Novel strains of mice deficient for the vesicular acetylcholine transporter: insights on transcriptional regulation and control of locomotor behavior

Defining the contribution of acetylcholine to specific behaviors has been challenging, mainly because of the difficulty in generating suitable animal models of cholinergic dysfunction. We have recently shown that, by targeting the vesicular acetylcholine transporter (VAChT) gene, it is possible to generate genetically modified mice with cholinergic deficiency. Here we describe novel VAChT mutant lines. VAChT gene is embedded within the first intron of the choline acetyltransferase (ChAT) gene, which provides a unique arrangement and regulation for these two genes. We generated a VAChT allele that is flanked by loxP sequences and carries the resistance cassette placed in a ChAT intronic region (FloxNeo allele). We show that mice with the FloxNeo allele exhibit differential VAChT expression in distinct neuronal populations. These mice show relatively intact VAChT expression in somatomotor cholinergic neurons, but pronounced decrease in other cholinergic neurons in the brain. VAChT mutant mice present preserved neuromuscular function, but altered brain cholinergic function and are hyperactive. Genetic removal of the resistance cassette rescues VAChT expression and the hyperactivity phenotype. These results suggest that release of ACh in the brain is normally required to "turn down" neuronal circuits controlling locomotion.     

Expression of multiple mRNA species for choline acetyltransferase in human T-lymphocytes

Acetylcholine (ACh) is synthesized by choline acetyltransferase (ChAT) in cholinergic neurons. However, both ACh and mRNA for ChAT are expressed in mononuclear leukocytes and various human leukemic T-cell lines. Multiple ChAT mRNA species (R-, N0-, N1-, N2-, and M-types) having an identical coding region and different 5'-noncoding regions have been discovered in human brain and spinal cord. These mRNAs are transcribed by a combination of use of different promoter regions and alternative splicing. However, which types of ChAT mRNA species are expressed in T-lymphocytes remains to be elucidated. In the present study, we used two human leukemic T-cell lines, CCRF-CEM (CEM) and MOLT-3, which express the same ChAT mRNA as that in the nervous system. Major mRNA species in CEM were N2- and M-type, and to a lesser extent N1-type, while MOLT-3 expressed only N2-type. Neither CEM nor MOLT-3 expressed R-type mRNA. We previously found a lack of mRNA expression encoding vesicular acetylcholine transporter (VAChT) in CEM and MOLT-3, which mediates ACh transport to synaptic vesicles in cholinergic neurons. These findings suggest that the mechanisms regulating ChAT mRNA expression in T-lymphocytes differ from those in cholinergic neurons.     

Coordinate Expression of the Vesicular Acetylcholine Transporter and Choline Acetyltransferase Following Septohippocampal Pathway Lesions

Abstract: The gene for the vesicular acetylcholine transporter (VAChT) was recently cloned and found to be located within a 5' noncoding intron of the gene for choline acetyltransferase (ChAT). There appear to be several shared and unique promoters for each gene, suggesting that control of expression of these two genes can be either coordinated or independent. Two lesions, axotomy and immunotoxin, directed at the well defined septohippocampal cholinergic pathway were used to determine VAChT and ChAT protein expression in the degenerating terminal fields in the hippocampus and the cell bodies of the medial septum nucleus after injury. Two weeks after lesioning, decreases of up to 90% in VAChT were found in the affected hippocampus by immunoblotting and immunocytochemistry, similar to ChAT activity. The number of VAChT- and ChAT-immunopositive neurons in the medial septum decreased by up to 95%. Eight weeks following axotomy, the number of VAChT- and ChAT-immunopositive neurons had increased to almost 50% in fimbria-fornix-lesioned animals, indicating coordinate reexpression of both cholinergic markers in recovered neurons. There was no recovery of either VAChT or ChAT immunoreactivity after the irreversible immunotoxin lesions. Thus, with use of immunological techniques, there appears to be coordinate expression of VAChT and ChAT in the septohippocampal pathway following either unilateral fimbria-fornix or bilateral immunotoxin lesion.     

