Immunohistochemical localization of choline acetyl-transferase in the human cerebellum

Guinea pig antiserum specific to purified bovine choline acetyltransferase was found to cross-react with human enzyme. The peroxidase-antiperoxidase immunohistochemical method was then used to demonstrate the localization of choline acetyltransferase in formalin-fixed and paraffin-embedded human cerebellum from normal as well as from Huntington's disease brains. Choline acetyltransferase was localized exclusively in the mossy fibers and the glomeruli of the cerebellar folia. These immunohistochemical findings reveal the distribution of cholinergic axons and their terminals. The results are not only similar to our previous studies using the same method on the localization of choline acetyltansferase in rabbit cerebellum, but also de onstrate that some mossy fibers are cholinergic as suggested by others.     

The central cholinergic system studied by choline acetyltransferase immunohistochemistry in the cat

An atlas of the distribution of cholinergic cell bodies, fibers, and terminals, as well as cholinoceptive cells, in the central nervous system of the cat (excluding the cerebellum) is presented from results obtained in immunohistochemical work on choline acetyltransferase. Cholinergic cell bodies are observed in more than forty areas, and cholinoceptive cells in sixty discrete areas of brain sections from the spinal cord to the olfactory bulb. The atlas is presented in seventy cross-sectional drawings of cat brain extending from the olfactory bulb to the upper cervical spinal cord.     

Cholinergic innervation of the cerebellum of rat, rabbit, cat, and monkey as revealed by choline acetyltransferase activity and immunohistochemistry.

The cholinergic innervation of the cerebellar cortex of the rat, rabbit, cat and monkey was studied by immunohistochemical localization of choline acetyltransferase (ChAT) and radiochemical measurement of regional differences in ChAT activity. Four antibodies to ChAT were used to find optimal immunohistochemical localization of this enzyme. These antibodies selectively labeled large mossy fiber rosettes as well as finely beaded terminals with different morphological characterization, laminar distribution within the cerebellar cortex, and regional differences within the cerebellum. Large "grape-like" classic ChAT-positive mossy fiber rosettes that were distributed primarily in the granule cell layer were concentrated, but not exclusively located in three separate regions of the cerebellum in each of the four species studied: 1) The uvula-nodulus (lobules 9 and 10); 2) the flocculus-ventral paraflocculus, and 3) the anterior lobe vermis (lobules 1 and 2). No intrinsic cerebellar neurons were labeled. No cells in either the inferior olive (the origin of cerebellar climbing fibers) or in the locus coeruleus (an origin of noradrenergic fibers) were ChAT-positive. Thin, finely beaded axons, similar to cholinergic axons of the cerebral cortex of the rat, were observed in both the granule cell layer and molecular layer of the cerebellar cortex of the rat, rabbit and cat. The regional differences in ChAT-positive afferent terminations in the cerebellar cortex was for the most part confirmed by regional measurements of ChAT activity in the rat, rabbit, and cat. The three cholinergic afferent projection sites correspond to regions of the cerebellar cortex that receive vestibular primary and secondary afferents. These data imply that a subset of vestibular projections to the cerebellar cortex are cholinergic.     

Cholinergic innervation of the human cerebellum

Cholinergic innervation of the human cerebellum was investigated immunocytochemically by using a polyclonal rabbit antiserum against choline acetyltransferase. Immunoreactive structures were found throughout the cerebellar cortex but were localized predominantly in the vermis, flocculus, and tonsilla. These included (1) a population of Golgi cells in the granular layer; (2) a subpopulation of mossy fibers and glomerular rosettes; (3) thin, varicose fibers closely associated with the Purkinje cell layer and the molecular layer; and (4) a relatively dense network of fibers and terminals contributing to the glomerular formations in the granular layer. In the cerebellar nuclei, some cells stained positively for choline acetyltransferase, and a terminal field pattern could be detected with a distinct but sparse network of varicose fibers. Acetylcholine appears to be a primary transmitter in the vestibulocerebellar pathways at several levels, which may account for the potent effects of muscarinic antagonists in diminishing vestibular vertigo in humans. © 1993 Wiley-Liss, Inc.     

