Localization of choline acetyltransferase in human brain stem neurons

Bulletin of Experimental Biology and Medicine -     

Immunohistochemical localization of choline acetyltransferase using a monoclonal antibody: a radioautographic method

Monoclonal antibodies to rat striatal choline acetyltransferase were produced by fusion of sensitized mouse lymphocytes with murine plasmacytoma (NS1) cells. Two stable anti-choline acetyltransferase lines were established by limiting dilution cloning. Specificity of antibody was established by the following criteria: (1) on an enzyme linked immunosorbant assay, antibodies reacted against choline acetyltransferase which was highly purified; (2) by immunoprecipitation, monoclonal antibody bound to its antigen and precipitated choline acetyltransferase activity from solution, when used in conjunction with rabbit antimouse IgG; and (3) monoclonal antibody was shown to specifically localize cholinergic neurons. The monoclonal antibody to choline acetyltransferase was radiolabeled in culture by incubating hybridomas in medium containing 3H-labeled amino acids. This 3H-labeled antibody was used for radioautography on cryostat sections of rat peripheral and central nervous systems. In a sampling of areas, highly specific labeling of cholinergic structures was afforded at both light and electron microscopic levels. Double labeling of tyrosine hydroxylase, a catecholaminergic marker, and choline acetyltransferase was carried out by reacting sections first with the 3H-labeled antibody to choline acetyltransferase and then with rabbit antibody to tyrosine hydroxylase. The choline acetyltransferase label was radioautographically processed and tyrosine hydroxylase was visualized by the peroxidase-antiperoxidase method. The combined techniques of peroxidase and radioautographic histochemistry provide permanent electron dense labels which can be examined simultaneously within a single histologic section.     

Acetylcholinesterase and choline acetyltransferase staining of neurons in the opossum esophagus

The purpose of the present investigation was to identify and compare cholinergic intramural neurons in the lower esophageal sphincter and esophageal body by histochemical staining for acetylcholinesterase and the enzyme that synthesizes acetylcholine, choline acetyltransferase. Opossums were anesthetized and their abdominal cavity was opened by a midline incision to expose the esophagogastric junction. The lower esophageal sphincter was identified manometerically and localized in situ with markers. Tissues were removed, rapidly frozen in freon cooled with liquid nitrogen and serial cryostat sections were obtained from the lower esophageal sphincter and esophageal body. Sections were stained with one of the above histochemical procedures and adjacent sections were stained with Solachrome cyanin , which differentially stains nerve elements from muscle fibers. The muscle of the lower esophageal sphincter and esophageal body was stained with nonspecific cholinesterase with some selectivity of intensity of reaction in the various smooth muscle layers. All identifiable plexus neurons in the esophagus stained for nonspecific cholinesterase and acetylcholinesterase. Nerve fiber tracts were also stained for acetylcholinesterase within the longitudinal and circular layers of the tunica muscularis. Reaction for choline acetyltransferase showed no staining in the muscle layers or nerve fiber tracts of either part of the esophagus studied; however, selected neurons within the myenteric plexus of both regions (approximately 38%) were reactive. There was no significant difference in the number of positive choline acetyltransferase neurons in the lower esophageal sphincter or esophageal body.     

Distribution of cholinergic neurons and fibers in the hypothalamus of the rat using choline acetyltransferase as a marker

The distribution of choline-acetyltransferase-like immunoreactive structures in the rat hypothalamus and preoptic area was examined by using avidin-biotin immunocytochemistry. We found that the hypothalamus is richly innervated by the cholinergic neuron system. Sites containing cholinergic neurons of varying density were: medial and lateral preoptic areas, septohypothalamic nucleus, median preoptic area, lateral hypothalamus including the perifornical area, anterior hypothalamic nucleus, arcuate nucleus, dorsomedial hypothalamic nucleus, posterior hypothalamic nucleus, dorsal and ventral premammilary nuclei, neuropil mediodorsal to the anterior hypothalamic nucleus, neuropil ventral to the anterior hypothalamic nucleus and ventromedial hypothalamic nucleus, neuropil between lateral hypothalamus and ventromedial hypothalamus, and neuropil between dorsal premammilary nucleus and posterior hypothalamic nucleus. There were also many varicose and non-varicose fibers in the preoptic area and hypothalamus. Two kinds of varicose fibers, one with strong immunoreactivity and the other with weak immunoreactivity, were seen. Non-varicose fibers were also detected in the optic chiasma and habenulo-interpeduncular tract. These fibers were passing fibers.     