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.     

Identification and Transgenic Analysis of a Murine Promoter that Targets Cholinergic Neuron Expression

Abstract : Choline acetyltransferase (ChAT) is a specific phenotypic marker of cholinergic neurons. Previous reports showed that different upstream regions of the ChAT gene are necessary for cell type-specific expression of reporter genes in cholinergic cell lines. The identity of the mouse ChAT promoter region controlling the establishment, maintenance, and plasticity of the cholinergic phenotype in vivo is not known. We characterized a promoter region of the mouse ChAT gene in transgenic mice, using β-galactosidase ( LacZ ) as a reporter gene. A 3,402-bp segment from the 5'-untranslated region of the mouse ChAT gene (from -3,356 to +46, +1 being the translation initiation site) was sufficient to direct the expression of LacZ to selected neurons of the nervous system ; however, it did not provide complete cholinergic specificity. A larger fragment (6,417 bp, from -6,371 to +46) of this region contains the requisite regulatory elements that restrict expression of the LacZ reporter gene only in cholinergic neurons of transgenic mice. This 6.4-kb DNA fragment encompasses 633 bp of the 5'-flanking region of the mouse vesicular acetylcholine transporter (VAChT), the entire open reading frame of the VAChT gene, contained within the first intron of the ChAT gene, and sequences upstream of the start coding sequences of the ChAT gene. This promoter will allow targeting of specific gene products to cholinergic neurons to evaluate the mechanisms of diseases characterized by dysfunction of cholinergic neurons and will be valuable in design strategies to correct those disorders.     

Single dose exposure of sarin and physostigmine differentially regulates expression of choline acetyltransferase and vesicular acetylcholine transporter in rat brain

Choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT) are the key components of cholinergic system apart from acetylcholinesterase. Effects of subcutaneous exposures of 0.25 and 0.5 LD(50) sarin and 0.75 mg/kg physostigmine on immunoreactivity levels of these two proteins (ChAT and VAChT) were studied. Immunoreactivity levels of ChAT decreased significantly after 1 and 3 days in cortex and 3 days of 0.25 LD(50) sarin administration in cerebellum. While 0.5 LD(50) sarin exposure caused significant down regulation after 2.5 h to 7 days in cortex and 1 and 3 days in cerebellum with respect to controls. Physostigmine at 0.75 mg/kg dose showed enhanced levels of ChAT after 1 day which decreased significantly after 3 and 7 days both in cortex and cerebellum compared to controls. VAChT level decreased significantly after 1 day in cortex and 3 and 7 days in cerebellum after 0.25 LD(50) sarin administration, while 0.5 LD(50) sarin significantly lowered VAChT immunoreactivity level after 2.5 h and 7 days in cortex and 2.5 h and 1 day in cerebellum. Physostigmine at 0.75 mg/kg dose showed significant enhanced immunoreactivity levels of VAChT after 1, 3, and 7 days in cortex and 3 days in cerebellum. Results show that acetylcholinesterase inhibition by sarin caused reduction in cholinergic neurotransmission at cholinergic proteins expression levels, while physostigmine caused differential expression of key cholinergic proteins. Moreover, cortex, which receives greater cholinergic innervations, is more susceptible to anticholinesterase effect on cholinergic gene expression. These changes can explain delayed neurocognitive changes during anticholinesterases induced chronic neurotoxicity.     

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.     

