Choline Acetyltransferase mRNA Research Articles

Localization of choline acetyltransferase and acetylcholine in the chorion of early human pregnancy.

This report concerns the activity of choline acetyltransferase (ChAT), and the localization of ChAT and acetylcholine (ACh) in the human chorion during the 5th to 10th weeks of gestation. Radio-enzyme, immunohistochemical, and in situ hybridization assays were used. We found that ChAT activity increases as a function of gestational age between the 5th and the 9th weeks of pregnancy. At 5 weeks' gestation, ChAT was detected by immunohistochemical means mainly in cytotrophoblast and villous stromal cells, particularly in the cytotrophoblastic shell and cell columns. With the development of blood vessels at the 6th week, their endothelial cells also expressed ChAT. The amount of ChAT immunoreaction product deposits decreased in the villous cytotrophoblast by the 9th and 10th weeks when the layer of the cell became flat. By comparison, ChAT protein was detected only rarely in the syncytiotrophoblast layer during the gestation period studied. The ChAT gene transcript was demonstrated in most of the constitutive cells of the chorion. However, in contrast to the results of the immunohistochemical assays, positive hybridization signals for ChAT mRNA were evenly distributed over both cytotrophoblast and syncytiotrophoblast. On the other hand, the distribution of ACh immunoreaction products was almost coincident with that of ChAT.

Kami-untan-to (KUT) improves cholinergic deficits in aged rats

The Effects of the kampo (Japanese herbal) medicine, kami-untan-to (KUT)' on the central cholinergic system and passive avoidance behavior in aged rats (2 years old) were examined. When KUT was orally administered to aged rats, mean latency was significantly increased on passive avoidance test of step through type and choline acetyltransferase (ChAT) activity was increased in the frontoparietal cortex. Oral administration of KUT also increased ChAT mRNA levels in the basal forebrain and nerve growth factor (NGF) mRNA levels in the frontoparietal cortex as demonstrated by the reverse transcription-polymerase chain reaction (RT-PCR) method. These results suggest that oral administration of KUT improves age-related disturbance of memory via increasing effects of ChAT and NGF. KUT may be useful as a potential therapeutic agent in diseases caused by cholinergic deficit such as senile dementia of the Alzheimer type.

P-970 - Expression of choline acetyltransferase mRNA in T-lymphocytes.

P-970 - Expression of choline acetyltransferase mRNA in T-lymphocytes.

1010 Induction of choline acetyltransferase mRNA in human T-lymphocytes during the immune responses

In this study, we evaluated whether astrocytic and microglial activation mediates below-level neuropathic pain following spinal cord injury. Male Sprague–Dawley (225–250 g) rats were given low thoracic (T13) spinal transverse hemisection and behavioral, electrophysiological and immunohistochemical methods were used to examine the development and maintenance of below-level neuropathic pain. On postoperation day 28, both hind limbs showed significantly decreased paw withdrawal thresholds and thermal latencies as well as hyperexcitability of lumbar (L4-5) spinal wide dynamic range (WDR) neurons on both sides of spinal dorsal horn compared to sham controls (* P<0.05). Intrathecal treatment with propentofylline (PPF, 10 mM) for 7 consecutive days immediately after spinal injury attenuated the development of mechanical allodynia and thermal hyperalgesia in both hind limbs in a dose-related reduction compared to vehicle treatments (* P<0.05). Intrathecal treatment with single injections of PPF at 28 days after spinal injury, attenuated the existing mechanical allodynia and thermal hyperalgesia in both hind limbs in a dose related reduction (* P<0.05). In electrophysiological studies, topical treatment of 10 mM PPF onto the spinal surface attenuated the neuronal hyperexcitability in response to mechanical stimuli. In immunohistochemical studies, astrocytes and microglia in rats with spinal hemisection showed significantly increased GFAP and OX-42 expression in both superficial and deep dorsal horns in the lumbar spinal dorsal horn compared to sham controls (* P<0.05) that was prevented in a dose-related manner by PPF. In conclusion, our present data support astrocytic and microglial activation that contributes to below-level central neuropathic pain following spinal cord injury.