Immunohistochemical localization of amelogenin in human odontogenic tumors, using a polyclonal antibody against bovine amelogenin

In the present study, we investigated the localization of amelogenin in odontogenic tumors, using an anti-amelogenin polyclonal antibody. In order to make the antibody, antisera against an amelogenin fraction obtained from the enamel matrix of unerupted bovine tooth was raised in rabbits. By Western blot analysis, a main band of 25 kDa and six minor bands (6.8, 12, 18, 20, 23, and 27 kDa) were detected under nonreducing conditions. Immunoreactivity for the amelogenin was observed in ameloblasts and in the immature enamel matrix of 4-day-old rats. In odontogenic tumors, positive reactions for amelogenin were localized in limited areas in adenomatoid odontogenic tumor, calcifying odontogenic cyst, primary intraosseous carcinoma and odontoma. The strongest immunoreactions were shown in enamel matrices in odontomas. Small mineralized foci in epithelial nests showed positive reactions, and a few reactions were observed in epithelium adjacent to the mineralized foci. In calcifying odontogenic cysts, some ghost cells in the lining epithelium were strongly stained. The results indicate that the present antibody for amelogenin is useful for the determination of odontogenic tumors, especially in those in which small mineralized foci are present in the epithelial nests.     

82-kDa choline acetyltransferase is in nuclei of cholinergic neurons in human CNS and altered in aging and Alzheimer disease

Cholinergic neurons express choline acetyltransferase (ChAT) which synthesizes acetylcholine. We show here for the first time that primate-specific 82-kDa ChAT is expressed in nuclei of cholinergic neurons in human brain and spinal cord; isoform-specific antibodies were used to compare localization patterns and temporal expression of the more abundant 69-kDa ChAT and primate-specific 82-kDa ChAT in necropsy tissues. The 82-kDa ChAT co-localizes with 69-kDa ChAT in well-characterized cholinergic areas, but is also found in the claustrum which does not contain 69-kDa ChAT. Cholinergic neuron function changes with increasing age and are targeted in neurodegenerative diseases such as AD, thus we compared expression and subcellular localization of 69- and 82-kDa ChAT in necropsy brain samples from control subjects of varying ages and from Alzheimer disease (AD) subjects. The 82-kDa ChAT protein was expressed in cholinergic neurons in brain from birth until the eighth decade of life and in AD, but the subcellular staining pattern and proportion of neurons that were immunopositive changed with increasing age and in AD.     

Immunohistochemical localization of two types of choline acetyltransferase in neurons and sensory cells of the octopus arm

Cholinergic structures in the arm of the cephalopod Octopus vulgaris were studied by immunohistochemistry using specific antisera for two types (common and peripheral) of acetylcholine synthetic enzyme choline acetyltransferase (ChAT): antiserum raised against the rat common type ChAT (cChAT), which is cross-reactive with molluscan cChAT, and antiserum raised against the rat peripheral type ChAT (pChAT), which has been used to delineate peripheral cholinergic structures in vertebrates, but not previously in invertebrates. Western blot analysis of octopus extracts revealed a single pChAT-positive band, suggesting that pChAT antiserum is cross-reactive with an octopus counterpart of rat pChAT. In immunohistochemistry, only neuronal structures of the octopus arm were stained by cChAT and pChAT antisera, although the pattern of distribution clearly differed between the two antisera. cChAT-positive varicose nerve fibers were observed in both the cerebrobrachial tract and neuropil of the axial nerve cord, while pChAT-positive varicose fibers were detected only in the neuropil of the axial nerve cord. After epitope retrieval, pChAT-positive neuronal cells and their processes became visible in all ganglia of the arm, including the axial and intramuscular nerve cords, and in ganglia of suckers. Moreover, pChAT-positive structures also became detectable in nerve fibers connecting the different ganglia, in smooth nerve fibers among muscle layers and dermal connective tissues, and in sensory cells of the suckers. These results suggest that the octopus arm has two types of cholinergic nerves: cChAT-positive nerves from brain ganglia and pChAT-positive nerves that are intrinsic to the arm.     

