Cholinergic neurotoxicity induced by ethylcholine aziridinium (AF64A) in neuron-enriched cultures

The sequence of events in neuronal changes induced by the cholinotoxin ethylcholine aziridinium (AF64A) was studied. Neuron-enriched cultures derived from 8-day-embryonic chick cerebra were treated with AF64A at concentrations of 10(-5), 10(-4) and 10(-3) M. Choline acetyltransferase (ChAT) was used as an index of cholinergic neurons. Changes in cell morphology, the immunocytochemical and biochemical presence of ChAT, and DNA and protein content were assessed. Neuron-enriched cultures exposed to AF64A showed a dose-dependent response; after 24 h of exposure to 10(-3) M toxin all cells were dead, whereas a concentration of 10(-5) M did not alter culture morphology or DNA and protein contents. Despite the lack of cytological changes and the presence of ChAT immunoreactivity, biochemically assessed ChAT activity was reduced 36% in 10(-5) M treated cultures. Thus, the implicated decrease in acetylcholine synthesis in these cells cannot entirely account for the neuronal degeneration. Simultaneous exposure of cultures to both AF64A and 10 times higher concentrations of choline chloride delayed or diminished the neurotoxic changes. The protective effect of high choline concentrations was interpreted as evidence of competition between choline and AF64A for the high affinity choline transport system and as constituents in the cell membrane. Examination of the temporal sequence of cytotoxic changes in 10(-4) M exposed cultures revealed that disruption of neuronal aggregates and fragmentation of neurites occurred between 4 and 8 hours of exposure. After 24 h, some neurons survived but with attenuated arbors; in contrast, astrocytes appeared intact, suggesting that glial cells are more resistant than neurons to the toxic effects of AF64A. These findings suggest this culture model may be useful to further elucidate the mechanisms of AF64A drug action and study differentiation of cultured neuronal populations in the absence of cholinergic cells.     

Neuron-enriched cultures derived from spinal cord of 10-day-old chick embryos: Influence of neuropeptides on neuronal survival, proliferation and cholinergic expression

The developmental regulation of cell proliferation, survival and cholinergic expression by growth hormone-releasing hormone (GHRH) and somatostatin (SRIF) was investigated in neuronenriched cultures derived from 10-day-old embryonic chick spinal cord. In this study, ³H-thymidine incorporation into DNA was assessed, using two different applications, in order to determine both cellular proliferation and survival. The rate of neuroblast proliferation in both control and neuropeptide-treated cultures increased or remained the same up to day 6. However, in neuropeptide-treated cultures the magnitude of cell proliferation remained at levels higher than those observed in controls through day 6 and was most significant in SRIF-treated cultures at C4. In all groups, proliferation markedly declined by day 8. Survival of neuronal cells labelled at C4 remained high up to day 12 in all three groups, then drastically declined by day 17. Neuronal survival in the neuropeptide-treated cultures was also higher than in controls. Cholinergic expression, as assessed by activity of choline acetyltransferase (ChAT), responded differentially to neuropeptide treatment. Cultures treated with GHRH (100 nM) exhibited a long term significant enhancement in ChAT activity throughout the culture period, whereas those treated with SRIF (50 nM) expressed a transient decline in ChAT activity. Videometric analysis showed that both neuropeptides enhanced neuronal aggregation, neuritic arborization and neuritic length. These findings lead us to suggest that GHRH and SRIF may provide neurotrophic signals important not only for neuronal proliferation and survival but also for cholinergic neuronal expression. Furthermore, we propose that GHRH possesses specific cholinotrophic properties, whereas SRIF may act as a general neurotrophic factor.     

Enzymatic activities during differentiation of the human neuroblastoma cells, LA-N-1 and LA-N-2

The presence of 10(-5) M retinoic acid (RA) in the culture medium of LA-N-1, a catecholaminergic cell line, and LA-N-2, a cholinergic cell line, enhanced their morphological differentiation. Tyrosine hydroxylase (TH) activity of the LA-N-1 cells was increased in the RA-treated cells compared with control cultures at day 4 and remained elevated. Choline acetyltransferase (ChAT) activity in the LA-N-2 cells gradually increased until 8 days in vitro (DIV) both in the untreated control and the RA treated cultures. This activity in control and treated cells decreased gradually to a constant level of activity. The ChAT activity at 8 DIV of RA-treated LA-N-2 cells was increased 2.1-fold (P less than 0.001) as compared to the control cultures. This increase in ChAT activity was accompanied by a 73% decrease of acetylcholinesterase (AChE) activity in LA-N-2 cells by 8 DIV. AChE activity of LA-N-1 cells was unchanged during the time course of the experiment. Phospholipase-A2 (PL-A2) activity in RA-treated LA-N-2 cells was increased at day 4 as compared with the control cultures. There were no differences observed in phospholipase-D (PL-D), choline kinase and GPC-phosphodiesterases activities in RA-treated and -untreated LA-N-1 and LA-N-2 cells.     

