Choline Acetyltransferase mRNA Levels Research Articles

Autonomic microganglion cells: a source of acetylcholine in the rat carotid body.

Hypoxic chemosensitivity of peripheral arterial chemoreceptors and the ventilatory response to O2 deprivation increases with postnatal development. Multiple putative neurotransmitters, which are synthesized in the carotid body (CB), are thought to mediate signals generated by hypoxia. Acetylcholine (ACh) is believed to be a major excitatory neurotransmitter participating in hypoxic chemosensitivity. However, it is not known whether ACh originates from type I cells in the CB. In these studies, we tested the hypothesis that choline acetyltransferase (ChAT) and vesicular ACh transporter (VAChT) mRNAs are expressed in the CB and that mRNA levels would increase with postnatal maturation or exposure to hypoxia. Semiquantitative in situ hybridization histochemistry and immunohistochemistry were used to localize cholinergic markers within neurons and cells of the rat CB, the nodose-petrosal-jugular ganglion complex, and the superior cervical ganglion up to postnatal day 28. We show that the pattern of distribution, in tissue sections, is similar for both ACh markers; however, the level of VAChT mRNA is uniformly greater than that of ChAT. VAChT mRNA and immunoreactivity are detected abundantly in the nodose-petrosal-jugular ganglion complex in a number of microganglion cells embedded in nerve fibers innervating the CB for all postnatal groups, whereas ChAT mRNA is detected in only a few of these cells. Contrary to our hypothesis, postnatal maturation caused a reduction in ACh trait expression, whereas hypoxic exposure did not induce the upregulation of VAChT and ChAT mRNA levels in the CB, microganglion, or within the ganglion complex. The present findings indicate that the source of ACh in the CB is likely within autonomic microganglion cells and cholinergic nerve terminals.

Nigral GABAergic inhibition upon cholinergic neurons in the rat pedunculopontine tegmental nucleus.

We investigated, in a midbrain parasagittal slice preparation of Wistar rats (postnatal day 9-17), the synaptic inhibition of neurons in the pedunculopontine tegmental nucleus (PPN), which was mediated by gamma (gamma)-amino-butyric acid (GABA). Whole-cell patch-clamp recording was used, in combination with a single-cell reverse transcription-polymerase chain reaction amplification technique, to record synaptic potentials and to identify the phenotype of the recorded PPN neuron. In the presence of the ionotropic glutamate receptor antagonists, 6-cyano-2, 3-dihydroxy-7-nitro-quinoxaline-2, 3, dione, and dl-2-amino-5-phosphonovaleric acid, single electrical stimuli were applied to the substantia nigra pars reticulata (SNr), one of the basal ganglia output nuclei. Stimulation of the SNr evoked inhibitory postsynaptic potentials (IPSPs) in 73 of the 104 neurons in the PPN. The IPSPs were abolished with a GABAA receptor antagonist, bicuculline. Inhibitory postsynaptic currents of the neurons were reversed in polarity at approximately -93.5 mV, which was close to the value of the equilibrium potential for chloride ions of -88.4 mV. Single-cell reverse transcription-polymerase chain reactions revealed that approximately 30% (9/32) of the PPN neurons that received inhibition from the SNr expressed detectable levels of choline acetyltransferase mRNA. These findings show that output from the SNr regulates the activity of cholinergic PPN neurons through GABAA receptors.

Effects of ageing and long-term hormone replacement on cholinergic neurones in the medial septum and nucleus basalis magnocellularis of ovariectomized rats.

Ovariectomized aged rats, some of which received long-term hormone replacement with oestrogen or oestrogen plus progesterone, were evaluated for the number and size of basal forebrain cholinergic neurones, as well as relative levels of choline acetyltransferase (ChAT) and trkA mRNA, in order to determine whether effects on basal forebrain cholinergic cell survival and function correspond with differences in cognitive performance previously described. The results show that ageing combined with long-term loss of ovarian function produced substantial reductions in the levels of ChAT and trkA mRNA in the medial septum and nucleus basalis magnocellularis, relative to much younger ovariectomized controls. In contrast, no significant effects on the number or size of the cholinergic cells were detected, indicating that loss of ovarian function does not cause a loss of cholinergic neurones with age. Long-term hormone replacement had no apparent effect on the number of ChAT-positive neurones detected, and did not prevent the reductions in ChAT and trkA mRNA associated with ovariectomy and ageing. Collectively, the data suggest that ageing combined with long-term loss of ovarian function has a severe negative impact on basal forebrain cholinergic function, but not on cholinergic cell survival per se.