Immunohistochemical localization of choline acetyltransferase of a peripheral type in the rat larynx

As shown in the accompanying paper, choline acetyltransferase, so far the best histochemical marker for identifying cholinergic structures, has at least one alternative splice variant. The variant, termed pChAT because of its preferential expression in peripheral organs, encouraged us to study peripheral, probably cholinergic, cells and fibers by immunohistochemistry using an antiserum against a peptide specific for pChAT. We chose the larynx of the rat, since cholinergic innervation in this organ has been well established by physiological studies, but not sufficiently by chemical neuroanatomy. Neuronal somata positive for pChAT were found in the intralaryngeal ganglia. Our double staining study indicated that these somata always possessed acetylcholinesterase activity, while the reverse did not hold true. Nerve fibers positive for pChAT were distributed widely in the intrinsic laryngeal muscles, laryngeal glands, blood vessels and laryngeal mucosa. In the intrinsic laryngeal muscles, pChAT-positive terminals were apposed closely to motor end-plates which were stained positively for acetylcholinesterase activity. Denervation experiments revealed that there were three types of pChAT-positive fibers in the larynx: (1) special visceral efferent fibers to the intrinsic laryngeal muscles, which decreased dramatically in number after vagotomy; (2) parasympathetic postganglionic fibers near the laryngeal glands and blood vessels, which appeared unaffected after vagotomy or cervical sympathectomy: and (3) afferent fibers innervating the laryngeal mucosa, which reduced markedly in number after vagotomy performed distal, but not proximal, to the nodose ganglion. Such afferent fibers remained unchanged following the neonatal capsaicin treatment, suggesting their independence from those containing substance P.     

Immunohistochemistry of choline acetyltransferase in the guinea pig brain

Choline acetyltransferase (ChAT) was localized immunohistochemically within the brain of the guinea pig using a monoclonal antibody. ChAT was found in the cytoplasm of cell bodies and primary dendrites of neurons located in striatum, basal forebrain, cranial nerve motor nuclei and scattered cells in the pons. The greatest numbers of immunoreactive neurons were located in the diagonal band of Broca, medial septum and striatum. Distinct immunoreactive fibers were not visible using this antibody, although a diffuse immunostaining was present in the same nuclear regions as well as in the nerve roots of cranial nerve nuclei and the interpeduncular nuclei. Results of the present study agree closely with other previous reports of acetylcholine distributions.     

Ultracytochemical observations of choline acetyltransferase neurons in the rat neostriatum

Summary The fine structural localization of ChAc activity was studied in the rat neostriatum by a ultracytochemical method. The reaction products of ChAc activity were seen in the cisternal structures and plasma membrane of some medium-sized neurons. Some boutons with ChAc-positive vesicles were observed to make an axo-dendritic or axo-somatic symmetrical synapse.     

