the synthesis and release of substance P (SP) in the sensory vagus nerve, cultures of neonatal rat nodose ganglia were developed. In microexplant cultures, obtained from small fragments of nodose ganglia, SP was present in low amounts: after 3 weeks, 141 +/- 36 pg per well, 10 ganglia equivalents per well. To enhance neuron survival, nodose ganglia were enzymatically dissociated using neutral protease. Estimated survival at 5 days was 20-30%, with 800-1200 surviving neurons per plated ganglion, and decreased slowly thereafter. Specific SP immunostaining was present in 10-20% of neurons, mostly of small diameter (18-22 micron). SP content was low for 5 days then rose progressively after 14 days to 80-150 pg per plated ganglion. The addition of nerve growth factor (NGF, 100 ng/ml) to the culture medium did not alter neuron survival. However, SP content was doubled in the presence of NGF, or fell rapidly to one-half control levels following its withdrawal: e.g. following 12 days in culture with NGF 1185 +/- 176 pg/well vs NGF withdrawn day 8-12, 592 +/- 118 pg/well, mean +/- S.D., P less than 0.01. Somatostatin, present in one-sixth the amount of SP, was unaltered by NGF. In subsequent studies, plating of neurons onto previously dissociated rat atriacytes increased survival by 50% but did not alter SP content per surviving neurons. These studies demonstrate that SP is present in dissociated cultures of rat vagal sensory neurons; the quantities and estimated net synthesis rate correspond to previous observations in vivo. The studies also demonstrate that SP content but not neuron survival are regulated by NGF in nodose ganglion neurons. This model may prove valuable for the study of SP and other sensory neuropeptides in this important class of visceral afferent neurons.     

Control of the Na, K -pump by nerve growth factor is essential to neuronal survival

Recently we have shown that nerve growth factor (NGF) controls the performance of the Na⁺, K⁺ -pump in its target ganglionic neurons in suspension cultures. In the present study, enriched neuronal preparations of embryonic day 8 (E8) chick dorsal root ganglia (DRG) were obtained by means of a differential attachment procedure using tissue culture plastic dishes. Neurons were routinely seeded into polyornithine-coated 16 mm culture wells in the presence of NGF. After 18 h, cultures were switched to media with or without NGF, and containing either⁸⁶Rb⁺ (as a tracer for K⁺) or²²Na⁺ (as a tracer for Na ions). Over the next 12–15 h the cultures were assessed for numbers of surviving neurons and accumulated radioactivity. Cultured E8 chick DRG neurons fail to maintain their intracellular K⁺ concentration when deprived of NGF over 4–6 h. The NGF-deprived and K⁺- depleted neurons reaccumulate K⁺ within minutes of delayed NGF administration. The occurrence of this K⁺ response in culture to added NGF parallels the response occurring in E8 neuronal suspensions, including the time of onset of irreversibility. Similar experiments performed with²²Na⁺ indicate corresponding ionic behaviors for cultured E8 DRG neurons. These NGF-controlled ionic responses in monolayer cultures occur for E7 and E10 neurons, but not E14 neurons and parallel the survival response to NGF of the same neurons. Blocking the pump performance by NGF deprivation leads to neuronal death. Identical results are obtained by addition of oubain or omission of external K⁺ in the presence of NGF. Partial reduction of pump performance by any one of these treatments leads to partial survival of the neuronal population in a precisely predictable manner. Therefore, control of the pump by NGF is an essential component of the NGF action on neuronal survival.     

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.     

Neuroprotection of cultured foetal rat hippocampal cells against glucose deprivation: are GABAergic neurons less vulnerable or more sensitive to TCP protection?

In the rat brain, hippocampal neurons are particularly sensitive to secondary excitotoxic injury induced by ischaemia or hypoglycaemia. To determine some distinctive features of vulnerability among neuronal phenotypes in the hippocampus following a metabolic insult, we used an in vitro model of mild glucose deprivation. Primary cultures from the rat hippocampus (21 days in vitro) were deprived of glucose for 4 h and then were returned to the standard medium for 24 or 48 h. Survival of the GABAergic neuronal population was evaluated both by measuring [3H]GABA uptake and by counting GAD65-immunostained cells. This was compared with the survival of the total neuronal population evaluated by counting the neurofilament-200-immunostained cells. Glucose deprivation for 4 h followed by a recovery period of 48 h induced a decrease of 59% and 40% in the number of GAD65- and neurofilament-200-immunostained cells, respectively. Thus, GABAergic neurons were slightly more vulnerable to glucose deprivation than the other neurons in the hippocampal cell cultures. When the excitotoxic component of cellular death was blocked in the presence of TCP, an NMDA-antagonist, the survival of GABAergic neurons was almost complete after 48 h of recovery. In contrast, measurements of the release of lactate dehydrogenase in the medium indicated that TCP largely protected hippocampal cells after 24 h but was ineffective after 48 h. This observation was confirmed by immunostaining data which showed that after 48 h TCP did not significantly increase the survival of neurofilament-200-immunostained cells. These results indicate that after glucose deprivation and a recovery period of 48 h, GABAergic neurons in hippocampal cell cultures are not more resistant than other neurons but they are more sensitive to TCP protection.     

