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.     

Immunocytochemical study of a temperature-sensitive choline acetyltransferase mutant of Drosophila melanogaster

Using a monoclonal antibody to choline acetyltransferase (ChAT), we have identified immunoreactive synaptic terminals in the neuropil regions of the cephalic ganglion of Drosophila melanogaster. This study demonstrates the distribution of antibody-labeled structures within the optic lobe, and then investigates the immunoreactivity altered by mutation in two temperature-sensitive ChAT alleles, chats-1 and chats-2. The general structure of the optic lobe was first observed by means of the silver impregnation technique. Then the presence of ChAT immunoreactivity was determined by the application of antibody [1G4] conjugated with HRP to frozen sections, followed by the 3,3'-diamino-benzidine tetratinct layers, which correspond to the three synaptic layers of the laminarneurons, in the medulla. Also, staining appeared in four distinct layers in the lobula. In addition, weaker staining was observed in the lamina, which corresponds to the retinula cell terminals. Somal layers were not stained. In Canton-S (wild-type), the three medullar layers stain distinctly at both 19 degrees C and 30 degrees C. In chats-1 at 19 degrees C, the stain appeared in the same layers as that of Canton-S, but with somewhat lower density. In chats-2 at 19 degrees C, the density of the stain was even lower. The densities of the stain in these mutants were further decreased after exposing the flies to 30 degrees C. The decreases were dependent on the length of exposure to the higher temperature. The decrease in stain of the specimens obtained after 24 hours exposure to 30 degrees C was clearly recognizable in both chats-1 and chats-2.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Distributions of choline acetyltransferase and acetylcholinesterase activities in the retinal layers of the red-tailed hawk and road runner.

The activities of choline acetyltransferase and acetylcholinesterase were assayed in submicrogram samples from layers of red-tailed hawk and road runner retina. Both enzyme activities were concentrated in and near the inner plexiform layer. Within the inner plexiform layers of both species, activities of each enzyme were concentrated in two bands, one in each half of this layer. Little choline acetyltransferase activity was found superficial to the middle third of the inner nuclear layer. The distributions of acetylcholinesterase activities corresponded well to those of choline acetyltransferase, except in the outer plexiform layer and the outer margin of the inner nuclear layer of the hawk. These distributions of enzyme activities indicate that populations of amacrine cells in the retinae of these species are cholinergic. In addition to these same cells and presumably cholinoceptive amacrine and ganglion cells, acetylcholinesterase activity in the hawk was associated with a population of horizontal cells that may be unrelated to synaptic cholinergic neurotransmission. Choline acetyltransferase activities associated with amacrine somata and processes were about four times greater in the hawk than in the road runner, suggesting important differences in the density and function of cholinergic elements between species. Possible synaptic relationships in the inner plexiform layer consistent with the interspecies differences in enzyme activities are considered.     

Distribution of choline acetyltransferase immunoreactive axons and terminals in the rat and ferret brainstem.

A survey was made of the density of the cholinergic innervation of different parts of the brainstem of the rat and ferret. Sections of rat and ferret brainstems were stained for choline acetyltransferase (ChAT) immunoreactivity by using a sensitive immunocytochemical method. Adjacent sections were stained for acetylcholinesterase activity or Nissl substance. The density of the distribution of fine calibre, varicose ChAT-positive axons, assumed to represent cholinergic terminals, was categorised arbitrarily into high, medium, or low. A high density of ChAT-positive terminals was found in all or parts of these structures: interpeduncular nucleus, superficial grey layer of the superior colliculus (ferret), intermediate layers of the superior colliculus, lateral part of the central grey (rat), an area medial to the parabigeminal nucleus (rat), pontine nuclei, ventral tegmental nucleus (rat), midline pontine reticular formation, and an area ventral to the exit point of the 5th nerve (ferret). A medium density of ChAT-positive terminals was observed in all or parts of: the substantia nigra zona compacta (ferret), ventral tegmental area (ferret), superficial grey layer of the superior colliculus, intermediate and deep layers of the superior colliculus, lateral central grey, area medial to the parabigeminal nucleus, inferior colliculus, dorsal tegmental nucleus, ventral tegmental nucleus (ferret), pontine nuclei, ventral nucleus of the lateral lemniscus (ferret), midline pontine reticular formation, ventral cochlear nucleus, dorsal cochlear nucleus, lateral superior olive, spinal trigeminal nuclei, prepositus hypoglossal nucleus, lateral reticular nucleus, paragigantocellular nucleus, and the dorsal column nuclei including the cuneate, external cuneate, and gracile nuclei. A low density of ChAT-positive terminals was seen throughout the remainder of the brainstem of the rat and ferret, but these terminals were absent from the medial superior olive, substantia nigra zona reticulata (rat), and the central part of the ferret lateral superior olive. A pericellular-like distribution of ChAT-positive terminals was observed in the ventral cochlear nucleus and in association with some of the cells of the nucleus of the mesencephalic tract of the trigeminal nerve. A climbing fibre type arrangement of ChAT-positive terminals was found in the substantia nigra zona compacta (ferret) and medial reticular formation. In general, the distribution of staining for AChE activity reflected that of the distribution of ChAT immunoreactivity in the brainstem, except in a few regions where there were also species differences in the distribution of ChAT-positive terminals, e.g., in the superficial grey layer of the superior colliculus and in the substantia nigra.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of enucleation on choline acetyltransferase activity in layers of goldfish optic tectum