Rat fibroblast growth factor receptor-4 mRNA in the brain is preferentially expressed in cholinergic neurons in the medial habenular nucleus

We examined the cellular localization of fibroblast growth factor receptor (FGFR)-4 mRNA preferentially expressed in the medial habenular nucleus by in situ hybridization. FGFR-4 mRNA was expressed in the ventral part of the medial habenular nucleus. FGFR-4 mRNA was colocalized with choline acetyltransferase mRNA to a high degree (more than 90%), indicating that FGFR-4 mRNA-expressed cells are cholinergic neurons. The present finding indicates that FGFR-4 in the brain has a function specific to cholinergic neurons in the ventral part of the medial habenular nucleus.

Specific Vesicular Acetylcholine Transporter Promoters Lie within the First Intron of the Rat Choline Acetyltransferase Gene

The sequence encoding the vesicular acetylcholine transporter (VAChT) has recently been localized within the first intron of the choline acetyltransferase (ChAT) gene in various species. In rat, we previously identified a class of VAChT mRNAs that may originate from the same promoter region as two ChAT mRNAs. Here, we demonstrate by a detailed analysis of the 5'-noncoding region of the VAChT gene, that two specific VAChT promoters lie within the first intron of the ChAT gene. Two VAChT mRNAs are generated from these promoters. These results demonstrate that the promoter regions of these two genes are intermingled, which highlight the unique organization of the ChAT/VAChT gene locus.

NGF mRNA is expressed by GABAergic but not cholinergic neurons in rat basal forebrain

Nerve growth factor (NGF) supports the survival and biosynthetic activities of basal forebrain cholinergic neurons and is expressed by neurons within lateral aspects of this system including the horizontal limb of the diagonal bands and magnocellular preoptic areas. In the present study, colormetric and isotopic in situ hybridization techniques were combined to identify the neurotransmitter phenotype of the NGF-producing cells in these two areas. Adult rat forebrain tissue was processed for the colocalization of mRNA for NGF with mRNA for either choline acetyltransferase, a cholinergic cell marker, or glutamic acid decarboxylase, a GABAergic cell marker. In both regions, many neurons were single-labeled for choline acetyltransferase mRNA, but cells containing both choline acetyltransferase and NGF mRNA were not detected. In these fields, virtually all NGF mRNA-positive neurons contained glutamic acid decarboxylase mRNA. The double-labeled cells comprised a subpopulation of GABAergic neurons; numerous cells labeled with glutamic acid decarboxylase cRNA alone were codistributed with the double-labeled neurons. These data demonstrate that in basal forebrain GABAergic neurons are the principal source of locally produced NGF.

Increase in alpha-CGRP and GAP-43 in aged motoneurons: a study of peptides, growth factors, and ChAT mRNA in the lumbar spinal cord of senescent rats with symptoms of hindlimb incapacities.

Sprague-Dawley rats develop progressive motor dysfunctions during the third year of life. We use this as a model to examine possible neuronal mechanism(s) that may cause motor impairments occuring during aging. In this study we have used indirect immunofluorescence histochemistry (IF) and in situ hybridization histochemistry (ISH) to study quantitatively and qualitatively the staining pattern and mRNA expression of calcitonin gene-related peptide (alpha-CGRP), growth-associated protein 43 (GAP-43), and acidic fibroblast growth factor (aFGF) in spinal lumbar motoneurons of young adult (2-3 months) and aged (30 months) Sprague-Dawley rats. In addition, mRNAs encoding choline acetyltransferase (ChAT), beta-CGRP, and cholecystokinin (CCK) were analyzed. All aged rats used in this study disclosed symptoms of hindlimb incapacity, ranging from mild weight-bearing insufficiency to paralysis of the hind limbs. The symptoms were confined to the musculature of the hindlimb and hip regions. Only a small number (approximately 15%) of the large motoneurons that innervate the hindlimb muscles were lost in those aged rats that had clinical symptoms of hindlimb motor incapacities. The remaining motoneurons expressed ChAT mRNA at levels similar to those of young adult rats. The vast majority of these motoneurons showed increased mRNA levels for alpha-CGRP and GAP-43. Aged motoneurons contained more CGRP like immunoreactivity (LI), but the number of immunoreactive neurons was smaller than in adult rats. GAP-43-LI could be detected in motoneurons in aged, but not in adult, rats. GAP-43-LI was always colocalized with CGRP-LI in aged motoneurons. Studies of individual aged rats revealed that the increase of GAP-43 mRNA-positive cell bodies occurred in cases with the most severe clinical symptoms, whereas the increase in alpha-CGRP was even evident in rats with mild symptoms. No alterations in content of aFGF-LI or aFGF mRNA could be detected in the aged rat, and the content of CCK and beta-CGRP mRNAs was also normal. The usefulness of this rat model for studies of neuromuscular aging and possible functional roles for GAP-43 and CGRP in plastic and regenerative processes during aging are discussed.