Rat embryonic septal neurons survive and express cholinergic properties in isolation and without nerve growth factor.

We studied survival and expression of cholinergic properties in embryonic septal neurons grown in very low density microcultures (1-7 cells per Terasaki well). Even in cultures containing only a single neuron, at least 10% of plated neurons survived for 2 weeks or more in medium containing fetal calf serum or an acid-stable fraction (55,000 Da) of horse serum. Of these surviving neurons, 30-40% stained positively for acetylcholinesterase (AChE) or nerve growth factor (NGF) receptor, even though the culture medium lacked detectable levels of NGF, brain-derived neurotrophic factor, and fibroblast growth factor. Addition of NGF or an antibody against NGF had no effect on either neuronal survival or the percentage of neurons staining positively for AChE or NGF receptor after 18-20 days in vitro. There was no cell division in medium containing the serum fraction, but when 10% fetal calf serum was present cell division occurred in some of the cultures, and in half of these cases at least one of the clonal progeny became AChE-positive. These results demonstrate that some embryonic septal cells can survive at least 2 weeks and develop cholinergic neuronal properties in the absence of other cells or NGF.     

Reversible physiological alterations in sympathetic neurons deprived of NGF but protected from apoptosis by caspase inhibition or Bax deletion

Cell death in nervous system development and in many neurodegenerative diseases appears to be apoptotic or programmed. Withdrawal of nerve growth factor (NGF) from cultures of superior cervical ganglia neurons (SCG) is an excellent model of programmed cell death (PCD), producing apoptosis within 24-48 h. This death can be prevented by treatment with caspase inhibitors or deletion of the proapoptotic Bax gene. Since inhibition of apoptosis is an attractive strategy for the therapy of many neurological diseases and little is known about the function of neurons when apoptosis has been aborted, we examined the electrophysiological properties of NGF-deprived SCG neurons from rats and mice, saved by the caspase inhibitor boc-aspartyl(OMe)fluoromethyl ketone (BAF) or by Bax deletion. Compared to NGF-maintained controls, the resting membrane potentials of BAF-saved neurons were depolarized by 9 mV and the action potentials were prolonged by over 50%. Nicotinic cholinergic current density was depressed by about 50%. Electrophysiological parameters returned to normal within 4 days after NGF restoration. Neurons from Bax-deficient mice were altered differently by NGF withdrawal. There were no detectable changes in resting or action potentials. However, nicotinic current density was reduced just as in BAF-saved rat neurons. There were no observable changes in the processes of individual neurons after 6 days of NGF deprivation in the presence of BAF. Our results indicate that neurons are physiologically altered during pharmacological inhibition of PCD, but fully recover after trophic support is returned.     

Developmental change in the nerve growth factor action from induction of choline acetyltransferase to promotion of cell survival in cultured basal forebrain cholinergic neurons from postnatal rats

Nerve growth factor (NGF), a well-characterized target-derived growth factor, has been postulated to promote neuronal differentiation and survival of the basal forebrain cholinergic neurons. In the present paper, we demonstrate that a developmental change in NGF action occurs in postnatal rat basal forebrain cholinergic neurons in culture. Firstly, NGF acts as maturation factor by increasing choline acetyltransferase (ChAT) activity and acts later as a survival factor. In dissociated cell cultures of septal neurons from early postnatal (P1-4) rats, ChAT activities were increased by the addition of NGF. That is, ChAT activities in P1 septal cells cultured for 7 days was increased 4-fold in the presence of NGF at a concentration of 100 ng/ml. However, the number of the acetylcholinesterase (AChE)-positive neurons was not significantly different between these groups. In contrast, septal neurons from P8 to P14 rats showed different responses to NGF. Although the P14 septal neurons in culture for 7 days without NGF lost about half of the ChAT activity during a 7-day cultivation, cells cultured with NGF retained the activity at the initial level. The number of AChE-positive neurons counted in cultures with NGF was much greater than the number without NGF. These results suggest that, during the early postnatal days, the action of NGF on the septal cholinergic neurons in culture changes from induction of ChAT activity to the promotion of cholinergic neuronal cell survival. During this developmental period in vivo, septal neurons are terminating their projections to the hippocampal formation. Similar NGF-regulated changes in cholinergic neurons were observed in cultured postnatal neurons from vertical limb of diagonal band. An analogy has been pointed out between the neuronal death of the basal forebrain cholinergic neurons and a similar neuronal death in senile dementia, especially Alzheimer's type. The work reported here might present a possibility that NGF could play a role in preventing the loss of the basal forebrain cholinergic neurons in this disease.     