Exploring the regulation of the expression of ChAT and VAChT genes in NG108-15 cells: implication of PKA and PI3K signaling pathways.

Involvement of different protein kinases regulated by cAMP and implication of muscarinic receptors in the regulation of choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT) mRNA levels and ChAT activity has been studied in NG108-15 cells. Dibutyryl cAMP enhanced both ChAT and VAChT mRNA levels and stimulated ChAT activity. Muscarinic stimulation or inhibition did not change ChAT activity or the receptor subtype mRNA pattern. MEK1/2 did not affect the regulation of ChAT and VAChT mRNA levels. However, PKA plays a major role in regulating ChAT and VAChT mRNA levels, because H89 decreased both. Strikingly, inhibition of PI3K by LY294002 had two opposite effects: ChAT mRNA level was decreased and VAChT mRNA level was increased. Such a result consolidates the observation that ChAT and VAChT genes, despite their unusual organization in a single "cholinergic locus," can be differentially or synergistically regulated, depending on the activated signaling pathways.

More than one way to toy with ChAT and VAChT

Expression of choline acetyltransferase (ChAT) and of the vesicular acetylcholine transporter (VAChT) is required for the acquisition and the maintenance of the cholinergic phenotype. The ChAT and VAChT genes have been demonstrated to share a common gene locus and this suggests a coordinate regulation of their expression. In the present work, we examined the effects of several differentiating treatments on the modulation of ChAT and VAChT expression at the mRNA and protein levels in growing and differentiating NG108-15 cells. In cells grown in the presence of serum, all the agents tested—retinoic acid, dexamethasone and dibutyrylcyclicAMP (dbcAMP)—induced an increase of ChAT and VAChT mRNA levels but with different efficacy. Treatment with dbcAMP plus dexamethasone resulted in the largest increase of VAChT mRNA amount while retinoic acid mostly enhanced ChAT mRNA level. However, while ChAT activity was increased by all agents, no change in the VAChT protein level was detected. In cells differentiated by serum deprivation, only retinoic acid was effective in inducing an increase of VAChT and ChAT mRNA and ChAT activity, while we observed a downregulation by dbcAMP and dexamethasone. Treatment with the antimitotic agent cytosine arabinoside led to an increase of ChAT activity which was further largely enhanced by the addition of dbcAMP plus dexamethasone, but to only a slight change in VAChT activity. We further showed that complex glycosylation processes which might play a role in targeting and/or stability of the membrane protein VAChT are deficient in these cells. Indeed, in transient transfection assays with the reporter soluble enzyme luciferase placed under regulatory and promoter regions of the VAChT gene, we observed a modulation of luciferase expression by differentiating agents. These data illustrate the complexity of the processes which participate to the expression of the ChAT and VAChT genes, both at the transcriptional and posttranslational levels.

Thyroid hormone and retinoids affect motoneuron phenotype and reaction after axotomy in the spinal cord of adult rats

Motoneuron phenotype in the spinal cord is regulated by an intrinsic genetic program, extrinsic environmental signals and target-derived molecules. Axonal lesions trigger a phenotype switch to foster repair phenomena and axonal re-growth. We have investigated the influence of the long-term treatment with thyroid hormone and all trans retinol palmitate (RA) on motoneuron phenotype and spinal cord reaction to axotomy in adult male rats. Neurochemical markers, investigated by in situ hybridization and immunocytochemistry, included choline acetyltransferase (ChAT), calcitonin gene-related peptide (CGRP) and neurotrophin low affinity receptor p75. Treatment was administered for 56 days and then mid-thigh sciatic axotomy was performed on a number of animals from each experimental groups; the rats were examined 9 days after surgery. The results indicate that: (1) Number and size of ChAT-immunoreactive neurons in the lumbar tract of the spinal cord was reduced in hypothyroid compared to control rats, whereas steady-state level of ChAT mRNA in labelled motoneurons failed to be modified by hypo and hyperthyroidism, but was increased by RA administration; (2) none of the administered treatments did alter CGRP mRNA level, whereas all of them influenced the axotomy-induced changes of motoneuron phenotype; (3) in hyperthyroid rats ChAT mRNA level of lumbar motoneurons not reduced homolateral to lesion while the number of ChAT-IR profiles was pronouncedly reduced; (4) up-regulation of p75 induced by peripheral nerve lesion was reduced in RA-treated rats. These data indicate that the motoneuron phenotype is regulated by transcription factors, which also play a role in phenotype switch regulation after axotomy.

