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Hiroshi Kimura - One of the best experts on this subject based on the ideXlab platform.
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acetylCholine synthesis by Choline Acetyltransferase of a peripheral type as demonstrated in adult rat dorsal root ganglion
Journal of Neurochemistry, 2007Co-Authors: Jean-pierre Bellier, Hiroshi KimuraAbstract:pChAT is a splice variant of a peripheral type encoded alternatively by the gene for Choline Acetyltransferase of the common type (cChAT), the enzyme responsible for acetylCholine synthesis. Immunohistochemistry using pChAT antiserum has successfully visualized many known peripheral Cholinergic cells, whereas most cChAT antibodies failed to do so. As, however, accumulating evidence indicates that pChAT expression also occurs in various non-Cholinergic neurons, we examined possible acetylCholine production by pChAT in rat dorsal root ganglion as a model. The present study indicated that the ganglion neurons possessed pChAT, but never cChAT, mRNA and protein. Our detailed analysis further showed that, despite low enzyme activities of both Choline Acetyltransferase and acetylCholinesterase, the level of acetylCholine in the ganglion was as high as to that in various brain regions receiving Cholinergic innervation. By using immunoprecipitation methods, we here provide evidence that pChAT definitely has enzyme activity enough to supply physiological concentrations of acetylCholine in the ganglion. We propose that pChAT contributes both to acetylCholine neurotransmission in physiologically identified Cholinergic cells and to functions yet unknown in non-Cholinergic neurons. Thus pChAT provides a new window on the role of neuronal acetylCholine.
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the production of antibodies that distinguish rat Choline Acetyltransferase from its splice variant product of a peripheral type
Neurochemistry International, 2007Co-Authors: Shin Kimura, Akinori Matsuo, Jean-pierre Bellier, Ikuo Tooyama, Hiroshi KimuraAbstract:To produce antibodies that permit the immunohistochemical discrimination of Choline Acetyltransferase of the common type (cChAT) from its splice variant of a peripheral type (pChAT), we immunized rabbits with a cChAT specific recombinant protein encoded by ChAT exons 7 and 8 of the rat cChAT gene. Successful antibody production was proved by Western blotting on rat brain and on HEK293 cells expressing green fluorescent protein (GFP), cChAT-GFP and pChAT-GFP. By immunohistochemistry our antiserum clearly labeled known Cholinergic structures in rat brain, but gave no positive staining in the trigeminal ganglion which contained many neurons positive with pChAT antiserum.
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expression of a splice variant of Choline Acetyltransferase in magnocellular neurons of the tuberomammillary nucleus of rat
Neuroscience, 2003Co-Authors: Hiroshi Kanayama, Osamu Yasuhara, Akinori Matsuo, Yoshinari Aimi, Jean-pierre Bellier, Ikuo Tooyama, J I Nagy, Kenji Fukui, Hiroshi KimuraAbstract:Abstract A splice variant of Choline Acetyltransferase mRNA has recently been identified in the pterygopalatine ganglion of rat. An antibody against this variant protein (designated pChAT) was demonstrated to immunolabel peripheral Cholinergic neurons. In the present study, we investigated the expression of pChAT in rat brain. Amongst the brain regions examined, magnocellular neurons in the tuberomammillary nucleus of the posterior hypothalamus were immunohistochemically labelled with anti-pChAT antibody, whilst no immunolabelling was detected in Cholinergic neurons in the basal forebrain or striatum. RT-PCR analysis confirmed the expression of pChAT mRNA in the posterior hypothalamus. The distribution of pChAT-positive neurons in the tuberomammillary nucleus was compared with that of neurons positive for adenosine deaminase, which is contained in all neurons of this nucleus. After colchicine treatment to inhibit axonal transport of enzyme, virtually all pChAT-positive cells contained adenosine deaminase. Conversely, about 85% of adenosine deaminase-positive cells contained pChAT in the ventral area, whilst 19% of adenosine deaminase-positive cells were pChAT-positive in the dorsal area. Long axonal projections of pChAT-positive cells in the tuberomammillary nucleus were shown by retrograde labelling of these cells after injection of cholera-toxin B subunit into the cerebral cortex. This study demonstrates that a splice variant of Choline Acetyltransferase is expressed in the tuberomammillary nucleus of rat. The results raise the possibility that some of the known diverse projection areas of this nucleus may have a Cholinergic component.
Richard J Beninger - One of the best experts on this subject based on the ideXlab platform.