Astrocyte overgrowth in the brain stem and spinal cord of mice affected by spinal atrophy, wobbler

Biochemical and immunohistochemical analyses of the central nervous system (CNS) have been performed on the wobbler (WR) mouse, a mutant affected by a spinal atrophy. Total polypeptide patterns as well as specific neuronal and glial marker proteins were analyzed, using 2-dimensional gel electrophoresis, immunoblotting and ELISA, and the determination of enzyme activities. Despite the loss of motoneurons, the specific activities of choline acetyltransferase were not lower in WR than in control CNS samples. On the other hand, the level of glial acidic fibrillary protein (GFAP) was increased 3- to 5-fold in the spinal cord and brain stem of WR mice but not in the cerebellum. Immunohistochemistry indicated that the higher content of GFAP was due to a dense network of astrocytic processes extending over the whole cross-section of the spinal cord. This finding indicates that astrocyte overgrowth may be a fundamental feature of the wobbler disease, rather than occurring locally in response to motoneuron degeneration.     

Topography of choline acetyltransferase Immunoreactive Neurons and fibers in the rat spinal cord

The topography of choline acetyltransferase immunoreactivity was studied in the rat spinal cord with a monoclonal antibody. Cholinergic fibers were most prominent in lamina III of the dorsal horn and originated from cholinergic neurons within the spinal cord. Lamina X, which was rich in cholinergic neurons and fibers, provided cholinergic interconnections between the dorsal, intermediate and ventral gray. Within the ventral gray, choline acetyltransferase immunoreactive boutons were found on motor neurons. This study suggests that the cholinergic innervation of the spinal cord arises from neurons intrinsic to the spinal cord. The cholinergic neurons within the spinal cord may provide several, overlapping levels of regulation of spinal cord neurons.     

Cholinergic deficits in aged rat spinal cord: restoration by GM1 ganglioside

Cholinergic neurons of spinal cord are central for the processing of motor, autonomic, and sensory modalities. Aging is associated with a variety of motor and autonomic symptoms that might be attributed, in part, to impaired spinal cord function. We found that cholinergic neurochemistry is diminished in the spinal cord of 22–24-month-old rats compared with 3-month-old rats. Choline acetyltransferase, high-affinity choline transport and hemicholinium-3 binding to the choline carrier were reduced in the aged spinal cord. The activity of the choline transporter and the hemicholinium-3 binding were decreased in all spinal segments, cervical, thoracic, lumbar and sacral. Hemicholinium-3 binding was reduced in ventral and dorsal horns along all spinal segments. The activity of choline acetyltransferase was decreased only in cervical and lumbar cord. Treatment of aged animals with GM1 induced the recovery of the presynaptic cholinergic markers in the aged spinal cord.     

Calcitonin gene-related peptide and calcitonin immunoreactivity in brain and spinal cord in Alzheimer-type dementia

Calcitonin gene-related peptide (CGRP) and calcitonin (CT) immunoreactivity were measured in hypothalamus, parahippocampal gyrus, pituitary and grey matter of the posterior and anterior spinal cord from five to six cases of Alzheimer-type dementia (ATD) and from five to six controls. CGRP was slightly increased and choline acetyltransferase decreased in the anterior grey of ATD spinal cord. No other significant differences were observed between the levels of the two peptides in the ATD and control tissues, even in the parahippocampal gyrus and posterior grey of the spinal cord which had reduced choline acetyltransferase activity in the ATD cases. These results show that CGRP and CT are not affected in ATD, either as a consequence of a direct effect on peptidergic neurons or secondary to the loss of choline acetyltransferase activity.     

Choline acetyltransferase expression does not identify early pathogenic events in fetal SMA spinal cord

We investigated the expression of choline acetyltransferase, a specific marker for cholinergic neurons, in control and spinal muscular atrophy fetuses and newborns. By immunoblot we observed at 12 and 15 weeks a similar pattern of choline acetyltransferase expression in spinal muscular atrophy with respect to controls, although at 22 weeks this expression was reduced, probably due to a smaller number of motor neurons in the spinal muscular atrophy spinal cord. By immunohistochemistry, the counting of positive and negative motor neurons for choline acetyltransferase immunostaining in control and spinal muscular atrophy fetuses showed a similar proportion at all stages analyzed. The choline acetyltransferase-negative motor neurons were of similar appearance in both groups. After birth, chromatolytic motor neurons were detected in spinal muscular atrophy, all of which were choline acetyltransferase-negative. Our results in spinal muscular atrophy fetuses indicate that choline acetyltransferase immunostaining does not identify early events in neuronal pathogenesis and suggest that the spinal muscular atrophy surviving motor neurons may not be dysfunctional during this period. Furthermore, spinal muscular atrophy choline acetyltransferase-negative motor neurons showed detectable pathological changes only after birth, indicating that choline acetyltransferase is a late marker for motor neuron degeneration and not a primary contributing factor in this process.     