Stimuli that induce a cholinergic neuronal phenotype of NG108-15 cells upregulate ChAT and VAChT mRNAs but fail to increase VAChT protein

The vesicular acetylcholine transporter (VAChT) and choline acetyltransferase (ChAT) are encoded by genes organized in a single gene locus, and coregulation of the transcription of the two genes has been repeatedly reported in cholinergic tissues. In the present study, different stimuli were used to induce the differentiation of the hybridoma cells NG108-15 and we examined their effects on the modulation of VAChT and ChAT expression at the mRNA and protein levels. All agents upregulated the VAChT and ChAT mRNA levels, but to a different extent. ChAT activity was increased by retinoic acid, dexamethasone, and dibutyrylcyclic AMP (dbcAMP), and a synergistic effect was observed with a combined dexamethasone and dbcAMP treatment. Nonetheless, no changes in the VAChT protein level could be observed, as judged from ligand binding studies as well as from immunochemical detection. Hemicholinium-3-sensitive choline uptake, hemicholinium-3 binding, and acetylcholine content were increased by differentiating agents, with a rank order of potency comparable to their effects on ChAT activity. Prominent changes were observed in the expression of vesicular protein markers, particularly with the associated treatment dexamethasone and dbcAMP. Thus, it appears that although the different stimuli we have been using are able to stimulate neuronal features and activate the transcription of cholinergic genes, they did not contrive to increase the level of VAChT protein in these cells.

Upregulation of Tyrosine Hydroxylase and Downregulation of Choline Acetyltransferase in Lead-Exposed PC12 Cells: The Role of PKC Activation

The effects of lead (Pb) on the expression of tyrosine hydroxylase (TH) and choline acetyltransferase (ChAT) were compared in relation to Pb-activation of cPKC in the PC12 cells. Exposure to 0.53 μM Pb (0.1094 ppm) increased TH but reduced ChAT activity and mRNA levels. The increase of TH activity was detectable as early as 0.5 h of exposure, reached a maximum 150% of control after 2 h, and then diminished to a steady state 135% of control between 12 and 48 h of exposure. The decrease of ChAT activity was first detectable after 2 h of Pb exposure, reached a 45% reduction after 6 h, and remained stable thereafter through 48 h of exposure. PKC activity increased 200% after 2 h and then reverted to control levels by 48 h of Pb exposure. The increase of TH activity after 2 h but not 48 h of exposure exceeded that of its mRNA. PKC inhibitor Rö32-0342 suppressed TH activity increase after 2 h of Pb exposure by 80% without affecting TH mRNA. The decrease of ChAT activity correlated with the reductions in steady-state ChAT mRNA levels at 2 and 48 h of Pb exposure and Rö32-0342 had no effect on the Pb-induced decrease of either ChAT activity or mRNA. These results demonstrate that Pb alters TH and ChAT expression in PC12 cells in a reciprocal manner, i.e., upregulates the former but downregulates the latter. PKC is not involved in Pb-induced downregulation of ChAT but does mediate the early phase of Pb-induced augmentation of TH activity, presumably through postranslational modification (phosphorylation) of the enzyme. However, this effect is short-lived due to downregulation of PKC in the course of prolonged (48 h) Pb exposure. It is concluded that, in the course of prolonged exposure, both upregulation of TH and downregulation of ChAT reflect primarily the effects of Pb at the level of gene expression through mechanisms that are not related to Pb activation of PKC.