Brain-derived neurotrophic factor (BDNF) protects cultured rat cerebellar granule neurons against glucose deprivation-induced apoptosis

Summary. In the present study, cell death induced by glucose deprivation in primary cultures of cerebellar granule neurons was examined. Glucose deprivation-induced apoptotic cell death was demonstrated using the terminal transferase-mediated (TdT) deoxyuridine triphosphate (d-UTP)-biotin nick end labeling (TUNEL) method and DNA fragmentation assays. When the effects of different neurotrophins on the survival of cerebellar granule neurons after glucose deprivation were assessed, BDNF, but not NT-3 or NGF, was found to protect cerebellar granule neurons against glucose deprivation-induced cell death. In addition, BDNF treatment increased c-Fos immunoreactivity in the cerebellar granule neurons. These results are consistent with the hypothesis that neuronal death due to glucose deprivation has a significant apoptotic component and that neurotrophins can protect against hypoglycemic damage. Received December 17, 1997; accepted May 19, 1998     

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)     

Control of Na,K-pump activity in dorsal root ganglionic neurons by different neuronotrophic agents

Na⁺,K⁺-pump activity is indispensable for neuronal survival in vitro and a specific role in its regulation has been demonstrated for the NGF action on its target neurons. We have extended these earlier studies to include two other neuronotrophic agents: the chick eye-derived ciliary neuronotrophic factor (CNTF); and 12-O-tetradecanoyl-phorbol-13-acetate (TPA). CNTF and TPA individually supported the survival of an identical (and maximal) number of embryonic day 10 (E10) dorsal root ganglion (DRG) neurons as did NGF. E10 DRG neurons, seeded as monolayer cultures with⁸⁶Rb⁺ (as K⁺ tracer) but no trophic supplement in their medium, received NGF, CNTF, TPA, or no agent at 2, 4 or 6 h after seeding. The cultures were analyzed at 6 and 24 h for Na⁺,K⁺-pump performance and at 24 h for neuronal survival. Neurons receiving no agent lost their pump activity over the first 6 h and died over the 10–24 h incubation period. Both pump performance and survival were fully supported by any one of the 3 agents when provided at seeding time. Delayed presentation of NGF also led to full restoration of pump activity and survival support, as expected. In contrast, CNTF and TPA failed to correct the increasing pump deficits incurred with increasing times of trophic deprivation, and neuronal survival was proportionally reduced. Delayed addition of CNTF and TPA did, however, prevent further losses of both pump and viability. Close similarities were observed between pump failure and cell losses, demonstrating a linear correlation between pump performance and neuronal survival. Thus, neuronal survival is strongly correlated with the Na⁺,KK⁺-pump performance regardless of whether the DRG neurons are supported by NGF, CNTF or TPA. All 3 agents protect the neurons against pump losses (hence, against death), but only NGF appears to be able to restore pump function and cell viability.     

Synergistic Differentiation by Chronic Exposure to Cyclic AMP and Nerve Growth Factor Renders Rat Phaeochromocytoma PC12 Cells Totally Dependent upon Trophic Support for Survival

Chronic dibutyryl cAMP (dbcAMP) treatment was observed not only to potentiate the differentiating actions of nerve growth factor (NGF) in PC12 cells, but to render them completely dependent on trophic support for survival even in the presence of serum proteins. When both NGF and dbcAMP were withdrawn from doubly differentiated PC12 cultures, degenerative events occurred after a lag period of 12–18 h, and by 48 h ≤ 5–10% of the cells remained viable. Reduction in [³H]dopamine uptake, an index of cell function and neurite integrity, paralleled cell demise. At the cellular level, ∼20–30% of the nuclei exhibited clear signs of chromatin fragmentation, as characterized by propidium iodide staining, suggesting that degeneration occurred by apoptosis. The cells could be rescued completely from degeneration by dbcAMP or by other cAMP analogues, whereas NGF and depolarization were also effective, but only partially. Phorbol 12-myristate-13-acetate failed to afford protection. If deprivation was interrupted, cell demise could be stopped by restoration of initial culture conditions. Degenerative changes produced by deprivation and recovery processes were not inhibited by macromolecular synthesis inhibitors, e.g. cycloheximide and actinomycin-D. However, chronic addition of cycloheximide prior to deprivation greatly impaired the differentiation of NGF/dbcAMP cells, allowing these cells to withstand trophic support withdrawal. Altogether our results indicate that the cAMP transduction pathway plays a crucial role not only in the differentiation but also in the survival of NGF/dbcAMP PC12 cells.     