Effect of intravenous transplantation of bone marrow mesenchymal stem cells on neurotransmitters and synapsins in rats with spinal cord injury

Bone marrow mesenchymal stem cells were isolated, purified and cultured in vitro by Percoll density gradient centrifugation combined with the cell adherence method. Passages 3-5 bone marrow mesenchymal stem cells were transplanted into rats with traumatic spinal cord injury via the caudal vein. Basso-Beattie-Bresnahan scores indicate that neurological function of experimental rats was significantly improved over transplantation time (1-5 weeks). Expressions of choline acetyltransferase, glutamic acid decarboxylase and synapsins in the damaged spinal cord of rats was significantly increased after transplantation, determined by immunofluorescence staining and laser confocal scanning microscopy. Bone marrow mesenchymal stem cells that had migrated into the damaged area of rats in the experimental group began to express choline acetyltransferase, glutamic acid decarboxylase and synapsins, 3 weeks after transplantation. The Basso-Beattie- Bresnahan scores positively correlated with expression of choline acetyltransferase and synapsins. Experimental findings indicate that intravenously transplanted bone marrow mesenchymal stem cells traverse into the damaged spinal cord of rats, promote expression of choline acetyltransferase, glutamic acid decarboxylase and synapsins, and improve nerve function in rats with spinal cord injury.     

Alzheimer's disease: choline acetyltransferase activity in brain tissue from clinical and pathological subgroups

Choline acetyltransferase activity was measured postmortem in five brain regions to determine if such activity provided biochemical support for clinical and pathological subgrouping of Alzheimer's disease. Seven patients with Alzheimer's disease were divided into groups based on age at onset, severity of neuropathological changes, history of myoclonus, family history of dementia, cerebellar amyloid plaques, and congophilic angiopathy. Thirty-two age-matched normal control subjects and 17 neurological control patients with Huntington's disease were also studied. Patients with early-onset and late-onset Alzheimer's disease did not differ in the clinical duration of their disease. Choline acetyltransferase activity was significantly lower in patients with early-onset Alzheimer's disease than in age-matched control subjects in frontal cortex, temporal cortex, hippocampus, and cerebellum. In contrast, choline acetyltransferase activity in patients with late-onset Alzheimer's disease was significantly lower than in age-matched control subjects only in hippocampus. There was a tendency for choline acetyltransferase activity to be lower in cortex from patients with early-onset Alzheimer's disease compared with cortex from the late-onset group, and this difference was significant in temporal cortex. Choline acetyltransferase activity was also measured in the substantia innominata from 9 patients with Alzheimer's disease and 5 age-matched control subjects. Subjects with early-onset Alzheimer's disease had significantly lower choline acetyltransferase activity in substantia innominata than did control subjects. Patients with Alzheimer's disease and a history of myoclonus had significantly lower choline acetyltransferase activity than did affected patients without myoclonus. Multivariate regression analysis showed myoclonus to be the single best predictor of low brain choline acetyltransferase activity. These results provide further evidence for clinical, pathological, and biochemical heterogeneity in Alzheimer's disease.     

γ-氨基丁酸能神经元参与热应激的初步形态研究

目的 从形态上证实γ-氨基丁酸(GABA)神经元参与脊髓中间外侧柱胆碱能调节反应,参与热应激时下丘脑对高温调节反应. 方法 出生后6周的C57BL小鼠按随机数字表法分为正常对照组和热刺激组,取颈胸端脊髓及下丘脑组织切片,采用免疫组化双标记方法观察小鼠胸脊髓中间外侧柱胆碱能神经元标志物胆碱乙酰转移酶(CHAT)和谷氨酸脱羧酶65(GAD65)的免疫标记情况以及热应激后下丘脑TRPV4及GAD65分别和c-FOS的免疫双标记.结果 中间外侧柱交感神经节前神经元存在ChAT和GAD65免疫荧光双标记.热激活后,下丘脑热敏感神经元与GABA能神经元存在双标记.结论 GABA能神经元可能参与中间外侧柱交感神经节前胆碱能神经元调节,以及参与下丘脑对高温的反应.     

Expression of cholinergic markers in the developing chick embryo spinal cord

The expression of cholinergic neurotransmission in the developing spinal cord was followed with pre- and postsynaptic cholinergic markers and histoautoradiographic determinations of cholinergic receptors and ultrastructural studies. Two distinct steps in the development of cholinergic markers were evident. The first step of cholinergic expression occurs early in development and its characterized by a sudden and large increase in choline acetyltransferase activity, whereas the high affinity choline uptake mechanism remains undetectable and few muscarinic receptors are detectable. This phenomenon possibly reflects the expression of high choline acetyltransferase gene activity in developing neurons which are still not synaptically connected. The second step is also characterized by a sharp rise of choline acetyltransferase activity. This rise parallels an active development of high affinity choline uptake and big increase in both number and density of muscarinic receptors. Also nicotine receptors increase, as revealed by histoautoradiography. Specific morphogenetic events, such as the increase of synaptic junctions with postsynaptic thickenings and numerous presynaptic clear vesicles, occur at the same time. Such interactions may contribute to the full maturity of cholinergic neurotransmission of the spinal cord.     