Choline acetyltransferase (ChAT) activity was determined in layers of optic tectum in control goldfish and in goldfish 4-20 days following unilateral enucleation. Significant changes in activity were found in the periventricular (PV) and superficial gray and white (SGW) layers. Within 4 days, ChAT activity in the PV layer on the lesioned side was about 75% of that on the control side. By 20 days, ChAT specific activity in the SGW layer on the lesioned side was about 150-160% of that on the control side. This increase in specific activity in the SGW layer was accounted for by the decrease in volume and in density of the layer after enucleation, so that the total amount of activity in the layer did not change significantly, indicating that the optic terminals contain little to no ChAT activity. ChAT activity in the optic tract was very low and did not decrease after enucleation. These data strongly indicate that the retinotectal pathway in goldfish is not cholinergic and, therefore, that the ChAT activity in the SGW layer is related to sources other than retinal ganglion cells. It is suggested that one such source might be neurons with somata in the PV layer.     

Differential cholinergic innervation within functional subdivisions of the human cerebral cortex: a choline acetyltransferase study.

The distribution of cholinergic fibers in the human brain was investigated with choline acetyltransferase immunocytochemistry in 35 cytoarchitectonic subdivisions of the cerebral cortex. All cortical areas and all cell layers contained cholinergic axons. These fibers displayed numerous varicosities and, on occasion, complex preterminal profiles arranged in the form of dense clusters. The density of cholinergic axons tended to be higher in the more superficial layers of the cerebral cortex. Several distinct patterns of lamination were identified. There were also major differences in the overall density of cholinergic axons from one cytoarchitectonic area to another. The cholinergic innervation of primary sensory, unimodal, and heteromodal association areas was lighter than that of paralimbic and limbic areas. Within unimodal association areas, the density of cholinergic axons and varicosities was significantly lower in the upstream (parasensory) sectors than in the downstream sectors. Within paralimbic regions, the non-isocortical sectors had a higher density of cholinergic innervation than the isocortical sectors. The highest density of cholinergic axons was encountered in core limbic structures such as the hippocampus and amygdala. These observations show that the cholinergic innervation of the human cerebral cortex displays regional variations that closely follow the organization of information processing systems.     

Effects of trimethyltin (TMT) on choline acetyltransferase activity in the rat hippocampus

Trimethyltin (TMT) destroys specific subfields of the hippocampus in the rat. TMT also increases choline acetyltransferase (ChAT) activity in CA1 of Ammon’s horn and the outer molecular layer of the dentate gyrus. This observation suggests that axonal sprouting occurs in the cholinergic septohippocampal system in response to TMT. However, neither does-response nor time course data are available for the effects of TMT on this enzyme. The effects of three dose levels of TMT on ChAT activity in CA1 and the dentate gyrus were determined in Experiment 1 and ChAT activity in these two areas was measured at six time points following exposure to TMT in Experiment 2. Only the highest dose of TMT (6 mg/kg) significantly increased ChAT activity. ChAT activity in the dentate gyrus increased significantly by 3 d after administration and continued to increase until 21 d after exposure. A significant increase was not observed in CA1 until 7 d after exposure to TMT. Asymptotic levels were still reached at d 21. These results indicate a steep dose-response curve for TMT-induced changes in ChAT activity in the hippocampal formation and that this marker of cholinergic activity is more sensitive to perturbation by TMT in the dentate gyrus than Ammon’s horn.     