Fibroblast growth factors regulate calcitonin gene-related peptide mRNA expression in rat motoneurons after lesion and in culture.

In this study, we have investigated the effect of fibroblast growth factors (bFGF and FGF-5) and brain derived neurotrophic factor (BDNF) on the expression of calcitonin gene-related peptide (CGRP) in rat motoneurons in vivo and in vitro. Following sciatic nerve transection in adult rats, the levels of alpha-CGRP and beta-CGRP mRNA were up- and down-regulated respectively in axotomized motoneurons, revealed by in situ hybridization histochemistry. Local administration of 1 microgram bFGF was able to entirely abolish the up-regulation of alpha-CGRP mRNA, and to further down-regulate beta-CGRP. These effects, albeit less pronounced, were still evident with 0.2 micrograms bFGF. In contrast, bFGF did not attenuate the lesion-induced decrease of choline acetyltransferase (ChAT) mRNA. Administration of BDNF did not significantly alter the expression of CGRP or ChAT mRNA in axotomized motoneurons. Both alpha- and beta-CGRP mRNAs could be detected by PCR in enriched motoneuron cultures prepared from rat embryos at embryonic day 14-15. Comparing the amplification of alpha- and beta-CGRP mRNAs with that of mRNA encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in parallel samples, we found that cultures treated with FGF-5 had a lower ratio of alpha- and beta-CGRP mRNA to GAPDH mRNA, than did control or BDNF-treated cultures. BDNF, on the other hand increased alpha-CGRP and decreased beta-CGRP mRNA levels, though these effects were moderate compared with the effects of FGF-5. The results obtained in this study suggest that members of the FGF family of growth factors influence the expression of CGRP in rat motoneurons, and that the increase of this neuropeptide induced by axotomy may, at least in part, be due to deprivation of these target-derived factors.

Coregulation of Two Embedded Gene Products, Choline Acetyltransferase and the Vesicular Acetylcholine Transporter

The gene encoding the vesicular acetylcholine transporter (VAChT) has recently been localized within the first intron of the gene encoding choline acetyltransferase (ChAT) and is in the same transcriptional orientation. These two genes, whose products are required for the expression of the cholinergic phenotype, could therefore be coregulated. We thus tested the effects on VAChT gene expression of the cholinergic differentiation factor/leukemia inhibitory factor and retinoic acid, both of which induce ChAT activity and increase ChAT mRNA levels in cultured sympathetic neurons. These factors increased both the number of binding sites for vesamicol, a specific ligand of VAChT, and VAChT immunoreactivity. This increase in the number of VAChT molecules resulted from an increase in the amount of VAChT mRNA, as assessed by reverse transcription-PCR and which paralleled that of ChAT mRNAs. These data suggest a functional role for ChAT and VAChT gene organization and are consistent with the existence of a coregulatory mechanism for the embedded ChAT and VAChT genes.

Adenosine A 2A receptors stimulate acetylcholine release from nerve terminals of the rat hippocampus

The nature of the adenosine receptors involved in the enhancement of acetylcholine release in the hippocampus was studied. The A 2A agonist, CGS 21680, increased the veratridine-evoked release of [ 3H]acetylcholine from hippocampal synaptosomes. This presynaptic effect of CGS 21680 was greater at 3–30 nM than at 100 nM. The excitatory effect of CGS 21680 was antagonised by the A 2 antagonist, DMPX (10μM), and by the A 2A antagonist, CSC (200 nM), but not by the A 1 antagonist, DPCPX (20 nM). We also found co-expression of A 2A and choline acetyltransferase mRNAs in the nucleus of the diagonal band and the medial septum, where the cholinergic cell bodies that project into the hippocampus are located. These results indicate that A 2A adenosine receptors are present in cholinergic nerve terminals in the hippocampus and that activation of these receptors enhances acetylcholine release.

