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.     

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.     

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.     

Postnatal development of cholinergic neurotransmitter enzymes in the mouse cerebellum. Biochemical,light microscopic and electron microscopic cytochemical investigations

The activity and distribution of the cholinergic neurotransmitter enzymes acetylcholinesterase (AChE) and choline acetyltransferase (ChAc) in the developing cerebellum of the mouse were investigated using biochemical assays and light microscopic histochemistry for AChE and ChAc, and electron microscopic histochemistry for AChE. Postnatal alterations in the levels of AChE and ChAc in the cerebellum of the mouse are characterized by a divergent pattern. During the first two postnatal weeks, AChE activity increases progressively, whereas ChAc activity remains low. Beyond day 14, when AChE activity is steady or gradually decreasing, ChAc increases sharply to reach a peak on day 32. Histochemically, AChE activity is associated with the glomeruli and the Golgi cells of the internal granular layer, the medullary layer, and the deep cerebellar nuclei. Purkinje cells exhibit transient staining between days 2 and 9. At the ultrastructural level, AChE staining is first demonstrated on day 6 within the Golgi cell soma, on day 9 at the mossy fiber–granule cell synapse, and on day 11 at the Golgi terminal–granule cell synapse. The staining intensity of these structures reaches that of the adult on day 19. The histochemical reaction for ChAc is localized to moderate number of presumed Bergmann glial cells, a few large cells of the deep cerebellar nuclei, small numbers of Golgi cells, and all immature Purkinje cells. The molecular layer, glomeruli, and the medullary layer fail to demonstrate ChAc activity. This distribution of ChAc sharply contrasts with the localization established by other methods previously and is interpreted in the light of the drawbacks of the histochemical procedure. The biochemical fluctuations in AChE activity, correlated with the histochemical and cytochemical data, suggest that the postnatal increases in the enzyme are related to the ingrowth of the mossy fibers and to the maturation of the AChE-positive Golgi cells. Histochemical evidence for the correlation between the ontogenetic increases in cerebellar ChAc activity and progressive mossy fiber innervation must await the application of the immunohistochemical method to the developing cerebellum.     

Cholinergic innervation of mossy cells in the rat fascia dentata

Hilar mossy cells are the main cells of origin of the commissural/associational projection to the inner molecular layer of the rat fascia dentata. In order to analyze the cholinergic innervation of hilar mossy cells, a light and electron microscopic double‐labeling technique was used. Immunolabeling for calcitonin gene‐related peptide (CGRP) was employed to identify mossy cells and immunocytochemistry for choline acetyltransferase (ChAT) was used to label cholinergic septohippocampal fibers. Cholinergic boutons were abundant around mossy cell somata and on their proximal dendrites. Electron microscopy confirmed that many of these boutons formed synapses with the CGRP‐positive mossy cells. These data demonstrate a direct innervation of hilar mossy cells by cholinergic septohippocampal afferents. This connectivity could contribute to the electrophysiological behavior of mossy cells during theta oscillations. Hippocampus 1999;9:314–320. © 1999 Wiley‐Liss, Inc.     

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.     

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.     

Choline acetyltransferase activity in mouse cerebellar cultures

The finding of the acetylcholine synthetic enzyme, choline acetyltransferase, has been reported in mouse cerebellar cultures, and it has been used as an index of neuronal survival and maturation. These results are curious in light of immunocytochemical studies which show that this enzyme is localized within mossy fiber terminals in glomerular structures of the cerebellar cortex. Since most mossy fibers are of extracerebellar origin, a significant population of mossy fiber terminals would not be expected to be present in cerebellar cultures. The origin of this acetylcholine synthetic activity has been examined in mouse cerebellar cultures. Two groups of explants, one with and the other without incorporated dorsal pontine tissue, were cultivated. Only cultures that included pons showed well developed glomerular structures with mossy fiber rosettes. Homogenates of the cultures were assayed for their ability to synthesize acetylcholine, and the synthesis was shown to be due to choline acetyltransferase by use of the specific inhibitor, (naphthylvinyl)pyridine. Cultures lacking dorsal pontine tissue had only low levels of enzyme activity, whereas those which included pons had 20–60 times greater synthetic activity. These results indicate that the choline acetyltransferase activity arises from pontine tissue in cerebellar cultures and are consistent with mossy fibers being the source of this enzyme.     

