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.     

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.     

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.     

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.     

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.     

Thyroid Hormone Causes Sexually Distinct Neurochemical and Morphological Alterations in Rat Septal-Diagonal Band Neurons

Sex differences were investigated in cholinergic neurons of the septal-diagonal band region of adult rats subjected to neonatal treatment with 3,3',5-triiodo-L-thyronine (T3). Neonatal hyperthyroidism resulted in a 44% increase in specific activity of choline acetyltransferase (ChAT; EC 2.3.1.6) in adult male rat septal-diagonal band region, whereas no change in ChAT activity could be detected in either dorsal or ventral hippocampus. An increase in muscarinic cholinergic receptors, as measured by [3H]quinuclidinyl benzilate [( 3H]QNB) binding, was discovered in both septum-diagonal band and dorsal hippocampus of the T3-treated male rats. Immunohistochemistry in the septal-diagonal band region indicated a more intense staining in the neonatally T3-treated adult male rats than in controls, with larger and more abundant ChAT-positive and nerve growth factor receptor (NGF-R)-positive varicosities. ChAT immunocytochemistry showed a substantial decrease in cell body area in the medial septum and in the vertical limb of the diagonal band of T3-treated male rats, while cell density increased twofold. Female littermates subjected to the same treatment showed no changes in any of the biochemical or immunohistochemical cholinergic markers. Only in the medial septum was morphology significantly altered in the female T3-treated rats in that ChAT-positive cell body area increased. These results indicate a marked sexual variation in the septal-diagonal band region with respect to the sensitivity of postnatally developing cholinergic neurons to the actions of excess thyroid hormone.     

The early expression of VAChT and VIP in mouse sympathetic ganglia is not induced by cytokines acting through LIFRbeta or CNTFRalpha

Sympathetic ganglia consist of noradrenergic and cholinergic neurons. The cholinergic marker protein vesicular acetylcholine transporter (VAChT) and the neuropeptide vasoactive intestinal peptide (VIP), co-expressed in mature cholinergic sympathetic neurons, are first detectable during embryonic development of rat sympathetic ganglia. However, the subpopulation of cholinergic sympathetic neurons which innervates sweat glands in mammalian footpads starts to express VAChT and VIP during the first postnatal weeks, under the influence of sweat gland-derived signals. In vitro evidence suggests that the sweat gland-derived cholinergic differentiation factor belongs to a group of neuropoietic cytokines, including LIF, CNTF and CT-1, that act through a LIFRbeta-containing cytokine receptor. To investigate whether the embryonic expression of cholinergic properties is elicited by a related cytokine, the expression of VAChT and VIP was analyzed in stellate ganglia of mice deficient for the cytokine receptor subunits LIFRbeta or CNTFRalpha. The density of VAChT- and VIP-immunoreactive cells in stellate ganglia of new-born animals was not different in LIFRbeta(-/-) and CNTFRalpha(-/-) ganglia as compared to ganglia from wild-type mice. These results demonstrate that the early, embryonic expression of VAChT and VIP is not induced by cytokines acting through LIFRbeta- or CNTFRalpha-containing receptors.     

Differential regulation of motor neuron survival and choline acetyltransferase expression following axotomy