Identified cholinergic neurones in the adult rat brain are enriched in GAP-43 mRNA: a double in situ hybridisation study

The cellular expression of growth associated protein-43 mRNA by identified choline acetyl transferase mRNA positive cells was investigated in the mature rat brain using a combined radioactive and non-radioactive in situ hybridisation technique. Cellular sites of growth associated protein-43 mRNA were detected using a 35S-oligonucleotide while choline acetyl transferase mRNA positive neurones were identified using two alkaline phosphatase-labelled probes. In the cholinergic cells of the corpus striatum, basal forebrain and laterodorsal tegmental nucleus a specific growth associated protein-43 hybridisation signal (silver grains) was detected, demonstrating that these choline acetyl transferase mRNA positive cells are enriched in growth associated protein-43 gene transcripts. By contrast, the large cholinergic cells of the motor nucleus of the trigeminal nerve did not express growth associated protein-43 mRNA. Quantification of the growth associated protein-43 hybridisation signal expressed by identified choline acetyl transferase mRNA positive cells showed regional variations in the relative cellular abundance of this transcript; cholinergic cells in the laterodorsal tegmental nucleus and corpus striatum expressed the strongest cellular hybridisation signal. Mean cross-sectional somatic area measurements of these growth associated protein-43/cholinergic positive cells confirmed the identity of these neurones as belonging to the cholinergic phenotype. A strong 35S-growth associated protein-43 hybridisation signal was detected also in numerous other non-choline acetyl transferase mRNA positive nerve cells in other regions of the brain, although the chemical phenotypes of these neurones were not determined. Our data reveal that expression of the growth-associated protein GAP-43 is maintained in identified cholinergic neurones in the postnatal rat brain, suggesting that this protein may subserve important functions in cholinergic and other neurones of the adult mammalian brain.

All-trans- and 9-cis-retinoic acid enhance the cholinergic properties of a murine septal cell line: evidence that the effects are mediated by activation of retinoic acid receptor-alpha.

We investigated the effects of retinoids on the cholinergic properties of a murine septal cell line, SN56. Treatment of the cells with all-trans-retinol (vitamin A), all-trans-retinal, all-trans-retinoic acid (t-RA), 9-cis-retinoic acid (9c-RA), or 13-cis-retinoic acid caused time- and concentration-dependent increases in choline acetyltransferase activity (up to 3.4-fold) and in intracellular acetylcholine levels (up to 2.5-fold, with respective EC50 values of 68, 50, 18, 15, and 56 nM). Furthermore, treatment with either t-RA or 9c-RA at 1 microM for 48 h resulted in an increase in the expression of choline acetyltransferase mRNA by threefold that of controls. These data and the presence of putative retinoic acid response elements in the 5' region of the murine choline acetyltransferase gene indicate that retinoids stimulate choline acetyltransferase transcription in murine cholinergic neurons. No additivity or synergism was observed between the effects of t-RA and 9c-RA on any of these cholinergic properties of SN56 cells, suggesting a common mechanism of action of the two retinoids. However, a combined treatment with t-RA and forskolin, which activates adenylate cyclase, resulted in an additive increase in acetylcholine content. Using an antagonist selective for the retinoic acid receptor-alpha subtype, Ro 41-5253, we found that the effects of t-RA and 9c-RA on acetylcholine levels were abolished. An agonist selective for retinoic acid receptor-alpha, Ro 40-6055, increased acetylcholine levels to a similar extent as t-RA and 9c-RA, and this effect was blocked by the antagonist.(ABSTRACT TRUNCATED AT 250 WORDS)

Patterns of Presynaptic Gene Expression Define Two Stages of Synaptic Differentiation