Cholinergic differentiation triggered by blocking cell proliferation and treatment with all- trans-retinoic acid

This study determined whether the effect of all- trans-retinoic acid ( t-RA) on markers of cholinergic differentiation in a murine septal cell line, SN56.B5.G4, differed depending upon the cell’s proliferative status. To develop a model of non-proliferating cells, aphidicolin, a DNA α-polymerase inhibitor, was used. Cessation of proliferation by aphidicolin increased intracellular choline and acetylcholine (ACh) levels in the absence of change to choline acetyltransferase (ChAT) activity and mRNA and vesicular ACh transporter (VAChT) mRNA. Importantly, the response to t-RA differed depending upon proliferative status. Consistent with previous reports, t-RA increased ChAT and VAChT mRNA, ChAT activity and intracellular ACh levels in proliferating SN56 cells with no effect on intracellular choline levels. When cells were treated with t-RA while undergoing proliferative arrest, an additive effect of combined treatment was observed on ACh levels; nevertheless, this was only accompanied by an increase in choline levels, VAChT and ChAT mRNAs, but not ChAT activity. Indeed, aphidicolin treatment completely suppressed the t-RA-induced increase in ChAT activity observed in proliferating cells. To explore the response to t-RA in post-mitotic cells, a sequential treatment of aphidicolin and t-RA was employed. t-RA treatment was ineffective in increasing ACh and choline levels, over and above that observed with the aphidicolin treatment alone. Comparable to the combined treatment, sequential treatment lead to an increase in ChAT mRNA without any increase in ChAT activity. In conclusion, both the magnitude and the mechanism(s) of action whereby t-RA enhances the cholinergic phenotype of SN56 cells is dependent upon the cell’s proliferative status.

Cortical and nigral deafferentation and striatal cholinergic markers in the rat dorsal striatum: different effects on the expression of mRNAs encoding choline acetyltransferase and muscarinic m1 and m4 receptors.

The regulation of the striatal m1 and m4 muscarinic receptor mRNA as well as the choline acetyltransferase (ChAT) mRNA expression by nigral dopaminergic and cortical glutamatergic afferent fibres was investigated using quantitative in situ hybridization histochemistry. The effects induced by a unilateral lesion of the medial forebrain bundle and a bilateral lesion of the sensorimotor (SM) cortex were analysed in the dorsal striatum 3 weeks after the lesions. Dopaminergic denervation of the striatum resulted in a marked decrease in the levels of m4 mRNA throughout the striatum, while the levels of muscarinic m1 mRNA and ChAT mRNA in cholinergic neurons were unaffected by the lesion. In contrast, following bilateral cortical ablation, the levels of the muscarinic m1 mRNA were significantly increased in the striatal projection area of the SM cortex, whereas the expression of m4 mRNA remained unchanged. Single cholinergic cell analysis by computer-assisted grain counting revealed a decreased labelling for ChAT mRNA per neuron following cortical ablation. However, in contrast to the topographical m1 mRNA changes, the decreased ChAT mRNA expression was evenly distributed within the striatum, suggesting an indirect cortical control upon striatal cholinergic interneurons. Altogether, these data suggest that dopaminergic nigral and glutamatergic cortical afferents modulate differentially cholinergic markers, at the pre- and post-synaptic levels. Beside the fact that nigral and cortical inputs exert an opposite control on cholinergic neurotransmission, our study further shows that this control involved different muscarinic receptor subtypes: the m4 and m1 receptors, respectively.

Effects of AF64A on gene expression of choline acetyltransferase (ChAT) in the septo-hippocampal pathway and striatum in vivo.

AF64A (ethylcholine mustard aziridinium ion) was stereotaxically administered bilaterally (1 nmol/side) into rat lateral cerebral ventricles. Choline acetyltransferase (ChAT) activity and ChAT mRNA levels were measured at predetermined time points in the septo-hippocampal pathway and striatum, both well identified as rich in cholinergic neurons. AF64A caused a rapid but transient increase in ChAT mRNA (167%, P < 0.05) and ChAT activity (164%, P < 0.01) in the septum. By day 7 post treatment, there was a significant decrease in ChAT mRNA (42.5% of control, P < 0.05) in the septum although the ChAT activity still stayed high. This decreased ChAT mRNA level in the septum lasted for at least four weeks, and was paralleled by a long-lasting decrease in ChAT activity in the hippocampus. In the striatum, on the other hand, there were no observed changes in either ChAT activity or ChAT mRNA. These data suggest that the long term effect of AF64A on the septo-hippocampal cholinergic pathway may, at least in part, be due to an action of AF64A on gene expression in the cholinergic neuron. The difference in the response to AF64A between the septo-hippocampal and striatal cholinergic systems might be due to their difference in neuron types.

The cholinergic gene locus is coordinately regulated by protein kinase A II in PC12 cells.