Epidural perfusion cooling protects against spinal cord ischemia in rabbits

The protective effect of regional epidural spinal cord cooling was evaluated in a rabbit spinal cord ischemia model. Hypothermia was performed by the continual perfusion of 2–4°C cold saline in the epidural space around the ischemic lumbar segments, 4 min before and during ischemia. The spinal cord was deeply hypothermic (21°C) throughout the whole ischemie period. Ischemia was induced by the occlusion of the abdominal aorta for 40 min under normothermic or hypothermie conditions. Recovery of motor and sensory functions, spinal cord-evoked potentials, and motor-evoked potentials were then evaluated up to 24 h postischemia. After this period, choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activities were measured, in particular, zones of the lumbar spinal cord. AChE was also investigated histochemically. Animals in the normothermic group displayed fully developed spastic paraplegia with near complete loss of spinal somatosensory and motor-evoked potentials. AChE histochemistry showed extensive necrotic changes affecting lumbosacral gray matter. These changes corresponding with the pronounced losses of ChAT and AChE activities indicated irreversible injury of the spinal cord. In contrast, after hypothermic ischemia, animals survived without any sign of neurological impairment with almost full recovery of the spinal cord-evoked potentials. ChAT and AChE activities in the gray matter showed near control values corresponding with histochemical analysis of fully preserved gray matter. Hypothermia under the present experimental conditions efficiently protected the spinal cord against ischemic injury.     

Production of spinal cord growth inhibitor by nonneuronal cells

Separation of chick embryo spinal cord cells into cell cultures of glial-enriched and neuronal-enriched elements by the method of selective adhesiveness indicated a greater production of growth inhibitor by the glial-enriched cultures. Glial-enriched cultures produced 177.2% more inhibitor per microgram cell protein than did unseparated spinal cord cell cultures, whereas neuronal-enriched cultures produced less. The glial-enriched cultures contained 28% of the choline acetyltransferase of neuronal-enriched cultures, few or no discernible neurons, and no cells binding dopamine or containing catecholamines by cytofluorescence. The predominant cell of the glial-enriched cultures was large, phase-light with ruffled borders, with parallel streak-like processes, and fibrillar cytoplasm. Cultures produced by serial passage of unseparated spinal cord cells also were found to be depleted in neurons and dopamine-binding cells, as well as in choline acetyltransferase activity, to 19% of control values. These serially passaged cultures, however, retained the ability to produce inhibitor as well as did primary cultures. Serially passaged cultures of spinal cord cells were composed of cells similar to those predominant in glial-enriched cultures. Nerve growth factor had no effect on inhibitor production. We suggest that the growth inhibitor of spinal cord cultures was not produced by neuronal cells but may be of glial origin.     

Central noradrenaline metabolism in cerebellar ataxic mice

Central noradrenaline (NA) metabolism was investigated in 4 types of ataxic mutant mice, weaver, reeler, staggerer and rolling mouse Nagoya (RMN) and hypocerebellar mice experimentally produced by neonatal treatment with cytosine arabinoside (ara-C). As neurochemical markers for synthesis, steady-state level and turnover of NA, we measured tyrosine hydroxylase (TH) activity, NA and total (free + conjugated) 3-methoxy-4-hydroxyphenylethyleneglycol (total MHPG) concentrations in the cerebellum, cerebral cortex and spinal cord. In the weaver and staggerer whose cerebellar weight loss was severe (40 and 21% of controls, respectively), the 3 neurochemical markers were all increased in the cerebellum, and a similar increase was observed in other regions. In the reeler, the 3 markers were also markedly increased in the cerebellum, but no similar pattern of increase was observed in other regions. In the RMN whose cerebellum showed the least weight loss (76% of control), only TH activity and MHPG concentration were increased in the cerebellum and spinal cord but not in the cerebral cortex. In ara-C-treated mice, the neurochemical markers were also increased in the cerebellum, but to a lesser extent in other regions. These results suggest that, although total contents and total activity of these markers per whole cerebellum were significantly decreased, the central NA metabolism was basically enhanced throughout the CNS of the cerebellar ataxic mice irrespective of their cause. The degree and extent of this enhancement appeared to be correlated with the degree of the cerebellar weight loss.     

