Cellular Acetylcholine Content and Neuronal Differentiation

Abstract

N18TG2 neuroblastoma clone is defective for biosynthetic neurotransmitter enzymes; its inability to establish functional synapses is overcome in the neuroblastoma X glioma 108CC15, where acetylcholine synthesis is also activated. These observations suggest a possible relation between the ability to produce acetylcholine and the capability to advance in the differentiation program and achieve a fully differentiated state. Here, we report the characterization of several clones after transfection of N18TG2 cells with a construct containing a cDNA for rat choline acetyltransferase (ChAT). The ability of these clones to synthesize acetylcholine is demonstrated by HPLC determination on cellular extracts. In the transfected clones, northern blot analysis shows increased expression of mRNAs for a specific neuronal protein associated with synaptic vesicles, synapsin I. Fiber outgrowth of transfected clones is also evaluated to establish whether there is any relation between ChAT levels and morphological differentiation. This analysis shows that the transfected clone 1/2, not expressing ChAT activity, displays a very immature morphology, and its ability to extend fibers also remains rather poor in the presence of "differentiation" agents such as retinoic acid. In contrast, clones 2/4, 3/1, and 3/2, exhibiting high ChAT levels, display higher fiber outgrowth compared with clone 1/2 in both the absence and the presence of differentiating agents.

5-Hydroxytryptamine 1A and 2B Serotonin Receptors in Neurite Outgrowth: Involvement of Early Growth Response Protein 1

“…Cells were also transfected with plasmids containing the following cDNA: ChAT, as previously described [5]; 5-HT 1A and 5-HT 2B receptors [30,31,32]; EGR-1 [13]; and Zn-EGR [27]. In this paper we will refer to the different cell lines as follows: N18TG2 cells = N18; N18TG2 + ChAT gene = N18-ChAT; N18TG2 + 5-HT 1A gene = N18-1A (clones 2 and 10); N18TG2 + 5-HT 2B gene = N18-2B (clones 6 and 7); N18TG2 + ChAT and 5-HT 1A genes = N18-ChAT-1A (clones 7 and 16); N18TG2 + ChAT and 5-HT 2B genes = N18-ChAT-2B (clones 1 and 6); N18TG2 + 5-HT 1A and EGR-1 genes = N18-1A-EGR-1; N18TG2 + Zn-EGR gene = N18-Zn-EGR.…”

“…ChAT activity was measured by a modification of the Fonnum method [38], as previously described [5]. …”

“…In fact, cholinergic and serotonergic neurons are generated early in mouse brain development [3,4]. A later event in neurogenesis is fiber formation, which involves growth factors as well as neurotransmitters such as acetylcholine and serotonin [2,5,6]. Considering the cholinergic system, acetylcholinesterase is expressed in neurons during development where and when cholinergic synapses are not yet present, and a relationship between acetylcholinesterase expression and neuronal differentiation has been demonstrated [7,8,9,10].…”

“…We have previously shown that overexpression of the choline acetyltransferase (ChAT; EC 2.3.1.6) gene in murine neuroblastoma N18TG2 cells, which do not synthesize neurotransmitters [24], induces acetylcholine biosynthesis, the modulation of several neuronal markers and an increase in fiber outgrowth through stimulation of the muscarinic receptors endogenously expressed in N18TG2 cells [5,25]. Biosynthesis and secretion of acetylcholine induce the activation of the muscarinic receptor M 3 subtype and enhance the expression of transcription factor early growth response 1 (EGR-1) [13], which is involved in differentiation and long-term potentiation [26,27].…”

Asymmetric regulation by estrogen at the cholinergic gene locus in differentiated NG108-15 neuronal cells

“…The NG108-15 cells are a hybrid cell line constructed by somatic fusion of rat C6 glioma cells and mouse N18TG2 neuroblastoma cells. N18TG2 cells themselves do not express the differentiated phenotypes in the presence of RA (Bignami et al 1997), but NG108-15 and N18TG2 treated with a medium conditioned over C6 cells can differentiate by retinoic acid into functional cholinergic cells (Diebler et al 1998;Dolezal et al 2001;Castell et al 2002), indicating that the differentiation of N18TG2 neuroblastoma requires the cooperation of C6 glioma. The possibility remains that E 2 acted directly or indirectly on differentiation-inducing factor(s) derived from C6 glioma, and prompted further increase of ChAT gene expression.…”