Immunohistochemical localization of choline acetyltransferase in rabbit spinal cord and cerebellum

Guinea pig antiserum specific for purified bovine choline acetyltransferase has been shown to cross-react with rabbit enzyme. We used the peroxidase-antiperoxidase immunohistochemical method to demonstrate the localization of choline acetyltransferase in formalin-fixed and paraffin-embedded sections of rabbit spinal cord and cerebellum. In the spinal cord, in agreement with our and others' previous results using immunofluorescent techniques, choline acetyltransferase was found in the cell bodies of the ventral horn motor neurons. In the cerebellum, choline acetyltransferase was localized exclusively in the mossy fibers and the glomeruli of the cerebellar folia. The immunohistochemical findings in the cerebellum reveal the morphological detail of cholinergic axons and their terminals. The results are consistent with published biochemical data on the cerebellar distribution of choline acetyltransferase.     

Ultrastructural localization of high-affinity choline transporter in the rat neuromuscular junction: enrichment on synaptic vesicles

In cholinergic neurons, Na(+)- and Cl(-)-dependent, hemicholinium-3-sensitive, high-affinity choline uptake system is thought to be the rate-limiting step in acetylcholine (ACh) synthesis. The system is highly regulated by neuronal activity; the choline uptake is increased by a condition in which ACh release is favored. Here we analyzed the ultrastructural localization of the high-affinity choline transporter (CHT) in the rat neuromuscular junctions with two separate antibodies. The majority (>90%) of immunogold labeling of CHT was observed on synaptic vesicles rather than the presynaptic plasma membrane. Less than 5% of the gold-silver particles were associated with the plasma membrane, and more than 70% of such particles were localized within or in close vicinity to presynaptic active zones. Our morphological data support the recent hypothesis that trafficking of CHT from synaptic vesicles to the plasma membrane couples neuronal activity and choline uptake.     

Immunohistochemical localization of choline acetyltransferase in the central nervous system of the locust

Immunohistochemistry of the locust central nervous system with antibody to choline acetyltransferase (ChAT) purified from the same species shows: first, there are relatively few immunoreactive cell bodies in the CNS; and second, sensory neuropiles, such as the ventral association centre and the ventral VAC (vVAC), the anterior ring tract, the tritocerebrum and the antennal lobe, are immunoreactive. That ChAT is contained in sensory neurones is suggested by immunoreactivity found in peripheral neurone cell bodies. These results indicate that acetylcholine serves primarily as a sensory transmitter in the locust.     

Immunohistochemical localization of serotonin and choline acetyltransferase in sensory neurones of the locust

Sensory neuronal cell bodies in the leg of locust, Schistocerca gregaria, were visualized with antibodies to locust choline acetyltransferase and with antibodies to serotonin by the avidin-biotin peroxidase technique. Two groups of sensory cells react with the antibody to choline acetyltransferase: One group is associated with external mechanoreceptors (i.e., hair-plate hairs and campaniform sensilla) and the other with internal proprioceptors (i.e., chordotonal organs and multiterminal receptors). Sensory cells which react with the antibody to serotonin are associated only with internal proprioceptors being found in both chordotonal organs and multiterminal receptors. In the metathoracic femoral chordotonal organ indirect evidence suggests that some sensory cells are reactive to both antibodies. Some multiterminal receptors react with anti-choline-acetyltransferase, while others react with antiserotonin. These results support the conclusion that most insect sensory neurones are cholinergic but some are serotoninergic.     

Immunocytochemical localization of choline acetyltransferase in rat cerebral cortex: a study of cholinergic neurons and synapses

Choline acetyltransferase (ChAT), the acetylcholine-synthesizing enzyme and a definitive marker for cholinergic neurons, was localized immunocytochemically in the motor and somatic sensory regions of rat cerebral cortex with monoclonal antibodies. ChAT-positive (ChAT+) varicose fibers and terminal-like structures were distributed in a loose network throughout the cortex. Some immunoreactive cortical fibers were continuous with those in the white matter underlying the cortex, and many of these fibers presumably originated from subcortical cholinergic neurons. ChAT+ fibers appeared to be rather evenly distributed throughout all layers of the motor cortex, but a subtle laminar pattern was evident in the somatic sensory cortex, where lower concentrations of fibers in layer IV contrasted with higher concentrations in layer V. Electron microscopy demonstrated that immunoreaction product was concentrated in synaptic vesicle-filled profiles and that many of these structures formed synaptic contacts. ChAT+ synapses were present in all cortical layers, and the majority were of the symmetric type, although a few asymmetric ones were also observed. The most common postsynaptic elements were small to medium-sized dendritic shafts of unidentified origin. In addition, ChAT+ terminals formed synaptic contacts with apical and, probably, basilar dendrites of pyramidal neurons, as well as with the somata of ChAT-negative nonpyramidal neurons. ChAT+ cell bodies were present throughout cortical layers II-VI, but were most concentrated in layers II-III. The somata were small in size, and the majority of ChAT+ neurons were bipolar in form, displaying vertically oriented dendrites that often extended across several cortical layers. Electron microscopy confirmed the presence of immunoreaction product within the cytoplasm of small neurons and revealed that they received both symmetric and asymmetric synapses on their somata and proximal dendrites. These observations support an identification of ChAT+ cells as nonpyramidal intrinsic neurons and thus indicate that there is an intrinsic source of cholinergic innervation of the rat cerebral cortex, as well as the previously described extrinsic sources.     