Methylmercury poisoning of the developing nervous system in the rat: decreased activity of glutamic acid decarboxylase in cerebral cortex and neostriatum

The specific activities of glutamic acid decarboxylase (GAD) and choline acetyltransferase (ChAT) were measured in 6 regions of the central nervous system in young rats, following chronic postnatal administration of methylmercuric chloride. These rats exhibited signs of neurological impairment which included visual deficits, ataxia, spasticity and myoclonus. At the onset of neurological impairment, there was a significant reduction in GAD activity in the occipital cortex (43%), frontal cortex (37%) and caudate-putamen (42%). Preceding the onset of neurological impairment, diminished GAD activity was detected only in the occipital cortex. In the cerebellum, thalamus and spinal cord, GAD activities were normal throughout the experiment. No significant differences in ChAT activity were detected in any of the 6 regions. These results are consistent with a preferential involvement of GABAergic neurons in methylmercury-induced lesions of the cerebral cortex and neostriatum.     

Immunohistochemical localization of choline acetyltransferase in rabbit forebrain

uinea pig antiserum specific to the purified bovine choline acetyltransferase was used to demonstrate the localization of this enzyme in rabbit forebrain by the peroxidase-antiperoxidase immunohistochemical method. Choline acetyltransferase was localized in olfactory bulb, olfactory tract, olfactory tubercle, piriform cortex, septum, diagonal band, basal ganglia, thalamus, hypothalamus, subthalamus, habenula, cerebral cortex, hippocampal region, corpus callosum, internal capsule, fornix, longitudinal striae and other areas. The findings reflect the distribution of cholinergic axons and, possibly, their terminals. These observations correlate well with biochemical determinations of choline acetyltransferase and with previously proposed cholinergic pathways.     

Distribution of high affinity choline transporter immunoreactivity in the primate central nervous system

A mouse monoclonal antibody (clone 62-2E8) raised against a human recombinant high-affinity choline transporter (CHT)-glutathione-S-transferase fusion protein was used to determine the distribution of immunoreactive profiles containing this protein in the monkey central nervous system (CNS). Within the monkey telencephalon, CHT-immunoreactive perikarya were found in the striatum, nucleus accumbens, medial septum, vertical and horizontal limb nuclei of the diagonal band, nucleus basalis complex, and the bed nucleus of the stria terminalis. Dense fiber staining was observed within the islands of Calleja, olfactory tubercle, hippocampal complex, amygdala; moderate to light fiber staining was seen in iso- and limbic cortices. CHT-containing fibers were also present in sensory and limbic thalamic nuclei, preoptic and hypothalamic areas, and the floccular lobe of the cerebellum. In the brainstem, CHT-immunoreactive profiles were observed in the pedunculopontine and dorsolateral tegmental nuclei, the Edinger-Westphal, oculomotor, trochlear, trigeminal, abducens, facial, ambiguus, dorsal vagal motor, and hypoglossal nuclei. In the spinal cord, CHT-immunoreactive ventral horn motoneurons were seen in close apposition to intensely immunoreactive C-terminals at the level of the cervical spinal cord. CHT immunostaining revealed a similar distribution of labeled profiles in the aged human brain and spinal cord. Dual fluorescent confocal microscopy revealed that the majority of CHT immunoreactive neurons contained the specific cholinergic marker, choline acetyltransferase, at all levels of the monkey CNS. The present observations indicate that the present CHT antibody labels cholinergic structures within the primate CNS and provides an additional marker for the investigation of cholinergic neuronal function in aging and disease.     

Choline acetyltransferase immunohistochemical staining in the goldfish (Carassius auratus) brain: Evidence that the Mauthner cell does not contain choline acetyltransferase

In the hatchetfish, the Mauthner cell (M-cell) is thought to be cholinergic based on electrophysiological studies using cholinergic agents and on the localization of acetylcholinesterase (AChE) and alpha-bungarotoxin to M-cell-giant fiber synapses. Immunocytochemical studies have shown that mammalian and non-mammalian cholinergic neurons stain positive for choline acetyltransferase (ChAT), the enzyme responsible for synthesizing acetylcholine. We processed tissue from the goldfish (Carassius auratus) for the immunohistochemical detection of ChAT using the monoclonal antibody AB8 and the peroxidase-antiperoxidase procedure. ChAT immunoreactivity was found in selected areas of the goldfish brain including the cranial nerve nuclei and the ventral horn motoneurons of the spinal cord. Interestingly, the M-cell soma which stains positive for AChE was ChAT negative. This immunohistochemical evidence does not support cholinergic functioning of the Mauthner cell.