Growth hormone-releasing hormone and somatostatin influence neuronal expression in developing chick brain. II. cholinergic neurons

In a previous study we have shown that in ovo administration of growth hormone-releasing hormone (GHRH) influences catecholaminergic neuronal expression as assessed by tyrosine hydroxylase activity (Dev. Brain Res., 49 (1989) 275-280). In this study we examined the effects of GHRH and somatostatin (SRIF) on cholinergic neuronal neurotransmitter expression both in ovo and in vitro. Chick embryos were administered GHRH or SRIF in ovo via the air sac on embryonic days 1, 3, 5 and 7, sacrificed at day 8 and the activity of choline acetyltransferase (ChAT) was assayed in whole brain homogenates. ChAT activity was significantly higher in peptide-treated embryos as compared to controls. Similar results were obtained when GHRH or SRIF (50 ng/50 microliters) was administered in a single dose at day 2 or 3; ChAT activity was significantly increased in peptide-treated versus control embryos. In contrast, embryos treated with GHRH or SRIF on day 1 or 5 or with growth hormone (100 ng/50 microliters) on day 3 of incubation showed no difference in ChAT activity as compared to controls. More direct effects of GHRH and SRIF were tested in neuron-enriched cultures derived from 3- (E3) or 6-day-old (E6) chick embryos. Cultures were grown in either serum-supplemented or serum-free medium for 6 days in the presence of GHRH or SRIF concentrations ranging from 0.01 to 100 nM. As observed in ovo, ChAT activity was increased in E3 cultures treated with peptides. In addition, the composition of the culture medium influenced the response to peptide treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dual effects of thrombin and a 14-amino acid peptide agonist of the thrombin receptor on septal cholinergic neurons

We have compared the effects of thrombin and of the 14-amino acid peptide agonist (TRAP-14) of the thrombin protease activated receptor (PAR) on cholinergic neurons in pure cultures of rat septal neurons and in co-cultures of septal neurons and glial cells. In pure septal cultures, low concentrations of thrombin (up to 10 nM) did not affect choline acetyltransferase (ChAT) activity, a marker of cholinergic neurons, or 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction, an index of cell viability. However, 100 nM thrombin decreased ChAT activity and MTT reduction by 44 and 17%, respectively. In co-cultures, a low concentration of thrombin (1 nM) increased ChAT activity (+75%), whereas a high concentration (100 nM) decreased it (−83%). At this high concentration, thrombin was neurotoxic, as indicated by a large decrease in MTT reduction (−80%). Thrombin effects on ChAT activity were mimicked by TRAP-14 both in pure septal cultures (no effect at 0.1 μM and −63% at 100 μM) and in co-cultures (+25% at 0.1 μM and −28% at 100 μ M). In contrast, this peptide did not affect MTT reduction. These dual effects of thrombin and TRAP-14 on ChAT activity in co-cultures, were also observed on pure cultures of septal cells supplied with NGF. The activation and inhibition by TRAP-14 of the expression of ChAT activity in septal neuron/glial cell cultures were inhibited by a 9-amino acid peptide antagonist of thrombin PAR. Thus, the effects of thrombin on cholinergic neurons seem to be mainly mediated by thrombin PAR and glial cells seem to play a major role in these thrombin actions.     

Ethanol neurotoxicity on neuroblast-enriched cultures from three-day-old chick embryo is attenuated by the neuronotrophic action of GABA