VAChT-Cre. Fast and VAChT-Cre.Slow: postnatal expression of Cre recombinase in somatomotor neurons with different onset

The cholinergic gene locus (CGL) consists of the genes encoding the choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter (VAChT). To establish a cholinergic-specific Cre-expressing mouse, we constructed a transgene expression vector (VAChT-Cre) with 11.3 kb human CGL in which a Cre-IRES-EGFP unit was inserted in the VAChT open reading frame. The activity of Cre, whose expression was driven by the VAChT promoter, was examined by crossing a reporter mouse (CAG-CAT-Z) in which expression of LacZ is activated upon Cre-mediated recombination. Transgenic lines with the VAChT-Cre construct displayed the restricted Cre expression in a subset of cholinergic neurons in the somatomotor nuclei and medial habenular nucleus, but absent in visceromotor and other central and peripheral cholinergic neurons. Cre expression was first observed at postnatal day 7 and later detected in approximately 40-60% of somatomotor neurons. Based on the onset of Cre expression, we generated two mouse lines (two alleles; VAChT-Cre. Fast and VAChT-Cre.Slow) in which Cre expression reaches maximal levels fast and slow, respectively. The use of VAChT-Cre mice should allow us to deliver Cre to a subset of postnatal motor neurons, thereby bypassing lethality and facilitating analysis of gene function in adult motor neurons.     

Expression of the cholinergic gene locus in the rat placenta

High amounts of acetylcholine (ACh) and its synthesising enzyme choline acetyltransferase (ChAT) have been detected in the placenta. Since the placenta is not innervated by extrinsic or intrinsic cholinergic neurons, placental ACh and ChAT originate from non-neuronal sources. In neurons, cytoplasmic ACh is imported into synaptic vesicles by the vesicular acetylcholine transporter (VAChT), and released through vesicular exocytosis. In view of the coordinate expression of VAChT and ChAT from the cholinergic gene locus in neurons, we asked whether VAChT is coexpressed with ChAT in rat placenta, and investigated this issue by means of RT-PCR, in situ hybridisation, western blot and immunohistochemistry. Messenger RNA and protein of the common type of ChAT (cChAT), its splice variant peripheral ChAT (pChAT), and VAChT were detected in rat placenta with RT-PCR and western blot. ChAT in situ hybridisation signal and immunoreactivity for cChAT and pChAT were observed in nearly all placental cell types, while VAChT mRNA and immunolabelling were detected in the trophoblast, mesenchymal cells and the visceral yolk sac epithelial cells. While ChAT is nearly ubiquitously expressed in rat placenta, VAChT immunoreactivity is localised cell type specifically, implying that both vesicular and non-vesicular ACh release machineries prevail in placental cell types.     

PACAP and NGF cooperatively enhance choline acetyltransferase activity in postnatal basal forebrain neurons by complementary induction of its different mRNA species

Both nerve growth factor (NGF) and pituitary adenylate cyclase activating polypeptide (PACAP) have neurotrophic effects on basal forebrain cholinergic neurons. They promote differentiation, maturation, and survival of these cholinergic neurons in vivo and in vitro. Here we report on the cooperative effects of NGF and PACAP on postnatal, but not embryonic, cholinergic neurons cultured from rat basal forebrain. Combined treatment with NGF, brain-derived neurotrophic factor (BDNF), neurotrophin-4 (NT-4), and PACAP induced an additive increase in choline acetyltransferase (ChAT) activity. There were no cooperative effects on the number of cholinergic neurons, suggesting that ChAT mRNA expression had been induced in each cholinergic neuron. Further analysis revealed that NGF and PACAP led to complementary induction of different ChAT mRNA species, thus enhancing total ChAT mRNA expression. These results explain the cooperative neurotrophic action of NGF and PACAP on postnatal cholinergic neurons.     

Presence of a non-neuronal cholinergic system and occurrence of up- and down-regulation in expression of M2 muscarinic acetylcholine receptors: new aspects of importance regarding Achilles tendon tendinosis (tendinopathy)