To elucidate the sequence of molecular events leading to nerve terminal differentiation, we have examined the regulation of expression of presynaptic protein genes during synapse formation in vivo. In the chick ciliary ganglion (CG), synaptophysin IIa and synaptophysin IIb mRNAs showed threefold increases relative to neurofilament-M mRNA during the time of target contact [Embryonic Day 7 (E7)-E9]. Expression of synaptotagmin I mRNA also increased severalfold over this time interval. Thus, mRNAs for three synaptic vesicle proteins are upregulated coordinately during synaptogenesis. In contrast, the major increase in choline acetyltransferase (ChAT) mRNA (four-to fivefold) occurred between E15 and E20, coincident with the maturation of synapses in the CG. Coincident with ChAT upregulation, there is a switch in the relative abundance of mRNAs encoding vesicle protein isoforms. In particular, mRNAs encoding synaptophysin IIb and synaptotagmin II (which is undetectable at E9) become predominant. Therefore, although synaptic vesicle protein mRNAs are upregulated in a first phase of differentiation at the time of synapse formation, a temporally distinct phase of presynaptic protein gene regulation, associated with the specific maturation of synapses, is also apparent.

Coordinate expression of vesicular acetylcholine transporter and choline acetyltransferase in sympathetic superior cervical neurones.

The neurotransmitter acetylcholine is synthesized by choline acetyltransferase (ChAT) and transported into synaptic vesicles by the vesicular acetylcholine transporter (VAChT). Recently it has been reported that the entire coding region of VAChT mRNA is located in the first intron of the ChAT gene. In this study, ChAT and VAChT mRNAs were analysed in cultured sympathetic neurones. Cholinergic differentiation factor/leukaemia inhibitory factor and ciliary neurotrophic factor induced strong expression of ChAT and VAChT mRNAs in parallel. RT-PCR analysis of ChAT mRNAs revealed that five types of ChAT transcripts which differed in the 5' non coding regions were increased. RT-PCR analysis of VAChT mRNA indicated that the cytokines induced only VAChT mRNA species which did not contain the R-exon, and not those containing the R-exon. The results indicate that ChAT and VAChT expressions are coordinately but differentially regulated in cultured sympathetic neurones.

AMPA-induced lesions of the basal forebrain differentially affect cholinergic and non-cholinergic neurons: lesion assessment using quantitative in situ hybridization histochemistry.

The direct and transynaptic effects of lesions of the basal forebrain induced by alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and ibotenic acid were investigated using quantitative in situ hybridization histochemistry. Probes complementary to the sequences of choline acetyltransferase mRNA, glutamate decarboxylase mRNA and preproenkephalin mRNA were used to assess direct lesion effects within the basal forebrain and probes for postsynaptic M-1 and M-3 muscarinic receptors were used to assess long-term changes in neocortical muscarinic receptor mRNA expression following cholinergic deafferentation. AMPA-induced basal forebrain lesions destroyed significantly more neurons that expressed choline acetyltransferase mRNA than ibotenic acid-induced lesions (90 versus 60%), but significantly fewer neurons which expressed either glutamate decarboxylase or preproenkephalin mRNA (61 versus 83% reduction in glutamate decarboxylase mRNA and 56 versus 79% reduction in preproenkephalin mRNA). AMPA-induced lesions did, however, destroy a significant proportion of the neurons which expressed glutamate decarboxylase and preproenkephalin mRNA (approximately 60%). The neurons spared following AMPA-induced lesions were typically situated dorsolaterally within the dorsal pallidum, although neurons expressing glutamate decarboxylase or preproenkephalin mRNA were frequently observed within the areas of greatest cholinergic neuronal loss, i.e. the region of the nucleus basalis magnocellularis. These findings suggest that there is a population of non-cholinergic pallidal neurons which are insensitive to AMPA but not to ibotenic acid, reflecting a possibly heterogeneous distribution of NMDA and non-NMDA subtypes of glutamate receptors within the rat basal forebrain. AMPA-induced lesions of the basal forebrain were, however, without significant effect on the levels of expression of M-1 and M-3 muscarinic receptor mRNAs in the cerebral neocortex.