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excitotoxic lesions of rat basal forebrain differential effects on Choline Acetyltransferase in the cortex and amygdala
Neuroscience, 1992Co-Authors: R J Boegman, J Cockhill, Khem Jhamandas, Richard J BeningerAbstract:Previous studies have shown that basal forebrain lesions using different excitotoxins produce similar decreases in cortical Choline Acetyltransferase, but differential effects on memory, However, basal forebrain choiinergic neurons send efferents to the amygdala and cortex. The present studies compared the effects of several excitotoxins on Choline Acetyltransferase levels in both of these structures. Lesions of the basal forebrain were made in rats by infusing different doses of either a-amine-3-hydroxy-5-methyl- 4-isoxazole propionic acid, ibotenic acid, quisqualic acid, quinolinic acid or N-methyl-u-aspartic acid and measuring Choline Acetyltransferase seven days later. All of the excitotoxins exerted a differential response on Cholinergic neurons of the basal forebrain projecting to the cortex or amygdala. Quinolinic acid was a more potent neurotoxin to Cholinergic neurons innervating the amygdala than those projecting to the cortex. In contrast, quisqualic acid and ~-amine-3-hydroxy-5-methyl-4-jsoxazole were more potent neurotoxins to the cortical projection. cc-Amine-3-hydroxy-5-methyl-4-isozazole propionic acid was the most potent excitotoxin for destroying Cholinergic neurons innervating either the cortex or amygdala. A parallel neurotoxic response was obtained in the cortex and amygdala following infusion of ibotenic acid or N-methyl-o-aspartic acid with little selectivity for Choline Acetyltransferase depletion in the cortex or amygdala. Histological analysis of the injection site revealed that a~tylCholinesterase-positive neurons were destroyed by the excitotoxins in a dose-dependent manner. Excitotoxins (ibotenic acid, quinolinic acid, N-methyl-D-aspartic acid) that produce the greatest impairments in memory were found to produce the greatest depletion of Choline Acetyltransferase in the amygdala. These results might suggest that Cholinergic neurons projecting to the amygdala play an _- important role in memory. Excitotoxic lesions of the magnocellular cbolinergic neurons in the nucleus basalis (nbm) have been used extensively in studies to examine the effects of Cholinergic deaffer~ntation in neurochemical and behavioural experiments.6 When infused inta the nbm axon sparing neurotoxic glutamate receptor agonists such as kainic, ibotenic, quisqualic or quinolinic acid produce reductions in cortical Choline acetyltrans- ferase (ChAT) activity of up to 80%.'.',9,'2 While these reductions are associated with decreases in perform- ance on memory tasks,
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Excitotoxic lesions of rat basal forebrain: Differential effects on Choline Acetyltransferase in the cortex and amygdala. Neurosci. 51:129–135
1992Co-Authors: R J Boegman, J Cockhill, Khem Jhamandas, Richard J BeningerAbstract:Abstract-Previous studies have shown that basal forebrain lesions using different excitotoxins produce similar decreases in cortical Choline Acetyltransferase, but differential effects on memory, However, basal forebrain choiinergic neurons send efferents to the amygdala and cortex. The present studies compared the effects of several excitotoxins on Choline Acetyltransferase l vels in both of these structures. Lesions of the basal forebrain were made in rats by infusing different doses of either a-amine-3-hydroxy-5-methyl-4-isoxazole propionic acid, ibotenic acid, quisqualic acid, quinolinic acid or N-methyl-u-aspartic acid and measuring Choline Acetyltransferase ven days later. All of the excitotoxins exerted a differential response on Cholinergic neurons of the basal forebrain projecting to the cortex or amygdala. Quinolinic acid was a more potent neurotoxin to Cholinergic neurons innervating the amygdala than those projecting to the cortex. In contrast, quisqualic acid and ~-amine-3-hydroxy-5-methyl-4-jsoxazole were more potent neurotoxins to the cortical projection. cc-Amine-3-hydroxy-5-methyl-4-isozazole propionic acid was the most potent excitotoxin for destroying Cholinergic neurons innervating either the cortex or amygdala. A parallel neurotoxic response was obtained in the cortex and amygdala following infusion of ibotenic acid or N-methyl-o-aspartic acid with little selectivity for Choline Acetyltransferase d pletion in the cortex or amygdala. Histological analysis of the injection site revealed that a~tylCholinesterase-positive n uron
Larry L Butcher - One of the best experts on this subject based on the ideXlab platform.