Topographic relations of cholinergic and noradrenergic neurons in the feline pontomesencephalic tegmentum: an immunohistochemical study

The immunohistochemical localization of the neurotransmitter synthesizing enzymes choline acetyltransferase, tyrosine hydroxylase and dopamine-beta-hydroxylase was examined in the feline pontomesencephalic tegmentum. Examination of adjacent sections stained for either choline acetyltransferase, tyrosine hydroxylase or dopamine-beta-hydroxylase immunoreactivity, as well as individual sections doubly stained for both choline acetyltransferase and tyrosine hydroxylase immunoreactivity, unequivocally demonstrated that noradrenergic and cholinergic neurons were extensively intermingled in the brainstem tegmentum of the cat. This contrasts with the situation in various other species, where neurons utilizing these two neurotransmitters are discretely localized in distinct nuclei. Furthermore, the present studies demonstrate the existence of two types of choline acetyltransferase immunoreactive neurons in the feline tegmentum: the magnocellular neurons of the pedunculopontine and laterodorsal tegmental nuclei which stain histochemically for NADPH diaphorase, plus a population of small spindle-shaped neurons in the medial and lateral parabrachial nuclei which do not stain positively for NADPH diaphorase. The data are discussed with respect to several influential hypotheses of sleep cycle control.     

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.     

Intense immunoreactivity for Mn-superoxide dismutase (Mn-SOD) in cholinergic and non-cholinergic neurons in the rat basal forebrain

The immunohistochemical localization of manganese (Mn)-superoxide dismutase (Mn-SOD) was studied in the rat basal forebrain using polyclonal antibodies to Mn-SOD. Neurons of the basal forebrain exhibit a high density of Mn-SOD immunoreactivity. Double immunostaining with a monoclonal antibody to choline acetyltransferase demonstrated that both cholinergic and non-cholinergic neurons in the basal forebrain are intensely immunoreactive for Mn-SOD.     

A cholinergic innervation of the bovine pineal gland visualized by immunohistochemical detection of choline acetyltransferase-immunoreactive nerve fibers

An immunohistochemical investigation of the bovine pineal gland was performed using both a rabbit polyclonal antibody and a rat monoclonal antibody against choline acetyltransferase (ChAT). A network of ChAT-immunoreactive (IR) nerve fibers was located throughout the pineal gland, both in the perivascular spaces and between the pinealocytes. Most of the intrapineal ChAT-IR nerve fibers were endowed with varicosities. In addition, some ChAT-IR intrapineal neurons were found, often located at the base of the gland near the pineal recess. Within the habenular nucleus and pineal stalk, ChAT-IR perikarya and nerve fibers were also present. Some of these fibers projected towards the pineal gland. A number of ChAT-IR nerves were also located in the posterior commissure and could be followed into the gland. At the caudal tip of the pineal gland, a bundle of ChAT-IR nerve fibers was observed to penetrate into the gland together with blood vessels. The presence of a cholinergic innervation of the bovine pineal gland, together with previous demonstration of the presence of choline acetyltransferase and muscarinic receptor binding sites in the bovine pineal gland, indicates a functional influence of a cholinergic nervous system on the pinealocyte.     

Putative cholinergic projections from the nucleus isthmi and the nucleus reticularis mesencephali to the optic tectum in the goldfish (Carassius auratus)

The nucleus isthmi of fish and amphibians has reciprocal connections with the optic tectum, and biochemical studies suggested that it may provide a major cholinergic input to the tectum. In goldfish, we have combined immunohistochemical staining for choline acetyltransferase with retrograde labeling of nucleus isthmi neurons after tectal injections of horseradish peroxidase. Seven fish received tectal horseradish peroxidase injections, and brain tissue from these animals was subsequently processed for the simultaneous visualization of horseradish peroxidase and choline acetyltransferase. In many nucleus isthmi neurons the dense horseradish peroxidase label obscured the choline acetyltransferase reaction product but horseradish peroxidase and choline acetyltransferase were colocalized in 54 cells from nine nuclei isthmi. The somata of nucleus reticularis mesencephali neurons stained so intensely for choline acetyltransferase that we could not determine whether they were labelled also with horseradish peroxidase. However, the large choline acetyltransferase-immunoreactive axons of nucleus reticularis mesencephali neurons stained intensely enough for us to follow them rostrally; the axons are clustered together until the level of the rostral tectum where two groupings form: one travels into the tectum and the other travels rostroventrally to cross the midline and enter the contralateral diencephalic preoptic area. We conclude therefore that cholinergic neurons project to the optic tectum from the nucleus isthmi as well as nucleus reticularis mesencephali in goldfish.     