Although it is well known that motor neuron survival following axotomy is enhanced with maturation, the ability of surviving neurons to express the cholinergic enzyme choline acetyltransferase (ChAT) following axotomy has not been closely examined. Moreover, the utility of the facial nucleus in studies of motoneuron response to injury and to trophic factors, coupled with the increasing importance of the mouse in gene targeting, compelled us to investigate the age dependence of neuronal survival and ChAT expression in the mouse facial nucleus following axotomy. We cut the facial nerve at postnatal day (P)4, 7, 14, 21, and 28 or in the adult and used Nissl staining and ChAT immunocytochemistry to quantitate survival and ChAT expression, respectively, following 1, 2, or 3 weeks' survival at each age. We confirm in this model that the rate and extent of motor neuron death following axotomy is reduced with increasing maturity. The surviving neurons maintain a high ChAT content through P21; however, axotomy from P28 through adulthood results in a striking reduction in ChAT immunoreactivity. That is, although axotomy at P21 results in 61% motor neuron survival, with virtually all of the surviving neurons being ChAT positive, axotomy in the adult results in 72% survival but only 9% of the neurons are ChAT positive. Thus, surviving motor neurons in the adult animals are only weakly cholinergic. These results indicate that a change in the regulation of ChAT expression occurs following P21 so that cell survival and enzyme levels are uncoupled. We suggest that the putative factor or factors that enhances motor neuron survival in maturity is not capable of maintaining ChAT expression. © 1995 John Wiley & Sons, Inc.     

Maturation and maintenance of cholinergic medial septum neurons require glucocorticoid receptor signaling

Glucocorticoids have been shown to influence trophic processes in the nervous system. In particular, they seem to be important for the development of cholinergic neurons in various brain regions. Here, we applied a genetic approach to investigate the role of the glucocorticoid receptor (GR) on the maturation and maintenance of cholinergic medial septal neurons between P15 and one year of age by using a mouse model carrying a CNS-specific conditional inactivation of the GR gene (GRNesCre). The number of choline acetyltransferase and p75NTR immuno-positive neurons in the medial septum (MS) was analyzed by stereology in controls versus mutants. In addition, cholinergic fiber density, acetylcholine release and cholinergic key enzyme activity of these neurons were determined in the hippocampus. We found that in GRNesCre animals the number of medial septal cholinergic neurons was significantly reduced during development. In addition, cholinergic cell number further decreased with aging in these mutants. The functional GR gene is therefore required for the proper maturation and maintenance of medial septal cholinergic neurons. However, the loss of cholinergic neurons in the medial septum is not accompanied by a loss of functional cholinergic parameters of these neurons in their target region, the hippocampus. This pinpoints to plasticity of the septo-hippocampal system, that seems to compensate for the septal cell loss by sprouting of the remaining neurons.     

Reformation of the severed septohippocampal cholinergic pathway in the adult rat by transplanted septal neurons

Summary Transplants containing developing cholinergic neurons were obtained from the septum-diagonal band area of rat fetuses and were implanted into a lesion of the septohippocampal cholinergic pathway or into a cavity of the occipital cortex in adult recipient rats. The growth of new cholinergic fibres from the implant into the hippocampal formation was followed with choline acetyltransferase (ChAT) determinations and acetylcholine esterase (AChE) histochemistry. A fimbrial lesion alone, transecting the septohippocampal pathway, caused an almost complete cholinergic denervation of the hippocampal formation that persisted throughout the five month experimental period. A septal transplant implanted into the cavity of the fimbrial lesion restored a new AChE-positive innervation pattern in the hippocampus and the dentate gyrus that closely mimicked the original innervation removed by the lesion. In parallel, there was a progressive recovery in the ChAT levels, starting in the septal end, and progressing in a temporal direction. A new cholinergic fibre supply could be established in the hippocampal formation also along an abnormal route, i.e. from the transplants implanted into a cavity in the occipital cortex (involving also the dorsal part of the entorhinal cortex). Provided the hippocampus previously had been denervated of its normal cholinergic innervation, a partly normal AChE-positive terminal pattern was thus re-established also from this abnormal position. If, on the other hand, the cholinergic afferents were left intact, the ingrowing fibres were restricted mainly to the outer portion of the dentate molecular layer, i.e. the terminal zone of the lesioned entorhinal perforant path fibres. This suggests that the growth of the sprouting AChE-positive fibres into the normal cholinergic terminal fields was blocked by the presence of an intact cholinergic innervation. It is concluded that regrowing cholinergic axons can be guided over large distances within the hippocampal formation, and that their patterning within the terminal fields is very precisely regulated by mechanisms released by deafferentation.     