Effects of nerve growth factor on the survival of primary cultured adult and aged mouse sensory neurons

This study investigated the effects of exogenous nerve growth factor (NGF) on the survival and differentiation in primary culture of sensory neurons isolated from adult (6 months) and aged (2 years) mice. For neurons prepared from adult mice, a concentration effect was evident during a 2 week culture period: Neuronal counts in cultures supplemented with 25 and 50 ng/ml NGF did not differ significantly from those of control cultures without exogenous NGF or those with anti-NGF included in the culture medium, whereas cultures supplemented with either 100 or 200 ng/ml NGF contained higher numbers of neurons throughout the culture period. Cultures prepared from aged mice contained less neurons than those from adult mice, although those supplemented with 100 ng/ml NGF retained higher neuronal numbers than cultures from aged mice which did not receive exogenous NGF. Neuronal diameters were measured to investigate whether specific subpopulations of neurons were more dependent on NGF; the results indicate that neurons of a medium-larger diameter were more prevalent than cells with a smaller diameter following NGF administration. A shape index was calculated for each culture regimen; with longer culture periods a higher proportion of spindle-shaped neurons was observed. © 1993 Wiley-Liss, Inc.     

Enhanced survival and morphological features of basal forebrain cholinergic neurons in vitro: role of neurotrophins and other potential cortically derived cholinergic trophic factors

The present study examined survival- and growth-enhancing effects of cortical cells on basal forebrain cholinergic neurons (BFCNs) in culture and the degree to which endogenous nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) contribute to those trophic effects. When fetal (17 days of gestation) basal forebrain (BF) cells were grown for 5 days in coculture with cortical neurons, staining for acetylcholinesterase (AChE) showed a threefold increase in the number of BFCNs relative to BF cultures without cortex. Most of these labeled cells also displayed enhanced somatic, dendritic, and axonal growth. Coculturing cortical neurons with BF cells taken from postnatal animals produced similar results but with a somewhat greater degree of morphologic enhancement. Function-neutralizing antibodies to NGF, BDNF, and NT-3 were employed to determine whether they would block the trophic effects of cortical neurons on postnatal BFCNs. Although no significant changes in numbers or morphological features of AChE(+) neurons were observed with treatment with individual antibodies, cocultures treated with a combination of all three antibodies displayed fewer morphologically enhanced AChE(+) cells and more nonenhanced cells; the total number of AChE(+) neurons was not significantly changed. Treatment of pure BF cultures with exogenous NGF, BDNF, and NT-3 increased the number of AChE(+) neurons but did not reproduce the morphologic enhancement of cortical cells on BFCNs. These results suggest that neurotrophins by themselves can increase survival of postnatal BFCNs in culture and may work in concert with other unknown cortically derived factors to enhance BFCN morphologic differentiation. The unidentified cortical factors may also have strong survival-enhancing effects on BFCNs that are independent of the known neurotrophins.     

Relationship between NMDA receptor expression and MPP+ toxicity in cultured dopaminergic cells

It has been suggested that excitotoxicity could be contributing to dopamine cell loss after methylphenylpyridinium ion (MPP+) exposure, although the literature regarding this is contradictory. Given that in cell culture excitotoxicity has been reported to be dependent on culture age, we postulated that these discrepant results might be explained by a difference in developmental expression of N-methyl-D-aspartate (NMDA) receptors. To test this, mesencephalic cells were cultured and the number of dopaminergic neurons (tyrosine hydroxylase-immunoreactive cells [TH-IR] cells) expressing the NMDA R1 subunit (NR1) was determined using double-label immunofluorescence microscopy. An increase in the percentage of TH-IR cells expressing NR1 occurred over time in culture and this correlated with the toxicity of NMDA. At 7 days in vitro (DIV 7), only 17% (n=167 cells/4 experiments) of TH-IR cells expressed NR1 and these cells were insensitive to NMDA toxicity. This increased to 80% (n=254 cells/6 experiments) by DIV 11 and cultures were now susceptible to NMDA-induced injury. Cultures grown for either 7 or 11 days were treated for 48 hr with increasing concentrations of MPP= (0.5-20 microM) and the loss of dopaminergic neurons was determined by cell counting. Cultures at DIV 7 were more sensitive to MPP= than 11-day-old cultures (LD50= approximately 0.75 microM vs. 15 microM, respectively). Co-exposure to MK-801 (5 microM) did not protect against MPP+ toxicity in young cultures, but attenuated MPP+ toxicity in the older cultures, becoming statistically significant at 20 microM MPP+. These data indicate that the activation of NMDA receptors is not required for, but can contribute to, MPP(+)-induced neurodegeneration of dopaminergic cells in culture.