Regulation of TrkA and ChAT expression in developing rat basal forebrain: evidence that both exogenous and endogenous NGF regulate differentiation of cholinergic neurons

TrkA is a receptor tyrosine kinase whose activation transduces NGF signaling. TrkA expression has been demonstrated in NGF-responsive adult basal forebrain cholinergic neurons (BFCNs). Several lines of evidence have suggested that endogenous NGF plays a role in the development and differentiation of these neurons. We examined TrkA expression during development. TrkA mRNA and protein were present in basal forebrain neurons during the entire postnatal period; the distribution of neurons bearing these markers was identical to that for those containing choline acetyltransferase (ChAT) mRNA, suggesting that, as in the adult, TrkA gene expression is localized to BFCNs. The expression of TrkA and ChAT followed a very similar temporal pattern, suggesting regulation by the same factor(s). We discovered that NGF administration in vivo activated TrkA receptors, and increased both TrkA and ChAT mRNA; conversely, anti-NGF infusions suppressed expression of both genes. These results suggest that endogenous NGF regulates expression of TrkA and ChAT. Finally, while NGF infusion increased the size of developing BFCNs, NGF antibodies inhibited the normal developmental increase. The results are evidence that endogenous NGF acts on developing BFCNs to enhance gene expression and cellular differentiation.

The effect of mercury vapour on cholinergic neurons in the fetal brain: studies on the expression of nerve growth factor and its low- and high-affinity receptors

The effects of mercury vapour on the production of nerve growth factor during development have been examined. Pregnant rats were exposed to two different concentrations of mercury vapour during either embryonic days E6–E11 (early) or E13–E18 (late) in pregnancy, increasing the postnatal concentration of mercury in the brain from 1 ng/g tissue to 4 ng/g tissue (low-dose group) or 11 ng/g (high-dose group). The effect of this exposure in offspring was determined by looking at the NGF concentration at postnatal days 21 and 60 and comparing these levels to age-matched controls from sham-treated mothers. Changes in the expression of mRNA encoding NGF, the low- and high-affinity receptors for NGF (p75 and p140 trk, respectively) and choline acetyltransferase (ChAT) were also determined. When rats were exposed to high levels of mercury vapour during early embryonic development there was a significant (62%) increase in hippocampal NGF levels at P21 accompanied by a 50% decrease of NGF in the basal forebrain. The expression of NGF mRNA was found to be unaltered in the dentate gyrus. The expression of p75 mRNA was significantly decreased to 39% of control levels in the diagonal band of Broca (DB) and to approximately 50% in the medial septal nucleus (MS) whereas no alterations in the level of trk mRNA expression were detectable in the basal forebrain. ChAT mRNA was slightly decreased in the DB and MS, significantly in the striatum. These findings suggest that low levels of prenatal mercury vapour exposure can alter the levels of the NGF and its receptors, indicating neuronal damage and disturbed trophic regulations during development.

Expression of the Choline Acetyltransferase Gene Depends on Protein Kinase A Activity

Choline acetyltransferase activity is barely detectable in a mutant pheochromocytoma PC12 cell line, A123.7, which is deficient in protein kinase A activity. Northern blot and polymerase chain reaction analyses showed that this mutant cell line has dramatically reduced levels of choline acetyltransferase mRNA, which correlates with the low level of enzyme activity. Transient transfection analysis was used to assess the functionality, in these cells, of an enhancer element and a cholinergic-specific repressor element derived from the human choline acetyltransferase gene. The results show that the enhancer element is inactive in the protein kinase A-deficient cell line. Cotransfection experiments with plasmids expressing the catalytic subunit of protein kinase A support this conclusion. These data indicate that protein kinase A regulates expression of the choline acetyltransferase gene at the transcriptional level by controlling the activity of an enhancer element.

Expression of Choline Acetyltransferase mRNA and Protein in T-Lymphocytes.

Acetylcholine (ACh) is present in human blood and localized in T-lymphocytes which exhibit ACh-synthesizing activity. As the final step towards clarifying the origin of ACh present in the blood, we elucidated the enzyme involved in ACh synthesis in T-lymphocytes by detecting the mRNA for choline acetyltransferase (ChAT), which is known to catalyze ACh synthesis in the nervous system. The ChAT mRNA in the MOLT-3 human leukemic T-cell line was amplified by reverse transcription-polymerase chain reaction (RT-PCR) using specific order and reverse primers. A single specific RT-PCR product was observed on agarose gel. The sequence of the RT-PCR product of MOLT-3 was completely identical with nucleotide positions 322-973 of human brain ChAT cDNA. Western blot analysis with an antibody to ChAT confirmed the presence of ChAT protein in MOLT-3. These findings demonstrate that mRNA for the same ChAT as that in the nervous system is expressed in T-lymphocytes, and indicate that ACh synthesized by ChAT in T-lymphocytes is the origin of ACh in the blood.