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absence of p75ntr causes increased basal forebrain Cholinergic neuron size Choline Acetyltransferase activity and target innervation
The Journal of Neuroscience, 1997Co-Authors: Tracy T Yeo, Larry L Butcher, Jane Chuacouzens, Dale E Bredesen, Jonathan D Cooper, Janice S Valletta, William C Mobley, Frank M LongoAbstract:Emerging evidence suggests that the p75 neurotrophin receptor (p75NTR) mediates cell death; however, it is not known whether p75NTR negatively regulates other neuronal phenotypes. We found that mice null for p75NTR displayed highly significant increases in the size of basal forebrain Cholinergic neurons, including those that are TrkA-positive. Cholinergic hippocampal target innervation also was increased significantly. Activity of the Cholinergic neurotransmitter synthetic enzyme Choline Acetyltransferase (ChAT) was increased in both the medial septum and hippocampus. Upregulation of these Cholinergic features was not associated with increased basal forebrain or hippocampal target NGF levels. In contrast, striatal Cholinergic neurons, which do not express p75NTR, showed no difference in neuronal number, size, or ChAT activity between wild-type and p75NTR null mutant mice. These findings indicate that p75NTR negatively regulates Cholinergic neuronal phenotype of the basal forebrain Cholinergic neurons, including cell size, target innervation, and neurotransmitter synthesis.
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Cholinergic neurons in the rat central nervous system demonstrated by in situ hybridization of Choline Acetyltransferase mRNA
Neuroscience, 1992Co-Authors: Justin D. Oh, Ali Roghani, Nancy J. Woolf, Robert H Edwards, Larry L ButcherAbstract:Abstract Digoxigenin-labeled RNA probes and in situ hybridization histochemistry were used to examine Choline Acetyltransferase gene expression in the rat central nervous system. Hybridization signal was present only in brain sections processed with the antisense riboprobe. The sense probe did not yield labeling, further validating the specificity of tissue reactivity. Telencephalic neurons containing the mRNA for the Cholinergic synthetic enzyme were found in the caudate-putamen nucleus, nucleus accumbens, olfactory tubercule, islands of Calleja complex, medial septal nucleus, vertical and horizontal limbs of the diagonal band, substantia innominata, nucleus basalis, and nucleus of the ansa lenticularis. Some somata evincing hybridization signal were observed in the anterior amygdalar area, and an occasional such cell was seen in the basolateral and central amygdalar nuclei. Neurons in the cerebral cortex, hippocampus, and primary olfactory structures did not demonstrate hybridocytochemically detectable amounts of Choline Acetyltransferase mRNA. Thalamic cells were devoid of reactivity, with the exception of several neurons located primarily in the ventral two-thirds of the medial habenula. A few somata labeled with riboprobe were found in the lateral hypothalamus, caudal extension of the internal capsule, and zona incerta. Neurons in the pedunculopontine and laterodorsal tegmental nuclei were moderately reactive, whereas cells of the parabigeminal nucleus exhibited a very weak hybridization signal. No somata in the brainstem raphe nuclei, including raphe obscurus and raphe magnus, were observed to bind riboprobe. In contrast, motor neurons of the cranial nerve nuclei demonstrated relatively large amounts of Choline Acetyltransferase mRNA. Putative Cholinergic somata in the ventral horns and intermediolateral cell columns of the spinal cord were also labeled with riboprobe, as were a few cells around the central canal. We conclude that hybridocytochemistry with digoxigenin-labeled riboprobes confirms the existence of Cholinergic neurons (i.e. those that synthesize and use acetylCholine as a neurotransmitter) in most of the neural regions deduced to contain them on the basis of previous histochemical and immunocytochemical data. Notable exceptions are the cerebral cortex and hippocampus, which do not possess neurons expressing detectable levels of Choline Acetyltransferase mRNA.
Leon J. Thal - One of the best experts on this subject based on the ideXlab platform.