TAK-147, an acetylcholinesterase inhibitor, increases choline acetyltransferase activity in cultured rat septal cholinergic neurons

Although it is known that the brain can be injured by mechanical forces initiated at the moment of impact during trauma, it is not clear how the physical response of the brain dictates the injury patterns that occur in experimental models of traumatic brain injury. In this study, we investigated the mechanical response of the brain to a technique that creates a focal injury in the rat brain. Using a transient vacuum pulse applied to the exposed cortical surface, we found that the displacement of the cortex and the extent of in vivo blood-brain barrier breakdown were related significantly to the vacuum pressure level. The relationship between the response of the cortex and injury pattern points towards a new opportunity for control of the distribution and extent of injury patterns in animal models through a precise understanding of the model biomechanics, as well as potential improvements in means of preventing traumatic brain injury.     

Ultrastructural identification of cholinergic neurons in the external cuneate nucleus of the gerbil: acetylcholinesterase histochemistry and choline acetyltransferase immunocytochemistry

Summary Using acetylcholinesterase histochemical and choline acetyltransferase immunocytochemical localization methods, this study has provided conclusive evidence for the existence of cholinergic neurons in the external cuneate nucleus of gerbils. By light microscopy, both acetylcholinesterase and choline acetyltransferase labelling was confined to the rostral portion of the external cuneate nucleus. Ultrastructurally, acetylcholinesterase reaction products were found in the nuclear envelope, cisternae of rough endoplasmic reticulum and Golgi saccules of some somata and large dendrites as well as in the membranes of small dendrites, myelinated axons and axon terminals. These neuronal elements were also stained for choline acetyltransferase; immunoreactivity was associated with nuclear pores, nuclear envelope, perikaryal membrane and all the membranous structures within the cytoplasm. Of the total choline acetyltransferase-labelled neuronal profiles analysed, 79% were myelinated axons, 15% dendrites, 4% somata and 2% axon terminals. The immunostained axon terminals consisted of two types containing either round (Rd type; 62.5%) or pleomorphic (Pd type; 37.5%) vesicles. Both were associated directly with choline acetyltransferase-positive dendrites. In contrast to the paucity of choline acetyltransferase-labelled axon terminals, numerous choline acetyltransferase-positive myelinated axons were present. It may thus be hypothesized that most, if not all, of the external cuneate nucleus cholinergic neurons are projection cells; such cells may give rise to axonal collaterals which synapse onto their own dendrites for possible feedback control. Choline acetyltransferase-positive dendrites were contacted by numerous unlabelled presynaptic boutons, 60% of which contained round or spherical synaptic vesicles (Rd boutons) and 40% flattened vesicles (Fd boutons), suggesting that these neurons are under strong inhibitory control. The preferential concentration of cholinergic components in the rostral external cuneate nucleus may be significant in the light of the highly organized somatotopy in the external cuneate nucleus and its extensive efferent projections to medullary autonomic-related nuclei. Our results suggest that the cholinergic neurons may be involved in somatoautonomic integration.     

Characterization of cholinergic neurons in the rat neostriatum. A combination of choline acetyltransferase immunocytochemistry, Golgi-impregnation and electron microscopy

Immunocytochemistry with a monoclonal antibody against choline acetyltransferase has been used to characterise cholinergic neurons in the rat neostriatum. The light microscopic morphology, ultrastructure and synaptic input of these neurons was compared to that of the three types of large neuron found in Golgi preparations of the striatum. The cholinergic neurons are large and have long infrequently branching dendrites. Two of the immunoreactive neurons were also Golgi-impregnated and showed characteristics of the "classical" large neurons of the striatum. Examination in the electron microscope revealed that the synaptic input to perikarya and proximal dendrites is sparse, thus distinguishing them from another large type of neuron, found in the ventral regions of the striatum, whose dendrites and perikarya are ensheathed in synaptic boutons. It is concluded that one of the three morphologically distinguishable classes of large neuron in the striatum is a cholinergic neuron.     