Long-term ovarian hormone deprivation alters the ability of subsequent oestradiol replacement to regulate choline acetyltransferase protein levels in the hippocampus and prefrontal cortex of middle-aged rats

The role of oestrogen replacement therapy in preventing or delaying age-associated cognitive decline is controversial. Therapy success may critically depend on the time of treatment initiation following cessation of ovarian function. The present study aimed to assess, in middle-aged rats, whether the ability of oestradiol to modulate the cholinergic system depends on the timing of treatment initiation following ovariectomy. Using western blotting, protein levels of choline acetyltransferase (ChAT) were measured in the hippocampus and prefrontal cortex (PFC), which are both important areas with respect to cognitive function. In an initial experiment, we established the effects of oestradiol delivered via implanted capsules on ChAT levels in the hippocampus and PFC of young adult animals. In a second experiment, we tested the ability of the same oestradiol treatment paradigm to affect ChAT protein in 15-month-old middle-aged rats that had been ovariectomised either at the age of 10 months or at 15 months. In both experiments, rats were sacrificed 10 days after receiving implants and ChAT protein levels were measured. In both young adult and middle-aged animals, oestradiol treatment initiated immediately after ovariectomy significantly increased ChAT levels in the hippocampus but not in the PFC compared to cholesterol control treatment. However, when oestradiol treatment was initiated 5 months after ovariectomy, it failed to significantly increase ChAT levels in the hippocampus, but did so in the PFC. These data indicate that, after prolonged ovarian hormone deprivation, the ability of subsequent oestradiol treatment to modulate ChAT protein levels is altered in a site-specific manner.     

Distribution of cholinergic neurons in rat brain: demonstrated by the immunocytochemical localization of choline acetyltransferase

The neuroanatomical location and cytological features of cholinergic neurons in the rat brain were determined by the immunocytochemical localization of the biosynthetic enzyme, choline acetyltransferase (ChAT). Perikarya labeled with ChAT were detected in four major cell groups: (1) the striatum, (2) the magnocellular basal nucleus, (3) the pontine tegmentum, and (4) the cranial nerve motor nuclei. Labeled neurons in the striatum were observed scattered throughout the neostriatum (caudate, putamen) and associated areas (nucleus accumbens, olfactory tubercle). Larger ChAT-labeled neurons were seen in an extensive cell system which comprises the magnocellular basal nucleus. This more or less continuous set of neuronal clusters consists of labeled neurons in the nucleus of the diagonal band (horizontal and vertical limbs), the magnocellular preoptic nucleus, the substantia innominata, and the globus pallidus. Labeled neurons in the pontine tegmentum were seen as a group of large neurons in the caudal midbrain, dorsolateral to the most caudal part of the substantia nigra, and extended in a caudodorsal direction through the midbrain reticular formation into the area surrounding the superior cerebellar peduncle. The neurons in this latter group constitute the pedunculopontine tegmental nucleus (PPT). An additional cluster of cells was observed medially adjacent to the PPT, in the lateral part of the central gray matter at the rostral end of the fourth ventricle. This group corresponds to the laterodorsal tegmental nucleus. Large ChAT-labeled neurons were also observed in all somatic and visceral motor nerve nuclei. The correspondence of the distribution of ChAT-labeled neurons identified by our methods to earlier immunocytochemical and acetylcholinesterase histochemical studies and to connectional studies of these groups argues for the specificity of the ChAT antibody used.     