In the present study, using neuroblast-enriched cultures derived from three-day-old chick embryos (E3WE), we examined the morphological effects of ethanol and/or GABA, as well as the developmental profile of the cholinergic and GABAergic neuronal phenotypes, as assessed by the activities of choline acetyltransferase (ChAT) and glutamate decarboxylase (GAD). Cultures exposed to ethanol (50 mM) exhibited smaller and fewer aggregates than controls with a neuritic network that lacked fasciculation. In cultures treated with GABA (10⁻5 M) alone or ethanol + GABA the size and number of the neuronal aggregates was increased and also neuritic arborization and fasciculation was enhanced. Thus, addition of GABA restored the normal growth pattern in the ethanol-treated cultures. As previously shown, E3WE culture treated with ethanol alone showed a decrease in both ChAT and GAD activities compared to controls. Both cholinergic and GABAergic neuronal phenotypes were enhanced in cultures treated with GABA as assessed by increases in ChAT and GAD activities, respectively, compared to controls. Moreover, in cultures treated concomitantly with ethanol and GABA both ChAT and GAD activities were higher than in ethanol-alone-treated cultures. Thus, the presence of GABA in the ethanol-treated cultures counteracted the decline in ChAT and GAD activities observed in the ethanol-alone-treated cultures. We concluded that GABA through its neuronotrophic actions can rescue neuroblasts from ethanol insult and restore neuronal phenotypes.     

Critical periods to ethanol exposure during early neuroembryogenesis in the chick embryo: cholinergic neurons

The acute and chronic effects of ethanol on cholinergic neuronal expression were studied in the chick embryo during early neuroembryogenesis using choline acetyltransferase (ChAT) activity as a cholinergic marker. Ethanol administered to embryos in ovo on day 1 (E1) produced a 30% decrease in ChAT activity, while ethanol administration on day 3 elicited no significant change. Similar effects were produced by ethanol on ChAT activity in the spinal cord. The decrease in ChAT activity in both brain and spinal cord was not accompanied by a significant change in protein content. Of significance were our findings with chronic ethanol treatment: in embryos treated from E1 to E5 and sacrificed at E6, ChAT activity was decreased. In contrast, in embryos treated similarly but sacrificed at E8 ChAT activity was increased. These findings establish that the critical period of cholinergic neuronal sensitivity to ethanol is confined to E. Moreover, the increase in ChAT activity observed after chronic ethanol treatment indicates that the developing neurons have the capability to adapt to ethanol. This apparent adaptation results in overcompensation, as reflected by the increase in ChAT activity. Whether this overcompensation is at the expanse of another neuronal population remains to be investigated.     

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)     

Morphine alters astrocyte growth in primary cultures of mouse glial cells: evidence for a direct effect of opiates on neural maturation

To determine whether exogenous opiate drugs with abuse liability directly modify neural growth, the present study investigated the effects of morphine on astrocyte proliferation and differentiation in primary cultures of murine glial cells. The results indicate that morphine decreases glial cell production in a dose-dependent, naloxone-reversible manner. Most notably, gliogenesis virtually ceased in the presence of 10(-6) M morphine during the first week in culture, whereas 10(-8) M or 10(-10) M morphine caused an intermediate suppression of growth compared to control or 10(-6) M morphine treated cultures. Moreover, morphine treatment inhibited [3H]thymidine incorporation by glial fibrillary acidic protein (GFAP) immunoreactive, flat (type 1) astrocytes, suggesting that the decrease in glial cell production was due in part to an inhibition of astrocyte proliferation. Morphine also caused significant increases in both cytoplasmic area and process elaboration in flat (type 1) astrocytes indicating greater morphologic differentiation. In the above experiments, morphine-dependent alterations in astrocyte growth were antagonized by naloxone, indicating that morphine action was mediated by specific opioid receptors. These observations suggest that opiate drugs can directly modify neural growth by influencing two critical developmental events in astrocytes, i.e., inhibiting proliferation and inducing morphologic differentiation.     

Initial cholinergic differentiation in embryonic chick retina is responsive to insulin and cell-cell interactions

Previous work [Kyriakis et al., Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 7463-7467] had shown that insulin, when added during a window of binding from embryonic days 9-11, stimulates the normal developmental increase in choline acetyltransferase (ChAT) activity (a marker for cholinergic differentiation) in cultured embryonic chick retinal neurons. Here, we investigated the effect of insulin and IGF 1 on embryonic chick retinal neurons at the stage of development (embryonic day 6) when ChAT activity is first expressed. We investigated insulin peptide effects in retinal tissue developing in vitro as well as in cultures of retinal cells. We show that insulin also stimulated the initial embryonic increase in ChAT activity but had no stimulatory effect on glutamic acid decarboxylase activity (a marker for GABAergic differentiation), an enzyme whose activity also increases developmentally in the same retinal neurons. In fact, insulin inhibited the expression of GAD activity in the retina. The insulin-mediated increase in ChAT activity was independent of normal cell-cell interactions but could not replace them. Insulin also stimulated choline uptake but only after a two day delay, suggesting that the normal program for cholinergic differentiation in the chick retina was induced by insulin. IGF 1 did not have any effect on either cholinergic or GABAergic differentiation. We conclude that cholinergic differentiation in chick embryo retinal neurons is dependent on both insulin- and cell contact-mediated signals.     