Nerve growth factor increases choline acetyltransferase but not survival or fiber outgrowth of cultured fetal septal cholinergic neurons

Neurons dissociated from the septal area of fetal rat brains were grown in culture. Cholinergic neurons were identified by immunocytochemical visualization of choline acetyltransferase and cytochemical demonstration of acetyl cholinesterase. Choline acetyltransferase immunocytochemistry stained cell bodies and proximal processes while acetylcholinesterase cytochemistry visualized the entire neuron. Choline acetyltransferase-positive neurons could only be identified in cultures grown under conditions that produced the maximal choline acetyltransferase activity, measured biochemically. All of the choline acetyltransferase-positive neurons were double stained for acetylcholinesterase while only 6% of the acetylcholinesterase-positive cells were choline acetyltransferase negative in these cultures. These results indicate that acetylcholinesterase is a reliable marker for cholinergic cells in cultures of dissociated septal neurons. Being the more sensitive method, acetylcholinesterase staining was therefore used to identify cholinergic cells in cultures with choline acetyltransferase levels insufficient for immunocytochemical visualization of this enzyme. Addition of nerve growth factor or antibodies to nerve growth factor to the medium did not affect the number of cholinergic neurons surviving in culture. Furthermore, nerve growth factor and anti-nerve growth factor failed to influence the general morphological appearance and the number of processes of these neurons. However, nerve growth factor elevated the biochemically measured activity of choline acetyltransferase up to two-fold. The nerve growth factor-mediated increase in choline acetyltransferase activity was dose dependent with an ED50 of 10 ng/ml (4 X 10(-10) M). The increase was highly specific for nerve growth factor. It was blocked by anti-nerve growth factor, and epidermal growth factor, insulin and other control proteins failed to exert a similar effect. Nerve growth factor had to be present for at least 3 days in the culture medium to increase choline acetyltransferase activity, suggesting that the increase was due to an elevated choline acetyltransferase synthesis rather than to an activation of the enzyme.     

Lateralization of choline acetyltransferase (ChAT) activity in fetus and adult human brain

Choline acetyltransferase (ChAT) activity was measured in the first temporal gyrus (Brodmann area 22) and in the gyrus precentrale (Brodmann area 4) of both hemispheres in post-mortem human fetus and adult brains. In fetal brains the ChAT values were higher in the right first temporal gyrus in comparison to the left one (P < 0.05). Conversely in adult brains ChAT activity was higher in the left first temporal gyrus than in the controlateral one (P < 0.05). Absolute values of ChAT were similar in the right first temporal gyrus of fetus and adult brains. No asymmetry was found in the fetal and adult gyrus precentrale. These findings could be interpreted in terms of differential rates of development of cholinergic synapses in left and right human temporal lobes.     

Production of specific antibodies to choline acetyltransferase purified from pig brain

Choline acetyltransferase was purified from pig brain and a specific antiserum prepared in a rabbit. The final specific activity after a purification factor of 1,180,000 was 135 μmole × min^?1 × mg prot.^?1 with a yield of about 10%. The purity of this preparation was estimated to be 80% by sodium dodecyl sulphate-gel electrophoresis, the apparent molecular weight of the main band was 67,000. This preparation was used for initial immunization of a rabbit, which subsequently was injected with choline acetyltransferase preparations cut out of the electrophoresis gel. After 4 such additional injections, the rabbit antiserum could precipitate more than 95% of the enzymic activity present in a pig-brain homogenate by either direct or indirect immunoprecipitation. Double immunodiffusion showed that although a precipitation line could be seen with a choline acetyltransferase preparation of high specific activity, no such line was seen using a preparation having 4 times the specific activity of the starting homogenate. This result is in accordance with the prediction (Rossier, 1975) that the amount of choline acetyltransferase present in mammalian tissue extracts is too low to be visualized directly by double immunodiffusion tests.Such a specific antiserum against choline acetyltransferase should be useful as a tool to characterize unambiguously cholinergic neurons, in a way that has not been possible so far.     