The vesicular acetylcholine transporter (VAChT) gene and the choline acetyltransferase (ChAT) gene comprise the cholinergic gene locus. We have studied the coordinate regulation of these genes by cyclic AMP-dependent protein kinase (PKA) in the rat pheochromocytoma cell line PC12 and PC12 PKA-deficient mutants. Both ChAT and VAChT mRNA increased approximately fourfold after treatment of PC12 cells with dibutyryl cyclic AMP (dbcAMP). ChAT and PKA activity were also increased by dbcAMP. The basal levels of ChAT and VAChT mRNAs in the PKA-deficient cell lines were both about six times lower than in wild-type PC12 cells, and were induced less than twofold by addition of dbcAMP. H-89 and H-9, specific inhibitors for PKA, reduced ChAT and VAChT mRNA levels to approximately one-third that of untreated cells and ChAT activity to approximately one-fourth that of untreated PC12 cells. Activation of PKA type II, but not PKA type I, increased ChAT activity approximately threefold. Analysis of reporter gene constructs indicates that PKA affects gene transcription at an upstream site in the cholinergic gene locus. These results demonstrate that the expression of the ChAT and VAChT genes is regulated coordinately at the transcriptional level, and a signaling pathway specifically involving PKA II plays an important role in this process.

Impairment of Basal Forebrain Cholinergic Neurons Associated with Aging and Long-Term Loss of Ovarian Function

Recent studies suggest that women are at greater risk for Alzheimer's disease than men and that estrogen replacement can help to reduce the risk and severity of Alzheimer's-related dementia in postmenopausal women. We have hypothesized that the increased risk for Alzheimer's-related dementia is due, in part, to the loss of ovarian function in postmenopausal women and to the effects that decreased levels of ovarian hormones have on basal forebrain cholinergic function. In the present study, the effects of aging and ovariectomy on cholinergic neurons in the rat basal forebrain were examined to determine (1) whether aging differentially affects cholinergic neurons in the basal forebrain of males vs females, and (2) whether long-term loss of ovarian function produces deficits in basal forebrain cholinergic function beyond those associated with aging and sex. In part I of the study, gonadally intact male and female rats were sacrificed at 13, 19, and 25 months of age and the effects of aging on cholinergic neurons in the medial septum (MS) and nucleus basalis magnocellularis (NBM) were compared. In part II of the study, female rats were ovariectomized at 13 months of age and then sacrificed 3 and 6 months later along with gonadally intact, age-matched controls. Adjacent sections through the MS and NBM were processed for either immunocytochemical detection of choline acetyltransferase (ChAT) and p75NTR-like immunoreactivity or forin situhybridization detection and quantification of ChAT and trkA mRNA. Results from part I revealed no significant effects of age on the relative size or density of cholinergic neurons in the MS and NBM of gonadally intact animals. Likewise, no significant effects on the relative numbers of cholinergic neurons expressing p75NTR protein were detected. However, a significant decrease in trkA mRNA was detected in the MS of gonadally intact females, but not males, between 13 and 25 months of age. No significant effects of aging on ChAT mRNA were detected. Results from part II revealed significant decreases in both ChAT and trkA mRNA in the MS and NBM of female rats sacrificed 6 months, but not 3 months, following ovariectomy relative to age-matched, gonadally intact controls. Short-term estrogen replacement initiated 6 months following ovariectomy and administered for 3 days prior to sacrifice partially restored ChAT mRNA levels in the MS and trkA mRNA levels in the NBM. These findings suggest that ovarian hormones play a role in maintaining normal levels of ChAT and trkA expression in the MS and NBM. The fact that ChAT mRNA was decreased in the MS and NBM at 6 months following ovariectomy suggests that long-term loss of ovarian function produces a decrease in the functional status of basal forebrain cholinergic neurons projecting to the hippocampus and cortex. In addition, we hypothesize that the decreases in trkA mRNA detected both in the MS as a function of aging, and in the MS and NBM in response to ovariectomy, reflect decreases in the production of high affinity nerve growth factor (NGF) receptors, and a decrease in the responsiveness of the cholinergic neurons to endogenous NGF. This, in turn, may increase the susceptibility of the cholinergic neurons to the effects of aging and disease and thereby contribute to basal forebrain cholinergic decline. We conclude that long-term loss of ovarian function combined with aging has a negative impact on basal forebrain cholinergic neurons. These effects may contribute to the risk and severity of cognitive decline associated with aging and Alzheimer's disease in postmenopausal women.