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Choline Transporter 1 Maintains Cholinergic Function in Choline Acetyltransferase Haploinsufficiency
The Journal of neuroscience : the official journal of the Society for Neuroscience, 2004Co-Authors: Eugene P. Brandon, Tiffany J. Mellott, Donald P. Pizzo, Nicole G. Coufal, Kevin A. D'amour, Kevin T. Gobeske, Mark Lortie, Ignacio Lopez-coviella, Brygida Berse, Leon J. ThalAbstract:Choline Acetyltransferase (ChAT), the enzyme that synthesizes the neurotransmitter acetylCholine (ACh), is thought to be present in kinetic excess in Cholinergic neurons. The rate-limiting factor in ACh production is the provision of Choline to ChAT. Cholinergic neurons are relatively unique in their expression of the Choline transporter 1 (CHT1), which exhibits high-affinity for Choline and catalyzes its uptake from the extracellular space to the neuron. Multiple lines of evidence indicate that the activity of CHT1 is a key determinant of Choline supply for ACh synthesis. We examined the interaction of ChAT and ChT activity using mice heterozygous for a null mutation in the Chat gene (Chat+/-). In these mice, brain ChAT activity was reduced by 40-50% relative to the wild type, but brain ACh levels as well as ACh content and depolarization-evoked ACh release in hippocampal slices were normal. However, the amount of Choline taken up by CHT1 and ACh synthesized de novo from Choline transported by CHT1 in hippocampal slices, as well as levels of CHT1 mRNA in the septum and CHT1 protein in several regions of the CNS, were 50-100% higher in Chat+/- than in Chat+/+ mice. Thus, haploinsufficiency of ChAT leads to an increased expression of CHT1. Increased ChT activity may compensate for the reduced ChAT activity in Chat+/- mice, contributing to the maintenance of apparently normal Cholinergic function as reflected by normal performance of these mice in several behavioral assays.
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aberrant patterning of neuromuscular synapses in Choline Acetyltransferase deficient mice
The Journal of Neuroscience, 2003Co-Authors: Eugene P. Brandon, Donald P. Pizzo, Leon J. Thal, Weichun Lin, Kevin A Damour, Bertha Dominguez, Yoshie Sugiura, Silke Thode, Fred H Gage, Kuofen LeeAbstract:In this study we examined the developmental roles of acetylCholine (ACh) by establishing and analyzing mice lacking Choline Acetyltransferase (ChAT), the biosynthetic enzyme for ACh. As predicted, ChAT-deficient embryos lack both spontaneous and nerve-evoked postsynaptic potentials in muscle and die at birth. In mutant embryos, abnormally increased nerve branching occurs on contact with muscle, and hyperinnervation continues throughout subsequent prenatal development. Postsynaptically, ACh receptor clusters are markedly increased in number and occupy a broader muscle territory in the mutants. Concomitantly, the mutants have significantly more motor neurons than normal. At an ultrastructural level, nerve terminals are smaller in mutant neuromuscular junctions, and they make fewer synaptic contacts to the postsynaptic muscle membrane, although all of the typical synaptic components are present in the mutant. These results indicate that ChAT is uniquely essential for the patterning and formation of mammalian neuromuscular synapses.
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Choline Acetyltransferase activity and cognitive domain scores of alzheimer s patients
Neurobiology of Aging, 2000Co-Authors: Bruce A Pappas, Peter J Bayley, Barbara K Bui, Lawrence A Hansen, Leon J. ThalAbstract:Abstract Choline Acetyltransferase activity and cognitive domain scores of Alzheimer’s patients. Item scores from the Mattis Dementia Rating Scale (MDRS) and the Mini-Mental State Examination (MMSE) from 389 patients with probable Alzheimer’s disease were submitted to principal component analysis with orthogonal rotation. The optimal solution identified four factors that reflected the cognitive domains of attention/registration, verbal fluency/reasoning, graphomotor/praxis and recent memory. A subgroup of patients was identified for whom both the MDRS and the MMSE had been administered within the 12 months before death. Scores were assigned to these patients for the four factors. These cognitive-domain scores were then correlated with postmortem Choline Acetyltransferase (ChAT) activity in the medial frontal cortex, inferior parietal cortex, and hippocampus. ChAT activity in both the medial frontal and the inferior parietal cortex significantly correlated with scores on the graphomotor/praxis factor. Medial frontal ChAT also correlated significantly with the attention/registration scores. Hippocampal ChAT correlated significantly only with recent memory scores. These results are consistent with current animal research regarding the effect of selective Cholinergic lesions on behavior.
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nerve growth factor increases cortical Choline Acetyltransferase positive fiber staining without affecting cortical Cholinergic neurons
Brain Research, 1993Co-Authors: Ad J Dekker, Leon J. ThalAbstract:Lesions of the nucleus basalis magnocellularis (NBM) increased the number of neurons in the frontal neocortex staining for Choline Acetyltransferase (ChAT). Intracerebroventricular treatment with nerve growth factor (NGF; 10μg per day for 6 weeks) did not further increase this number. NGF increased the size of NBM neurons [Brain Res., 584 (1992) 55–63], but not those in the neocortex. However, NGF increased the area of ChAt-positive fiber staining in the neocortex. These data suggest that NGF enhances Cholinergic innervation to the neocortex by affecting residual NBM neurons, rather than cortical Cholinergic neurons.
