Mapping of the basal forebrain cholinergic system of the dog: A choline acetyltransferase immunohistochemical study

In an effort to produce a canine model of basal forebrain ischemia with memory deficits, we have shown that dogs possess a medial striate artery that perfuses basal forebrain territory, homologous to the human recurrent artery of Heubner. In the present study, we set out to delineate the precise topography of the cholinergic neurons in the canine forebrain, a neuronal system implicated in cognitive and memory functions. Floating coronal sections, derived from the head of the caudate nucleus to the rostral border of the hippocampus, were stained for choline acetyltransferase using a monoclonal antibody. Representative sections from one dog brain were drawn. These outlines were used for measurement of cell density, cell size, number of processes, and cell roundness. Choline acetyltransferase-positive neurons constituted four major subdivisions within the basal forebrain. A relatively dense population of cholinergic neurons was present in the medial septal nucleus (Ch1). A continuum of densely packed cells was also delineated within the vertical (Ch2) and horizontal (Ch3) nuclei of the diagonal band of Broca. A fourth group of heterogeneously packed cholinergic neurons represented the nucleus basalis magnocellularis (Ch4). Except for the caudal component of the Ch4 population, the forebrain cholinergic corticopetal system was located within the perfusion territory of the medial striate arteries. The Ch4 cell group in dogs is better defined than that of rodents but is not as sharply demarcated as in human and nonhuman primates. Our findings indicate that the dog may serve as an excellent model for assessing neurological and memory deficits, which, in humans, results from hypoperfusion of the recurrent artery of Heubner. © 1996 Wiley-Liss, Inc.     

Heterogeneous choline acetyltransferase staining in cholinergic neurons

We studied the features of choline acetyltransferase (ChAT) distribution in cholinergic structures of the central and peripheral nervous systems; specifically in the spinal cord and dorsal root ganglion (DRG) of embryo (E20), newborn, or adult rats using a goat polyclonal antibody. We found that this anti-ChAT antibody selectively labels both central and peripheral cholinergic structures. We observed cholinergic neurons with weak immunoreactivity. We demonstrated that at all stages of ontogeny that we studied, four groups of cholinergic neurons occurred in the cervical part of the spinal cord, viz., the small neurons of the dorsal horns, neurons of Rexed’s lamina X, neurons of the area of Rexed’s laminae VI–VII, and cells of the ventral horns. Interneurons of Rexed’s laminae VI–VII exhibited maximum ChAT reactivity as compared to other groups of cholinergic neurons of the spinal cord at all developmental stages we studied. We found an increase in the number of immunopositive cells of lamina X from E20 stage to the early postnatal and mature stages. In the peripheral cholinergic structures, i.e., the DRG, of the rat, all neurons expressed ChAT at all stages of ontogeny.     

The immunohistochemical localization of choline acetyltransferase in the cat brain

The distribution of neurons displaying choline acetyltransferase (ChAT) immunoreactivity was examined in the feline brain using a monoclonal antibody. Groups of ChAT-immunoreactive neurons were detected that have not been identified previously in the cat or in any other species. These included small, weakly stained cells found in the lateral hypothalamus, distinct from the magnocellular rostral column cholinergic neurons. Other small, lightly stained cells were also detected in the parabrachial nuclei, distinct from the caudal cholinergic column. Many small ChAT-positive cells were also found in the superficial layers of the superior colliculus. Other ChAT-immunoreactive neurons previously detected in rodent and primate, but not in cat, were observed in the present study. These included a dense cluster of cells in the medial habenula, together with outlying cells in the lateral habenula. Essentially all of the cells in the parabigeminal nucleus were found to be ChAT-positive. Additional ChAT-positive neurons were detected in the periolivary portion of the superior olivary complex, and scattered in the medullary reticular formation. In addition to these new observations, many of the cholinergic cell groups that have been previously identified in the cat as well as in rodent and primate brain such as motoneurons, striatal interneurons, the magnocellular rostral cholinergic column in the basal forebrain and the caudal cholinergic column in the midbrain and pontine tegmentum were confirmed. Together, these observations suggest that the feline central cholinergic system may be much more extensive than previous studies have indicated.     

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.     

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.     