Inhibitors of serine/threonine phosphatases increase membrane-bound choline acetyltransferase activity and enhance acetylcholine synthesis

The present investigation examines the effects of phosphatase inhibition on short-term regulation of cholinergic function, with particular emphasis on choline acetyltransferase, the enzyme which synthesizes acetylcholine. Rat hippocampal synaptosomes were treated with either okadaic acid (10 nM) or calyculin-A (50 nM) to inhibit protein phosphatases 1 and 2A for 20 min prior to subfractionation of nerve terminals and measurement of choline acetyltransferase activity, or quantification of high-affinity choline transport and acetylcholine synthesis. Inhibition of synaptosomal phosphatases did not alter total or salt-soluble choline acetyltransferase activity, but membrane-bound and water-soluble forms of the enzyme were selectively increased in okadaic acid-treated nerve terminals to 129±11% and 137±10% of control, respectively. High-affinity choline transport was reduced to 77±6% and 76±7% of control in calyculin-A- and okadaic acid-treated nerve terminals, respectively. Acetylcholine synthesis was reduced to 73±6% of control in calyculin-A-treated synaptosomes only; acetylcholine synthesis was at control levels in okadaic acid-treated cultures correlating with enhanced choline acetyltransferase activity in the water-soluble and nonionically membrane-bound fractions. These investigations indicate a role for phosphoprotein phosphatases in the regulation of acetylcholine synthesis in the cholinergic nerve terminal. The observed increases in choline acetyltransferase activity in two subcellular fractions appears to compensate for decreased choline precursor availability, allowing acetylcholine synthesis to be maintained at control levels. The uncoupling of choline transport and acetylcholine synthesis in this situation represents a unique functional role for a subfraction of choline acetyltransferase.     

Immunohistochemical localization of choline acetyltransferase during development and in Chats mutants of Drosophila melanogaster

The distribution of choline acetyltransferase (CAT) in the nervous system of Drosophila melanogaster was determined by indirect immunohistochemical procedures using a monoclonal antibody specific to the enzyme. Immunoreactivity was first detected in the nervous system of 16 hr embryos, and increased considerably by the end of embryogenesis. Neuropil was preferentially stained, though cell bodies could also be observed. Staining was prominent in the CNS of all 3 larval instars but decreased substantially during the mid-pupal stage. Prior to eclosion, the level of immunoreactivity increased and the adult staining pattern became discernible. In the adult brain, staining was extensive, with numerous structures, such as the optic lobes and mushroom bodies, staining strongly. The adult thoracic ganglia were also moderately immunoreactive. These results imply a wide distribution of cholinergic neurons in the CNS of Drosophila. Immunoreactivity was also determined for 2 temperature-sensitive CAT mutants, Chats1 and Chats2. These files exhibit reduced CAT activity at permissive temperature, 18 degrees C, which eventually falls to undetectable levels after incubation at nonpermissive temperature, 30 degrees C. Chats2 mutants, after incubation at either 18 or 30 degrees C displayed virtually no staining. This result indicated that the immunoreactivity observed in wild-type flies was specifically associated with the enzyme encoded by the Cha gene. The intensity of staining in Chats1 mutants incubated at 18 degrees C appeared greater than in control flies, even though CAT enzyme activity in Chats1 is lower. This suggests that the enzyme molecule itself is structurally altered in Chats1 mutants. After incubation at 30 degrees C, staining in Chats1 flies decreased but did not disappear.     

Localization of choline acetyltransferase (ChAT) immunoreactivity in the brain of a caecilian amphibian,Dermophis mexicanus (Amphibia: Gymnophiona)

The organization of the cholinergic system in the brain of anuran and urodele amphibians was recently studied, and significant differences were noted between both amphibian orders. However, comparable data are not available for the third order of amphibians, the limbless gymnophionans (caecilians). To further assess general and derived features of the cholinergic system in amphibians, we have investigated the distribution of choline acetyltransferase immunoreactive (ChAT-ir) cell bodies and fibers in the brain of the gymnophionan Dermophis mexicanus. This distribution showed particular features of gymnophionans such as the existence of a particularly large cholinergic population in the striatum, the presence of ChAT-ir cells in the mesencephalic tectum, and the organization of the cranial nerve motor nuclei. These peculiarities probably reflect major adaptations of gymnophionans to a fossorial habit. Comparison of our results with those in other vertebrates, including a segmental approach to correlate cell populations across species, shows that the general pattern of organization of cholinergic systems in vertebrates can be modified in certain species in response to adaptative processes that lead to morphological and behavioral modifications of members of a given class of vertebrates, as shown for gymnophionans.