Differences in neuronal and glial cell phenotypic expression in neuron-glia cocultures: influence of glia-conditioned media and living glial cell substrata.

Neuron-glia cocultures were prepared using, as a source for glial cells, either C6 glia (2B clone) of early (2B23) or late (2B111) passages or advanced passages of glial cells derived from primary cultures prepared from aged mouse cerebral hemispheres (MACH). Six-day-old chick embryo cerebral hemispheres (E6CH) were the source of neuron-enriched cultures. Glutamine synthetase (GS) activity was used as a marker for astrocytes and 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) activity was used as a marker for oligodendrocytes. GS activity was markedly enhanced in cocultures of E6CH neurons and 2B23 glioblastic cells, whereas GS activity was reduced in cocultures of E6CH neurons and 2B111 astrocytic glia. In contrast, CNP activity was enhanced in cocultures of C6 glial cells with E6CH neurons. Glial cells from aged mouse brain did not respond to coculturing with E6CH neurons. It appears from these findings that neuronal input enhances the differentiation of glioblastic cells to either astrocytic or oligodendrocytic expression, whereas it decreases the activity of committed astrocytes. In contrast, glial cells from aged mouse brain do not respond to neuronal input. Choline acetyltransferase (ChAT) activity, a marker for cholinergic neurons, was enhanced only when E6CH cultures were grown in conditioned medium (CM) from 2B23 glioblastic cells. In contrast, ChAT activity was markedly diminished when E6CH neurons were cocultured with MACH glial cells but not when grown in CM from MACH glial cells. Thus, humoral factors from immature glial cells appear to enhance cholinergic neuronal phenotypic expression whereas cell-cell membrane contacts with aged glial cells diminish cholinergic phenotypic expression. The findings present supportive evidence that neuron-glia interrelationships are age dependent.     

Influence of triiodothyronine (L-T3) on the morphological and biochemical development of fetal brain acetylcholinesterase-positive neurons cultured in a chemically defined medium

In cerebral hemisphere cultures initiated from 15-day-old rat embryos, the number of acetylcholinesterase-positive (AChE+) cells increased from 6.8 +/- 1.6 cells/well on day 3 to 112 +/- 16 cells/well on day 15. With time in culture, AChE+ cells increased both in size of the perikarya and neurite length. The addition of L-triiodothyronine (L-T3) at a concentration of 3 x 10(-8) M at the initiation of the culture had no effect on the number of AChE+ cells but significantly increased the size and neurite length of AChE+ neurons after 5 days in vitro. These morphological effects are associated with biochemical effects. L-T3 increased AChE activity in both a dose- and time-dependent manner (the stimulatory effect of L-T3 becomes significant between day 8 and day 15). Since a major part of AChE+ cells may be cholinergic neurons, we have also measured the effect of L-T3 on ChAT activity. L-T3 also increased ChAT activity in a dose and time dependent manner. Furthermore, treatment of cultures with L-T3 at different times in culture demonstrated the presence of a critical period which occurs in vitro around day 20, since the stimulatory effect of L-T3 on ChAT activity is lost after 20 days in vitro. Studies of the time necessary for L-T3 to increase both ChAT and AChE activities show that 2 days and 15 days, respectively, are required for L-T3 to significantly stimulate both enzyme activities. This in vitro analysis demonstrated the morphological effect of L-T3 on the size and the neurite length of AChE+ cells. These effects are associated with biochemical effects on ChAT and AChE activities. Thus, it appears that thyroid hormones regulate several steps of neuronal maturation.     

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.     

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.     

An interaction between thyroid hormone and nerve growth factor in the regulation of choline acetyltransferase activity in neuronal cultures, derived from the septal-diagonal band region of the embryonic rat brain