Ikuo Tooyama - One of the best experts on this subject based on the ideXlab platform.
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human neural stem cells over expressing Choline Acetyltransferase restore cognition in rat model of cognitive dysfunction
Experimental Neurology, 2012Co-Authors: Dongsun Park, Akinori Matsuo, Hong Jun Lee, Seong Soo Joo, Daekwon Bae, Goeun Yang, Yunhui Yang, Inja Lim, Ikuo TooyamaAbstract:A human neural stem cell (NSC) line over-expressing human Choline Acetyltransferase (ChAT) gene was generated and these F3.ChAT NSCs were transplanted into the brain of rat Alzheimer disease (AD) model which was induced by application of ethylCholine mustard aziridinium ion (AF64A) that specifically denatures Cholinergic nerves and thereby leads to memory deficit as a salient feature of AD. Transplantation of F3.ChAT human NSCs fully recovered the learning and memory function of AF64A animals, and induced elevated levels of acetylCholine (ACh) in cerebrospinal fluid (CSF). Transplanted F3.ChAT human NSCs were found to migrate to various brain regions including cerebral cortex, hippocampus, striatum and septum, and differentiated into neurons and astrocytes. The present study demonstrates that brain transplantation of human NSCs over-expressing ChAT ameliorates complex learning and memory deficits in AF64A-cholinotoxin-induced AD rat model.
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the production of antibodies that distinguish rat Choline Acetyltransferase from its splice variant product of a peripheral type
Neurochemistry International, 2007Co-Authors: Shin Kimura, Akinori Matsuo, Jean-pierre Bellier, Ikuo Tooyama, Hiroshi KimuraAbstract:To produce antibodies that permit the immunohistochemical discrimination of Choline Acetyltransferase of the common type (cChAT) from its splice variant of a peripheral type (pChAT), we immunized rabbits with a cChAT specific recombinant protein encoded by ChAT exons 7 and 8 of the rat cChAT gene. Successful antibody production was proved by Western blotting on rat brain and on HEK293 cells expressing green fluorescent protein (GFP), cChAT-GFP and pChAT-GFP. By immunohistochemistry our antiserum clearly labeled known Cholinergic structures in rat brain, but gave no positive staining in the trigeminal ganglion which contained many neurons positive with pChAT antiserum.
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expression of a splice variant of Choline Acetyltransferase in magnocellular neurons of the tuberomammillary nucleus of rat
Neuroscience, 2003Co-Authors: Hiroshi Kanayama, Osamu Yasuhara, Akinori Matsuo, Yoshinari Aimi, Jean-pierre Bellier, Ikuo Tooyama, J I Nagy, Kenji Fukui, Hiroshi KimuraAbstract:Abstract A splice variant of Choline Acetyltransferase mRNA has recently been identified in the pterygopalatine ganglion of rat. An antibody against this variant protein (designated pChAT) was demonstrated to immunolabel peripheral Cholinergic neurons. In the present study, we investigated the expression of pChAT in rat brain. Amongst the brain regions examined, magnocellular neurons in the tuberomammillary nucleus of the posterior hypothalamus were immunohistochemically labelled with anti-pChAT antibody, whilst no immunolabelling was detected in Cholinergic neurons in the basal forebrain or striatum. RT-PCR analysis confirmed the expression of pChAT mRNA in the posterior hypothalamus. The distribution of pChAT-positive neurons in the tuberomammillary nucleus was compared with that of neurons positive for adenosine deaminase, which is contained in all neurons of this nucleus. After colchicine treatment to inhibit axonal transport of enzyme, virtually all pChAT-positive cells contained adenosine deaminase. Conversely, about 85% of adenosine deaminase-positive cells contained pChAT in the ventral area, whilst 19% of adenosine deaminase-positive cells were pChAT-positive in the dorsal area. Long axonal projections of pChAT-positive cells in the tuberomammillary nucleus were shown by retrograde labelling of these cells after injection of cholera-toxin B subunit into the cerebral cortex. This study demonstrates that a splice variant of Choline Acetyltransferase is expressed in the tuberomammillary nucleus of rat. The results raise the possibility that some of the known diverse projection areas of this nucleus may have a Cholinergic component.