Estrogen-mediated regulation of cholinergic expression in basal forebrain neurons requires extracellular-signal-regulated kinase activity.

Beyond the role estrogen plays in neuroendocrine feedback regulation involving hypothalamic neurons, other roles for estrogen in maintaining the function of CNS neurons remains poorly understood. Primary cultures of embryonic rat neurons together with radiometric assays were used to demonstrate how estrogen alters the cholinergic phenotype in basal forebrain by differentially regulating sodium-coupled high-affinity choline uptake and choline acetyltransferase activity. High-affinity choline uptake was significantly increased 37% in basal forebrain cholinergic neurons grown in the presence of a physiological dose of estrogen (5 nM) from 4 to 10 days in vitro whereas choline acetyltransferase activity was not significantly changed in the presence of 5 or 50 nM estrogen from 4 to 10 or 10 to 16 days in vitro. Newly-synthesized acetylcholine was significantly increased 35% following 6 days of estrogen treatment (10 days in vitro). These effects are in direct contrast to those found for nerve growth factor; that is, nerve growth factor can enhance the cholinergic phenotype through changes in choline acetyltransferase activity alone. This is most surprising given that mitogen-activated protein kinase and extracellular-signal-regulated kinase1/2, kinases also activated in the signaling pathway of nerve growth factor, were found to participate in the estrogen-mediated changes in the cholinergic phenotype. Likewise, general improvement in the viability of the cultures treated with estrogen does not account for the effects of estrogen as determined by lactate dehydrogenase release and nerve growth factor-responsiveness. These findings provide evidence that estrogen enhances the differentiated phenotype in basal forebrain cholinergic neurons through second messenger signaling in a manner distinct from nerve growth factor and independent of improved survival.

Molecular mechanisms regulating NGF-mediated enhancement of cholinergic neuronal phenotype: c-fos trans-activation of the choline acetyltransferase gene.

Nerve growth factor (NGF) enhances expression of the cholinergic phenotype observed as increased choline acetyltransferase (ChAT) activity, immunoreactivity, and mRNA. In the present study, treatment of cultured rat embryonic basal forebrain neurons with anti-c-fos, prior to administering NGF, blocked NGF-mediated increases in ChAT activity by 67%; basal ChAT activity was not affected by the antisense oligonucleotide treatment. Reverse transcription-polymerase chain reaction (RT-PCR) revealed that anti-c-fos treatment resulted in not only blockade but enhancement of steady-state ChAT mRNA at different time points. These data suggest that c-fos is an important component in NGF-mediated changes in the cholinergic phenotype and support the hypothesis that c-fos plays a role in the regulation of transcription of the ChAT gene. Elucidation of mechanisms underlying this regulation may aid drug development in neurodegenerative disease.

Optimization of serum-free culture conditions for growth of embryonic rat cholinergic basal forebrain neurons.

The objective of the present study was to optimize conditions for culturing embryonic rat basal forebrain neurons in serum-free defined medium to be used in investigations of cholinergic neuron function and responsiveness to neurotrophic factors. It was determined that a combination of neurobasal medium (NB) and DMEM/F12 medium (DM:F12) maintained culture viability, basal choline acetyltransferase (ChAT) activity and responsiveness of these neurons to nerve growth factor (NGF) better than growth of neurons in either medium alone; all media tested contained N2 supplements. While NB which was developed initially for culturing embryonic rat hippocampal neurons supported the growth of basal forebrain neurons, they had reduced ChAT activity and did not respond to NGF with enhanced cholinergic neuronal enzyme activity. On the other hand, DM:F12 did not consistently support survival of the neurons until assay of ChAT activity on day 6 in vitro; surviving cultures were compromised in their cholinergic capacity either under basal or NGF-enhanced conditions. Cultures grown in the combined media responded to brain-derived neurotrophic factor (BDNF), but not ciliary neurotrophic factor (CNTF), at concentrations up to 100 ng/ml with increased ChAT activity as predicted from the literature. These findings suggest that the nutrient composition of the medium is important in promoting expression of the cholinergic neuronal phenotype and that growth factor supplementation alone is insufficient to compensate for inadequate nutrient composition.

NGF-induction of the expression of ChAT mRNA in PC12 cells and primary cultures of embryonic rat basal forebrain.

The objective of this study was to examine the role of nerve growth factor (NGF) in regulation of expression of the cholinergic phenotype. NGF was administered to PC12 cells or primary cultures of embryonic (E17) rat basal forebrain for 2 days, then steady-state levels of choline acetyltransferase (ChAT) mRNA was monitored. Expression of ChAT mRNA isoforms was investigated using reverse transcription-polymerase chain reaction (RT-PCR) to amplify different upstream regions of the ChAT transcripts, and Southern blot analysis was used to verify identity of the PCR products. An NGF-induced increase of 1.8- and 1.5-fold in steady-state level of the ChAT transcript containing the M-exon (M-ChAT) was observed in PC12 cells and embryonic rat basal forebrain neurons, respectively. Also, a 2-fold increase in ChAT protein as determined by western blot analysis was associated with an NGF-mediated increase of 1.7-fold in ChAT activity in rat basal forebrain neurons within the same cultures following 4 days of NGF treatment.

Differential effects of nerve growth factor on expression of choline acetyltransferase and sodium-coupled choline transport in basal forebrain cholinergic neurons in culture.

Nerve growth factor (NGF) treatment of primary cultures of embryonic day 17 rat basal forebrain differentially altered activity of choline acetyltransferase (ChAT) and high-affinity choline transport; ChAT specific activity was increased by threefold in neurons grown in the presence of NGF for between 4 and 8 days, whereas high-affinity choline transport activity was not changed relative to control. Dose-response studies revealed that enhancement of neuronal ChAT activity occurred at low concentrations of NGF with an EC50 of 7 ng/ml, with no enhancement of high-affinity choline transport observed at NGF concentrations up to 100 ng/ml. In addition, synthesis of acetylcholine (ACh) and ACh content in neurons grown in the presence of NGF for up to 6 days was increased significantly compared with controls. These results suggest that regulation of ACh synthesis in primary cultures of basal forebrain neurons is not limited by provision of choline by the high-affinity choline transport system and that increased ChAT activity in the presence of NGF without a concomitant increase in high-affinity choline transport is sufficient to increase ACh synthesis. This further suggests that intracellular pools of choline, which do not normally serve as substrate for ACh synthesis, may be made available for ACh synthesis in the presence of NGF.