Cholinergic innervation of the monkey amygdala: an immunohistochemical analysis with antisera to choline acetyltransferase

The organization of the cholinergic innervation of the macaque monkey amygdaloid complex was investigated by means of immunohistochemical techniques and either a polyclonal antiserum or a monoclonal antibody directed against the specific synthetic enzyme choline acetyltransferase (ChAT). Adjacent series of sections were processed histochemically for the demonstration of the degradative enzyme acetylcholinesterase (AChE) or for cell bodies with thionin. The density of ChAT immunoreactivity differed substantially among the various nuclei and cortical regions of the amygdala. In general, the distribution of ChAT immunoreactivity paralleled the pattern of AChE staining. One notable exception was the presence of AChE containing cell bodies in addition to AChE positive fibers within nearly all of the nuclear and cortical regions. In contrast, ChAT immunoreactivity was associated only with fibers and terminals. The highest density of ChAT immunoreactive fibers and terminals was consistently observed in the magnocellular subdivision of the basal nucleus. Staining was substantially less dense in the more ventrally situated parvicellular subdivision. Medially, in the adjacent accessory basal nucleus, immunoreactive fibers and terminals were densest in the magnocellular and superficial subdivisions and least prominent in the parvicellular subdivision. Of the deep nuclei, the lateral nucleus generally obtained the least ChAT immunoreactive terminals and processes. Only its more densely cellular ventrolateral portion contained appreciable fiber and terminal staining. One of the more distinctive patterns of ChAT immunoreactivity was seen in the nucleus of the lateral olfactory tract. Here, ChAT positive fibers formed pericellular basket plexuses around unstained cell bodies. This unique pattern of staining was used to delineate the boundaries of the nucleus and indicated that it is present for much of the rostrocaudal extent of the amygdala. Another region of conspicuous staining on the medial surface of the amygdala was the sulcal portion of the periamygdaloid cortex. This region, associated with the sulcus semiannularis and bordering the entorhinal cortex, consistently contained dense immunoreactivity. The central nucleus also presented a somewhat idiosyncratic pattern of ChAT staining. The lateral subdivision had a diffuse distribution of immunoreactivity in which focal patches of more densely stained terminals and occasional fine fibers were embedded. In contrast, the medial subdivision contained a larger number of thicker, stained fibers without diffuse background labeling.(ABSTRACT TRUNCATED AT 400 WORDS)     

Origin of cholinergic nerves to the rat major cerebral arteries: Coexistence with vasoactive intestinal polypeptide

The distribution and density of vasoactive intestinal polypeptide (VIP) immunoreactive and acetylcholinesterase (AChE)-containing nerves around the cerebral arteries was studied by using whole mounts with or without lesioning the sphenopalatine ganglia. Abundant VIP immunoreactive and AChE-containing nerves were observed around the cerebral blood vessels in normal rats especially in the anterior circulation of the cerebral arteries. VIP-immunoreactivity and AChE-staining was also demonstrated in neurons within the sphenopalatine ganglia. Lesions of the sphenopalatine ganglia resulted in a marked reduction of both VIP-immunoreactivity and AChE activity. In many neurons, coexistence of both VIP and AChE was revealed. These results demonstrate that cholinergic neurons from the sphenopalatine ganglia innervate the cerebral vasculature at the base of the brain, and that VIP and AChE coexists within the same fibers.     

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.     

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.     

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.     

Effects of postnatal hypoxia-ischemia on cholinergic neurons in the developing rat forebrain: choline acetyltransferase immunocytochemistry

We studied the effect of early postnatal hypoxia-ischemia on cholinergic neurons in the developing rat forebrain using immunohistochemistry for choline acetyltransferase (ChAT). In 7-day-old rat pups, hypoxia-ischemia was induced in one cerebral hemisphere by combining unilateral carotid ligation with exposure to 8% oxygen for 2.5 h. This procedure caused brain injury in the hemisphere ipsilateral to ligation, most prominent in the corpus striatum, hippocampus and overlying cortex. In animals sacrificed 2-3 weeks after the insult, at approximately 3 weeks of age, the density of cholinergic cell bodies was slightly higher in the lesioned rostral caudate-putamen than the opposite side (+12%, P less than 0.05). In the more caudal portion of caudate-putamen, this effect was greater. In contrast, the size of the cholinergic perikarya in the injured striatum was significantly reduced. Cholinergic neurons in the septum (Ch1, Ch2), globus pallidus and nucleus basalis (Ch4) were relatively unaffected. Considered together with previously reported neurochemical data, these observations suggest that the immature cholinergic neurons are less vulnerable to death from hypoxia-ischemia than other components of the striatum. However, differentiation of surviving cholinergic perikarya and possibly their axonodendritic processes may be disrupted by the early insult.     

Tamoxifen enhances choline acetyltransferase mRNA expression in rat basal forebrain cholinergic neurons

Novel estrogen-like molecules known as SERMs (selective estrogen receptor modulators) produce many of the beneficial estrogen-like actions without the detrimental side-effects. The SERM, tamoxifen, an estrogen-like molecule with both agonist and antagonist properties, is widely prescribed for the treatment of breast cancer. While the effects of tamoxifen are being evaluated in many peripheral tissues, its effects in the central nervous system (CNS) have been largely ignored. In the present study, we begin to evaluate the effects of tamoxifen in the rat basal forebrain, a region known to be highly responsive to estrogen. We compared the effects of short-term (24 h) tamoxifen treatment to that of estrogen on ChAT mRNA expression in cholinergic neurons. In addition, we examined the effect of tamoxifen in the presence and absence of estrogen. Our results indicate that tamoxifen enhances ChAT expression in a manner similar to that of estrogen in several basal forebrain regions. In contrast, tamoxifen exhibits antagonist properties with respect to estrogen-induction of progesterone receptor mRNA in the medial preoptic nucleus. These results indicate tamoxifen has estrogenic properties with respect to cholinergic neurons, suggesting a previously unidentified effect of this agent in the CNS.     