VAMP-1 and VAMP-2 gene expression in rat spinal motoneurones: differential regulation after neuronal injury.

Vesicle-associated membrane protein (VAMP; synaptobrevin) is involved in the molecular regulation of transmitter release at the presynaptic plasma membrane. VAMP exists in two isoforms, VAMP-1 and VAMP-2, which are transcribed from two separate genes and differentially expressed in the nervous system. In situ hybridization was used to examine whether VAMP isoform mRNA expression may be altered by experimental manipulations. The effect of nerve injury on VAMP-1 and VAMP-2 mRNA levels in motoneurones of the rat lumbar spinal cord was compared with lesion-induced changes in the expression of choline acetyl transferase (ChAT) and alpha-calcitonin gene-related peptide (alpha-CGRP) mRNA. After unilateral sciatic nerve transection (axotomy), VAMP-1 mRNA expression decreased significantly in parallel with a downregulation of ChAT mRNA in axotomized motoneurones compared with the corresponding motoneurones on the contralateral unlesioned side. There was a rapid decrease in VAMP-1 and ChAT mRNA levels at 2 days after axotomy, and at 7 days there was a 65% decrease in VAMP-1 mRNA and a 48% decrease in ChAT mRNA. VAMP-1 mRNA levels continued to decrease at 14 and 21 days, while ChAT mRNA levels had returned to normal at this time. In contrast, VAMP-2 and alpha-CGRP mRNA levels were upregulated in axotomized motoneurones. A significant increase for both VAMP-2 and alpha-CGRP mRNA levels was present 2 days after axotomy, and a maximum was reached after 7 days for alpha-CGRP mRNA (163%) and after 14 days for VAMP-2 mRNA (587%). Immunohistochemical analysis did not reveal any detectable changes in VAMP-1- or VAMP-2-like immunoreactivity in the motoneurone cell soma after axotomy. In the proximal end of the transected sciatic nerve, there was an increase in VAMP-1- and VAMP-2-LI, which was most prominent at 2 days after lesion. The results show that, in axotomized spinal motoneurones, VAMP-1 mRNA is downregulated and VAMP-2 mRNA is upregulated, indicating differential regulation of the two separate VAMP genes and differential roles for the two VAMP isoforms in the regulation of exocytosis after nerve injury.

Kami-Untan-To enhances choline acetyltransferase and Nerve growth factor mRNA levels in brain cultured cells

Effects of a Kampo (Japanese herbal) medicine "kami-untan-to (KUT)" on central cholinergic neurons were examined in vitro. When rat embryo basal forebrain cells were cultured in the presence of KUT (200 μg/ml) for three days, a significant increase of choline acetyltransferase (ChAT) activity was observed. When rat embryo astroglial cells were grown in culture in the presence of KUT (12.5-400 μg/ml) for over 24 hours, the amount of NGF secreted in quiescent astrocytes was increased in a dose-dependent manner; this was detected by the ELISA method. However, KUT did not affect the proliferation of astroglial cells. KUT-induced ChAT activity and NGF secretion were completely blocked in the presence of actinomycin D and/or cycloheximide without a loss in cell viability. The treatment with KUT increased levels of ChAT mRNA in basal forebrain cells and NGF mRNA in cerebral cortex cells, respectively, these results were confirmed by using the RT-PCR (reverse transcriptase-polymerase chain reaction) method. These results suggest that KUT-induced ChAT activity and NGF secretion occur by modulating the transcriptional stage of both proteins.

Brain-derived neurotrophic factor spares choline acetyltransferase mRNA following axotomy of motor neurons in vivo.