Selective cholinergic depletion in medial septum leads to impaired long term potentiation and glutamatergic synaptic currents in the hippocampus

Cholinergic depletion in the medial septum (MS) is associated with impaired hippocampal-dependent learning and memory. Here we investigated whether long term potentiation (LTP) and synaptic currents, mediated by alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA) and N-methyl-D-aspartate (NMDA) receptors in the CA1 hippocampal region, are affected following cholinergic lesions of the MS. Stereotaxic intra-medioseptal infusions of a selective immunotoxin, 192-saporin, against cholinergic neurons or sterile saline were made in adult rats. Four days after infusions, hippocampal slices were made and LTP, whole cell, and single channel (AMPA or NMDA receptor) currents were recorded. Results demonstrated impairment in the induction and expression of LTP in lesioned rats. Lesioned rats also showed decreases in synaptic currents from CA1 pyramidal cells and synaptosomal single channels of AMPA and NMDA receptors. Our results suggest that MS cholinergic afferents modulate LTP and glutamatergic currents in the CA1 region of the hippocampus, providing a potential synaptic mechanism for the learning and memory deficits observed in the rodent model of selective MS cholinergic lesioning.     

NGF induction of NGF receptor gene expression and cholinergic neuronal hypertrophy within the basal forebrain of the adult rat

Chronic infusion of nerve growth factor (NGF) into the forebrain of the adult rat produced increases in NGF receptor (NGF-R) mRNA hybridization, NGF-R immunoreactivity, choline acetyltransferase (ChAT) mRNA hybridization, and neuronal hypertrophy, when compared with vehicle infusion or noninfused rat brain. In situ hybridization showed NGF induction of NGF-R gene expression, documented by increases in the number of NGF-R mRNA-positive cells within the medial septum, diagonal band, and nucleus basalis magnocellularis. NGF also produced hypertrophy of ChAT mRNA-positive neurons. These results suggest that NGF produces cholinergic neuronal hypertrophy through induction of NGF-R gene expression within the basal forebrain.     

Immunohistochemical characterisation of cholinergic neurons in the anterior pelvic ganglion of the male pig

This study investigated immunohistochemical properties of cholinergic neurons in the anterior pelvic ganglion (APG) of juvenile male pigs (n=7). Cholinergic neurons were identified using antibodies against choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT). Immunoblotting was applied to verify the specificity of ChAT-immunostaining. Western blotting performed on APG tissue homogenates detected single immunoreactive protein with a molecular weight matching that of ChAT (71.6 kDa). It was found that many APG neurons expressed immunoreactivity to ChAT or VAChT (40% and 39% of the neurons, respectively). The analysis of adjacent sections from the ganglion revealed complete colocalization of ChAT and VAChT in these nerve cells. Furthermore, virtually all the ChAT-positive neurons were tyrosine hydroxylase (TH)-negative (non-adrenergic) but many of them displayed immunoreactivity to nitric oxide synthase (NOS), vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY) or somatostatin (SOM). There were also single nerve cell bodies that stained for neither ChAT nor TH. The comparison of the adjacent sections revealed that NOS, VIP, NPY and SOM were simultaneously co-expressed in the majority of the cholinergic somata. ChAT- or VAChT-positive varicose nerve terminals supplied nearly all neuronal profiles within the ganglion often forming loose basket-like formations surrounding the particular nerve cell bodies. The present study for the first time has revealed that nearly all non-adrenergic neurons in the porcine APG are cholinergic in nature, i.e. express immunoreactivity for ChAT and VAChT. Considering a high coincidence between the chemical coding of non-adrenergic (cholinergic) nerve fibres supplying some porcine male reproductive organs described in earlier papers and that of cholinergic pelvic neurons found in this study it is further concluded that pelvic ganglia are probably the major source of cholinergic innervation for the porcine urogenital system.     

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.