Limited information is available concerning the existence of a cholinergic system in the human Achilles tendon. We have studied pain-free normal Achilles tendons and chronically painful Achilles tendinosis tendons with regard to immunohistochemical expression patterns of the M(2) muscarinic acetylcholine receptor (M(2)R), choline acetyltransferase (ChAT), and vesicular acetylcholine transporter (VAChT). M(2)R immunoreactivity was detected in the walls of blood vessels. As evidenced via parallel staining for CD31 and alpha-smooth muscle actin, most M(2)R immunoreactivity was present in the endothelium. M(2)R immunoreactivity also occured in tenocytes, which regularly immunoreact for vimentin. The degree of M(2)R immunoreactivity was highly variable, tendinosis tendons that exhibit hypercellularity and hypervascularity showing the highest levels of immunostaining. Immunoreaction for ChAT and VAChT was detected in tenocytes in tendinosis specimens, particularly in aberrant cells. In situ hybridization revealed that mRNA for ChAT is present in tenocytes in tendinosis specimens. Our results suggest that autocrine/paracrine effects occur concerning the tenocytes in tendinosis. Up-regulation/down-regulation in the levels of M(2)R immunoreactivity possibly take place in tenocytes and blood vessel cells during the various stages of tendinosis. The presumed local production of acetylcholine (ACh), as evidenced by immunoreactivity for ChAT and VAChT and the detection of ChAT mRNA, appears to evolve in response to tendinosis. These observations are of importance because of the well-known vasoactive, trophic, and pain-modulating effects that ACh is known to have and do unexpectedly establish the presence of a non-neuronal cholinergic system in the Achilles tendon.     

Influence of retinoic acid and of cyclic AMP on the expression of choline acetyltransferase and of vesicular acetylcholine transporter in NG108-15 cells

Treatment of the cholinergic cell line NG108-15 with retinoic acid or cAMP results in an increase of choline acetyltransferase activity (ChAT) whereas none of these agents influences the amount of the vesicular acetylcholine transporter (VAChT) as judged from vesamicol binding and immunoblot studies. We suggest that immaturity of posttranslational events controlling the expression of VAChT protein is responsible for the apparent absence of coregulation of ChAT and VAChT protein expression.     

Demonstration of choline acetyltransferase of a peripheral type in the rat heart.

Cholinergic innervation of the heart has been analyzed using cholinergic markers including acetylcholinesterase, choline acetyltransferase (ChAT), and vesicular acetylcholine transporter (VAChT). In the present study we demonstrate putative cholinergic nerves in the rat heart using an antibody to ChAT of a peripheral type (pChAT), which is the product of a splice variant of ChAT mRNA and preferentially localized to peripheral cholinergic nerves. Expression of mRNAs for pChAT and the conventional form of ChAT (cChAT) were verified in the rat atrium by RT-PCR. Localization of both protein products in the atrium was confirmed by Western blotting. Virtually all neurons and small intensely fluorescent cells in the intrinsic cardiac ganglia were stained immunohistochemically for pChAT. The density of pChAT-positive fibers was very high in the conducting system, high in both atria, the right atrium in particular, and low in the ventricular walls. pChAT and VAChT immunoreactivities were closely associated in some fibers and fiber bundles in the ventricular walls. These results indicate that intrinsic cardiac neurons homogeneously express both pChAT and cChAT. Furthermore, innervation of the ventricular walls by pChAT- and VAChT-positive fibers provides morphological evidence for a significant role of cholinergic mechanisms in ventricular functions.     

The developmental expression of vasoactive intestinal peptide (VIP) in cholinergic sympathetic neurons depends on cytokines signaling through LIFRbeta-containing receptors

Sympathetic ganglia are composed of noradrenergic and cholinergic neurons. Cholinergic sympathetic neurons are characterized by the expression of choline acetyl transferase (ChAT), vesicular acetylcholine transporter (VAChT) and the vasoactive intestinal peptide (VIP). To investigate the role of cytokine growth factor family members in the development of cholinergic sympathetic neurons, we interfered in vivo with the function of the subclass of cytokine receptors that contains LIFRbeta as essential receptor subunit. Expression of LIFRbeta antisense RNA interfered with LIFRbeta expression and strongly reduced the developmental induction of VIP expression. By contrast, ganglion size and the number of ChAT-positive cells were not reduced. These results demonstrate a physiological role of cytokines acting through LIFRbeta-containing receptors in the control of VIP expression in sympathetic neurons.