Atlas of cholinergic neurons in the forebrain and upper brainstem of the macaque based on monoclonal choline acetyltransferase immunohistochemistry and acetylcholinesterase histochemistry

Choline acetyltransferase immunohistochemistry was used to map the cholinergic cell bodies in the forebrain and upper brainstem of the macaque brain. Neurons with choline acetyltransferase-like immunoreactivity were seen in the striatal complex, in the septal area, in the diagonal band region, in the substantia innominata, in the medial habenula, in the pontomecencephalic tegmentum and in the oculomotor and trochlear nuclei. The ventral striatum contained a higher density of cholinergic cell bodies than the dorsal striatum. All of the structures that contained the choline acetyltransferase positive neurons also had acetylcholinesterase-rich neurons. Choline acetyltransferase positive neurons were not encountered in the cortex. Some perikarya in the midline, intralaminar, reticular and limbic thalamic nuclei as well as in the hypothalamus were rich in acetylcholinesterase but did not give a positive choline acetyltransferase reaction. A similar dissociation was observed in the substantia nigra, the raphe nuclei and the nucleus locus coeruleus where acetylcholinesterase-rich neurons appeared to lack perikaryal choline acetyltransferase activity.     

Monoclonal antibodies to monkey brain choline acetyltransferase: production and immunohistochemistry

Two monoclonal antibodies to monkey brain choline acetyltransferase (ChAT) were obtained. Immunoblot analysis indicated that both antibodies revealed two adjacent protein bands on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis. ChAT was demonstrated immunohistochemically by one of the antibodies in the motoneurons in the monkey brainstem and spinal cord. This antibody also revealed ChAT-positive terminal-like structures in the neuropils of lamina IX of the cervical spinal cord and interpeduncular nucleus.     

Cat 'P300' and cholinergic septohippocampal neurons: depth recordings, lesions, and choline acetyltransferase immunohistochemistry.

The role of septohippocampal circuits in the generation of the P300 response in cats (n = 12) was explored in a series of depth recording, tract-tracing and lesion experiments. Systematic mapping of the hippocampus in 1-mm increments from rostral to caudal extent revealed large positive potentials, greater in amplitude to rare than to frequent stimuli, within the 200-500 ms range. Each map revealed maximal amplitude responses at diverse, widely distributed hippocampus loci. Furthermore, these electrical responses displayed polarity inversion within the hippocampus that was generally localized to the pyramidal cell layer; polarity inversion was also observed in the adjacent entorhinal cortex and amygdala. Injections of propidium iodide, a tract-tracing agent, into these inversion sites resulted in retrograde labeling of small clusters of choline acetyltransferase (ChAT)-positive neurons in the medial septal nucleus and vertical limb of the diagonal band. Aspiration lesions that bilaterally destroyed large amounts of caudal hippocampus from stereotaxic levels A4 to A1 resulted in a decreased number of cells expressing ChAT in the rostral basal nuclear complex. In only 2 cats was the preoperative presence of a significant vertex P300 absent postoperatively. In the majority of cases (5 of 8 animals), hippocampal aspiration produced an enhancement of the preoperative P300 potential. We conclude that cholinergic mechanisms are importantly, albeit not exclusively, involved in the mediation of P300 potentials in cats. Neurons mediating P300 responses appear to be organized in diverse clusters of septal and diagonal band cells. These septal cells may facilitate, and in turn be facilitated or inhibited as a function of hippocampal, or other, allocortical feedback loops.     

GM1 and NGF synergism on choline acetyltransferase and choline uptake in aged brain

In the brain of aged rats high affinity choline uptake (HAChU) of the striatum, hippocampus, and frontal cortex is lower than in young rats, while choline acetyltransferase (ChAT) activity is lower in striatum and frontal cortex. Infusion into the lateral cerebral ventricle with nerve growth factor (NGF) enhances the low values of these cholinergic markers in a dose- and region-dependent manner. GM 1 ganglioside infused into the lateral ventricle, at a dose that is ineffective alone, together with NGF synergistically enhances the effect of NGF on ChAT and HAChU activities in the brain of aged animals. The pharmacology of this GM 1/NGF synergism suggests potentiation of response.