Complete sequence of a cDNA encoding an active rat choline acetyltransferase: a tool to investigate the plasticity of cholinergic phenotype expression

A cDNA clone encoding the complete sequence of an active rat choline acetyltransferase (ChoAcTase; acetyl-CoA:choline O-acetyltransferase, EC 2.3.1.6) has been isolated. Analysis of the deduced amino acid sequence reveals 85% and 31% identity with the porcine and Drosophila melanogaster enzymes, respectively. To further elucidate the molecular basis of neurotransmitter-related phenotypic plasticity, the expression of ChoAcTase mRNA was compared with that of tyrosine hydroxylase [TH; tyrosine 3-monooxygenase, L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2], in neurons from superior cervical ganglia grown in the following conditions: 1) normal medium, 2) high K+ medium, and 3) normal medium supplemented with 50% muscle-conditioned medium (CM). TH mRNA was expressed in all three media; its level rose in high K+ and decreased strikingly in the presence of CM. ChoAcTase mRNA could be visualized in CM, but fell to undetectable levels in normal and high K+ media. These results suggest that translational or post-translational mechanisms do not play a major role for the modulation of neurotransmitter-associated phenotype.     

The cholinergic system of the human hindbrain studied by choline acetyltransferase immunohistochemistry and acetylcholinesterase histochemistry

A map of cholinergic cells of the human brainstem identified by immunohistochemistry of choline acetyltransferase (ChAT) is presented, along with a map of acetylcholinesterase (AChE)-containing cells and fibers. ChAT-positive structures belong to 4 brainstem systems: the cranial motor nuclei; the parabrachial complex; the reticular system; and the vestibular system. All motor nuclei of the cranial nerves, as well as the nucleus supraspinalis, are ChAT-positive. The positively staining structures of the parabrachial system include the nucleus tegmentali pedunculopontinus, and the nuclei parabrachialis medialis and lateralis. Nuclei of the reticular system containing some ChAT-positive cells include the nucleus reticularis pontis oralis and caudalis, the nucleus reticularis tegmenti pontis, the nucleus reticularis gigantocellularis, the nucleus reticularis lateralis and the formatio reticularis centralis (medulla). Structures of the vestibular and auditory systems which contain some ChAT-positive cells include the nucleus vestibularis lateralis, and the nuclei olivaris superioris medialis and lateralis. All ChAT-positive structures stain strongly for AChE. AChE-positive, ChAT-negative structures were noted in several sensory systems. The substantia nigra, locus coeruleus and raphe nuclei, known to contain non-cholinergic cells, also stain positively. The significance of the AChE-positive, ChAT-negative staining in most structures remains to be determined. A knowledge of the cholinergic systems of human brain may be important to an understanding of the pathology of a number of diseases.     

Evidence for the existence of substance P-like immunoreactive neurons in the human cerebral cortex: an immunohistochemical analysis

Cellular localization of substance P-like immunoreactive (SP-IR) structures in the pre- and postcentral gyri of the human cerebral cortex was examined by indirect immunofluorescence. SP-IR was localized mostly in bipolar and partly in multipolar cells in layers II and IV. SP-IR fibers were also noted in these gyri, especially in layers II and IV.     

Formation of cholinergic synapses by intrahippocampal septal grafts as revealed by choline acetyltransferase immunocytochemistry

The ultrastructural features of the contacts established by intrahippocampal grafts of foetal septal/diagonal band neurones in the dentate gyrus and the CA1 region of the previously denervated host hippocampus have been analysed with electron microscopic immunocytochemistry using a monoclonal antibody to choline acetyltransferase (ChAT). The results show that the grafted ChAT-positive neurones are capable of forming extensive synaptic contacts with neuronal targets in areas of the dentate gyrus and CA1 which normally receive such innervation. While all types of contacts normally found in association with the granule and pyramidal cell layers were also present in the graft-reinnervated specimens, the quantitative relationship between somatic and dendritic synapses was abnormal. Thus, the ChAT-immunoreactive synapses on cell bodies, which amounted to only a few percent in the normal animal, constituted over 60% in the grafted animals. Conversely, synapses on dendrites which constituted over 90% in the normal dentate were reduced to less than 40% in the grafted animals. The postsynaptic targets of the graft-derived cholinergic synapses included dendrites and cell bodies of dentate granule cells and CA1 pyramidal cells. This supports previous electrophysiological studies and indicates that the septal grafts may be able to modulate host hippocampal function via direct efferent connections onto the granule and pyramidal neurons in the host hippocampal formation.