Choline acetyltransferase immunoreactive sympathetic ganglion cells in a teleost,Stephanolepis cirrhifer

The present study showed neurons immunoreactive for choline acetyltransferase (ChAT) in the cranial sympathetic ganglia lying close to the trigeminal-facial nerve complex of the filefish. In these ganglia, less than 1% of ganglion cells were positive for choline acetyltransferase. Choline acetyltransferase-positive neurons were significantly larger than the randomly sampled neurons in this ganglion. The majority of choline acetyltransferase-positive neurons were negative for tyrosine hydroxylase, but many of them were positive for galanin (GAL). Some neurons were positive for both choline acetyltransferase and tyrosine hydroxylase, but these neurons were rarely immunoreactive for dopamine beta hydroxylase, suggesting that they are not adrenergic. In the cranial sympathetic ganglia and the celiac ganglia, many nerve fibers immunoreactive for galanin were seen, and varicose terminals were in contact selectively with neurons negative for both choline acetyltransferase and tyrosine hydroxylase, but not with those positive for choline acetyltransferase or tyrosine hydroxylase. Nerve fibers immunoreactive for choline acetyltransferase were found to be present in contact with the deep layer of chromatophores, which was observed only in the labial region. These results suggest that cholinergic postganglionic neurons are present in the filefish cranial sympathetic ganglia, and that they also contain galanin. As few cholinergic sympathetic neurons express tyrosine hydroxylase and none express dopamine beta hydroxylase, they are unlikely to synthesize noradrenaline or adrenaline.     

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.     

Secondary vestibular cholinergic projection to the cerebellum of rabbit and rat as revealed by choline acetyltransferase immunohistochemistry, retrograde and orthograde tracers.

Previously we have shown that four regions of the cerebellum, the uvula-nodulus, flocculus, ventral paraflocculus, and anterior lobe 1, receive extensive, but not exclusive, cholinergic mossy fiber projections. In the present experiment we have studied the origin of three of these projections in the rat and rabbit (uvula-nodulus, flocculus, ventral paraflocculus), using choline acetyltransferase (ChAT) immunohistochemistry in combination with a double label, retrogradely transported horseradish peroxidase (HRP). We have demonstrated that in both the rat and rabbit the caudal medial vestibular nucleus (MVN) and to a lesser extent the nucleus prepositus hypoglossus (NPH) contain ChAT-positive neurons. Neurons of the caudal MVN are double-labeled following HRP injections into the uvula-nodulus. HRP injections into the uvula-nodulus also labeled less than 5% of the neurons in the cholinergic vestibular efferent complex. Fewer ChAT-positive neurons in the MVN and some ChAT-positive neurons in the NPH are double-labeled following HRP injections into the flocculus. Almost no ChAT-positive neurons in the MVN and some ChAT-positive neurons in the NPH are double-labeled following HRP injections into the ventral paraflocculus. Injections of Phaseolus leucoagglutinin (PHA-L) into the caudal MVN of both the rat and rabbit demonstrated projection patterns to the uvula-nodulus and flocculus that were qualitatively similar to those observed using ChAT immunohistochemistry. We conclude that the cholinergic mossy fiber pathway to the cerebellum in general and the uvula-nodulus in particular is likely to mediate secondary vestibular information related to postural adjustments.     

Localization of Mn-superoxide dismutase (Mn-SOD) in cholinergic and somatostatin-containing neurons in the rat neostriatum

We used rabbit antisera against manganese (Mn)-superoxide dismutase for immunohistochemical studies of localization in the rat neostriatum. Immunostaining was intense in large-sized neurons and several medium-sized neurons, but it was moderate to weak in other cells. Double immunostaining with monoclonal antibody to choline acetyltransferase or somatostatin demonstrated large-sized, Mn-SOD immunoreactive neurons to be cholinergic, and some medium-sized neurons which were intensely immunoreactive for Mn-SOD to contain somatostatinergic.     