Choline acetyltransferase (ChAT) is a functional and specific marker gene for neurons such as primary motor neurons that synthesize and release acetylcholine as a neurotransmitter. In adult mammals, transection of the peripheral nerve results in a loss of immunoreactivity for ChAT in the injured motor neurons without affecting their cell number. Using a quantitative RNase protection assay, we have investigated dynamic changes in ChAT mRNA levels following axotomy of motor neurons in the brainstem of adult rats. One week after transection of the left hypoglossal nerve, levels of ChAT mRNA in the ipsilateral side of the hypoglossal motor nucleus decreased dramatically to around 10% when compared to the uninjured contralateral side. When cut axons were chronically exposed to brain-derived neurotrophic factor (BDNF) for 1 week, ChAT mRNA levels were maintained at 63% of control levels. Thus, BDNF can abrogate the injury-induced loss of ChAT mRNA in mature motor neurons in vivo. In contrast, neither neurotrophin 4/5 nor nerve growth factor could prevent the decrease in message. This effect of BDNF on ChAT mRNA levels following peripheral injury to motor neurons demonstrates the existence of regulatory pathways responsive to neurotrophic factors that can "rescue" or "protect" cholinergic gene expression.

Histone hyperacetylating agents stimulate promoter activity of human choline acetyltransferase gene in transfection experiment

Butyrate (5 mM), Trichostatin A (1 microM) or Trapoxin A (30 nM) increased choline acetyltransferase (ChAT) activity in cultured rat sympathetic neurons 3- to 8-fold in 2 days. On the contrary, the three drugs decreased ChAT activity in human CHP126 cells. Butyrate had little effect on ChAT mRNA level in these cells, suggesting post-transcriptional mechanisms for the decrease in ChAT activity. However, transient transfection experiments using CHP126 cells revealed that the M promoter, but not the R promoter, of human ChAT gene was activated 20- to 130-fold by the three hyperacetylating agents. A butyrate-responsive element was localized in the 1 kbp region upstream of exon M. Constructs containing in addition the genomic segment between exons M and 1 displayed maximal basal activity and inducibility by butyrate, suggesting the presence of butyrate-activated promoter/enhancer elements in this region. The stimulatory effects of butyrate and Trichostatin A were also observed in stably transfected CHP126 clones, suggesting that the chromatin environment was not preventing the induction of the endogenous ChAT gene by butyrate. Rather, the data suggest different chromatin organizations for the stable transgene and the endogenous ChAT gene.

Developmental age-dependent upregulation of choline acetyltransferase and vesicular acetylcholine transporter mRNA expression in neonatal rat septum by nerve growth factor

We examined the effect of intraventricular injection of nerve growth factor (NGF) on the choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT) mRNA expression in the septa of neonatal rats. Rat pups were injected with 2.5 S NGF or cytochrome-c (control) on postnatal days (PN) 4 and 18, and sacrificed 3 days after injections for analysis of ChAT and VAChT mRNA levels by dot-blot hybridization of total septal RNA. In the NGF-treated pups, the ChAT and VAChT mRNA levels were elevated 3- and 2-fold, respectively, at PN7, and 1.8- and 1.3-fold at PN21. These results indicate that (1) NGF upregulates the expression of both ChAT and VAChT genes, (2) NGF has a greater effect on the expression of ChAT mRNA than VAChT mRNA, and (3) the effect of exogenous NGF on the expression of both genes diminishes with developmental age.

Fluctuations in relative levels of choline acetyltransferase mRNA in different regions of the rat basal forebrain across the estrous cycle: effects of estrogen and progesterone

Quantitative in situ hybridization techniques were used to compare relative cellular levels of choline acetyltransferase (ChAT) mRNA in different regions of the female rat basal forebrain at different stages of the estrous cycle and at different time points after the administration of physiological levels of estrogen and progesterone. Significant fluctuations in relative levels of ChAT mRNA were detected during the course of the estrous cycle. In the medial septum (MS) and striatum, the highest levels of ChAT mRNA were detected on diestrus 1. Fluctuations in the nucleus basalis magnocellularis (NBM) were highly variable, with the highest levels detected on diestrus 2. In ovariectomized animals, significant increases in ChAT mRNA were detected in the MS, NBM, and striatum within 1-3 d after a single administration of estradiol. In addition, the effects of estradiol on ChAT mRNA expression in the NBM and striatum were significantly enhanced by the subsequent administration of progesterone. The magnitude and timing of the effects of steroid replacement were consistent with the magnitude and time course of the fluctuations detected during the course of the estrous cycle. These data demonstrate that estrogen and progesterone can increase basal forebrain levels of ChAT mRNA significantly in specific regions of the rat basal forebrain, that the magnitude and time course of the effects vary between different subpopulations of cholinergic neurons, and that the effects are associated with changes in the functioning of specific basal forebrain cholinergic neurons across the estrous cycle.

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.