Culture conditions have been established for growing neurons from the medial frontal part of the forebrain, containing the septum and the diagonal band of Broca, of 17-day-old rat embryos in a chemically defined medium. At 10 days in vitro, the cultures contained more than 96% nerve cells of which about 18% were cholinergic neurons, while the proportion of astrocytes was less than 1%. The majority of the cells that stained for acetylcholinesterase were bipolar but with different sizes and shapes. During development both the specific activity of choline acetyltransferase (ChAT) and the amount of protein increased markedly in the cholinergic cultures, ChAT activity rising much more than the protein content. Exposure of the cultures to nerve growth factor (NGF) or 3,3',5-triiodo-L-thyronine (T3) enhanced the expression of ChAT activity in a dose-dependent manner. The elevation of ChAT activity was due to an increase in the amount of enzyme per cholinergic cell, since, during the experimental period studied, neither treatment with NGF nor with T3 had significant effects on the total protein content of the cultures or on the number of cells, including the cholinergic neurons. When cultures were supplemented with both agents at maximal effective concentrations, the stimulation in ChAT activity was much greater than the sum of the individual effects. The observations indicate that subcortical cholinergic neurons, which are affected in Alzheimer's disease and in Down's syndrome, are subject to regulation by an interaction between thyroid hormone and local humoral factors such as NGF.     

Effects of corticosterone on chick embryonic retinal cells in culture

Corticosterone has been shown to affect several patterns of glial cell and neuronal development. We have previously reported that exogenously administered corticosterone preferentially accumulated into the retinas of 8-day-old chick embryos. Moreover, we observed that it affects muscarinic cholinergic binding. Thus, we investigated the effect of different concentrations of corticosterone on retinal cells in culture. Retinas were dissected from 8-day-old embryos, dissociated and cells plated on salt-precipitated collagen. At day 5, cultures were treated with corticosterone (from 10(-9) M to 10(-7) M) for 24 h. Controls received either Dulbecco's Modified Eagle Medium (DMEM) plus 10% fetal calf serum (FCS) or DMEM only. Results show that the main effect of the hormone was inhibition of neuronal process outgrowth. Also cell aggregation, flat cell proliferation and confluency are altered in hormone-treated cultures. All these effects are reversible and can be attributed to hormone effect and not to serum deprivation.     

Cholinergic neurons of fetal rat telencephalon in aggregating cell culture respond to NGF as well as to protein kinase C-activating tumor promoters

Serum-free aggregating cell cultures of fetal rat telencephalon treated with the potent tumor promoter phorbol 12-myristate 13-acetate (PMA) showed a dose-dependent, persistent stimulation of the enzymes choline acetyltransferase (ChAT), glutamic acid decarboxylase and glutamine synthetase. After elimination of the proliferating cells by treatment of the cultures with Ara-C (0.4 microM) only the cholinergic marker enzyme, ChAT, could be stimulated by tumor promoters. The non-promoting phorbol ester, 4 alpha-phorbol 12,13-didecanoate proved to be inactive in these cultures, whereas the potent non-phorbol tumor promoter, mezerein, produced an even greater stimulatory effect than PMA. Since PMA and mezerein are potent and specific activators of protein kinase C, the present results suggest a role for this second messenger in the development of cholinergic telencephalon neurons. Stimulation of ChAT required prolonged exposure (48 h) of the cultures to PMA and the responsiveness of the cholinergic neurons to the tumor promoters decreased with progressive cellular maturation. The cholinergic telencephalon neurons showed the same pattern of responsiveness for tumor promoters as for nerve growth factor (NGF). However, the combined treatment with NGF and either PMA or mezerein produced an additive stimulatory effect, suggesting somewhat different mechanisms of action.     

Retinoic acid induces cholinergic differentiation of NTera 2 human embryonal carcinoma cells

Retinoic acid (RA), a natural metabolite of vitamin A, influences the survival and neurotransmitter phenotype of several classes of vertebrate neurons during development. We now report that RA induces a subpopulation of NTera 2/clone D1 (NT2) human embryonal carcinoma cells to differentiate into postmitotic cells with cholinergic properties (NT2-N cells). After growth for 6 days in the presence of RA (10 μM) low levels of the acetylcholine-synthesizing enzyme choline acetyltransferase (ChAT) were detected in NT2 cell cultures. ChAT activity in the NT2 cell cultures continued to increase for at least an addition 22 days to a final activity of 50 pmol ACh synthesized/min/mg protein. Immunohistochemical staining of RA-treated cultures demonstrated that only those cells with a neuronal morphology (NT2-N cells) expressed the human ChAT protein. Since such cells comprised a small proportion (∼20%) of the population, the ChAT activity per neuronal cell was estimated to approach 250–300 pmol ACh/min/mg protein. Cultures composed of >95% NT2-N cells had significantly lower ChAT specific activities and this could be increased by either ciliary neurotrophic factor or leukemia inhibitory factor, but not by nerve growth factor. We conclude that NT2 cells provide a system in which to study the molecular events that underlie neurotransmitter choice during the differentiation of human cholinergic neurons.     

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.