Quantitative analysis of the choline acetyltransferase-immunoreactive axonal network in the cat primary visual cortex: II. Pre- and postnatal development

The pre- and postnatal development of cholinergic projections was investigated in the cat striate cortex by applying immunohistochemical methods based on a monoclonal antibody against choline acetyltransferase (ChAT). The earliest age investigated was gestational day 54. At this stage a sparse network of ChAT(+) fibers was distributed throughout the striate cortex. Subsequent postnatal maturation of ChAT(+) fibers was characterized by an increase in fiber density that started in layer VI and gradually progressed toward more superficial layers. By 4 weeks of age the density of ChAT(+) fibers and varicosities had reached adult levels in layers V and VI but was still subnormal in layers I-IV. The mature pattern of cholinergic innervation was established by 13 weeks of age. There was no evidence for developmental gradients in the anteroposterior and mediolateral directions within area 17. These results indicate that the cholinergic projection to striate cortex develops continuously in an inside-out sequence as is characteristic for most cortical maturation processes. There was no indication that striate cortex receives an especially dense cholinergic input during the critical period.     

GABAergic basal forebrain neurons project to the neocortex: the localization of glutamic acid decarboxylase and choline acetyltransferase in feline corticopetal neurons

Our objective was to determine whether GABAergic and cholinergic basal forebrain neurons project to the neocortex. The retrograde connectivity marker wheat germ agglutinin lectin-bound horseradish peroxidase was injected into the neocortex of adult cats. Histo- and immunohistochemical methods were combined to label sequentially connectivity and transmitter markers (glutamic acid decarboxylase; choline acetyltransferase) in forebrain neurons. The labels of each marker were identified by correlative light and electron microscopy. Two principal types of doubly labeled neurons were demonstrated. The connectivity marker was colocalized with glutamic acid decarboxylase or choline acetyltransferase. The neurons were located in the basal forebrain. Their ultrastructural, cellular, and regional organization supported 2 conclusions. (1) GABAergic basal forebrain neurons project to the neocortex. This is important new morphological evidence for the origin of inhibitory neocortical afferents from a subcortical brain site. (2) The GABAergic and cholinergic basal forebrain neurons projecting to the neocortex exhibit remarkable structural similarities. The transmitter diversity of these intertwined neocortical afferents may be significant for the pathology and treatment of human neurological disorders such as Alzheimer's disease.     

Hippocampal and midline thalamic fibers and terminals in relation to the choline acetyltransferase-immunoreactive neurons in nucleus accumbens of the rat: a light and electron microscopic study

The synaptic interactions between terminals of allocorticostriatal and thalamostriatal fibers and the cholinergic neurons in the nucleus accumbens were investigated using degeneration and dual labelling immunocytochemistry in Wistar rats. The presumptive cholinergic neurons were labelled with antibodies directed against choline acetyltransferase and the afferent fibers were labelled anterogradely with Phaseolus vulgaris-leucoagglutinin. Fibers from the subiculum of the hippocampal formation and from the midline and intralaminar thalamus project densely into the medial nucleus accumbens where they overlap a relatively dense population of choline acetyltransferase-immunoreactive neurons. Varicosities containing Phaseolus vulgaris-leucoagglutinin juxtapose the immunoreactive neurons. To study the possibility that the cholinergic neurons could be the synaptic targets of these incoming fibers, the subiculum, the fornix, and the midline/intralaminar thalamus were lesioned in separate animals and brain sections were immunoprocessed for choline acetyltransferase and studied with the electron microscope. In addition, dual-labelling electron microscopic immunocytochemistry was employed. In total, 164 synaptic terminals from the subiculum/hippocampus and 130 from the midline/intralaminar thalamus were examined; all formed asymmetrical synaptic specializations. No hippocampal endings were seen to contact the somata or primary dendrites of the choline acetyltransferase-immunoreactive neurons; however, three were found in synaptic contact with distal, immunolabelled dendritic shafts. Most hippocampal terminals established contacts with unlabelled spines. Fifteen percent of the thalamic endings were found to synapse on the somata and the primary and distal dendrites of the choline acetyltransferase-immunoreactive neurons. The remaining thalamic terminals established synaptic junctions with small unlabelled dendrites or spines. These findings have important implications not only for our understanding of the synaptic organization of the hippocampal and thalamic projections to the nucleus accumbens, but also for the contribution of the cholinergic neurons to the circuitry of this nucleus.