Allan I. Levey

Central cholinergic pathways in the rat: An overview based on an alternative nomenclature (Ch1-Ch6)

Monoclonal antibodies to choline acetyltransferase and a histochemical method for the concurrent demonstration of acetylcholinesterase and horseradish peroxidase were used to investigate the organization of ascending cholinergic pathways in the central nervous system of the rat. The cortical mantle, the amygdaloid complex, the hippocampal formation, the olfactory bulb and the thalamic nuclei receive their cholinergic innervation principally, from cholinergic projection neurons of the basal forebrain and upper brainstem. On the basis of connectivity patterns, we subdivided these cholinergic neurons into six major sectors. The Ch1 and Ch2 sectors are contained within the medial septal nucleus and the vertical limb nucleus of the diagonal band, respectively. They provide the major cholinergic projections of the hippocampus. The Ch3 sector is contained mostly within the lateral portion of the horizontal limb nucleus of the diagonal band and provides the major cholinergic innervation to the olfactory bulb. The Ch4 sector includes cholinergic neurons in the nucleus basalis, and also within parts of the diagonal band nuclei. Neurons of the Ch4 sector provide the major cholinergic innervation of the cortical mantle and the amygdala. The Ch5-Ch6 sectors are contained mostly within the pedunculopontine nucleus of the pontomesencephalic reticular formation (Ch5) and within the laterodorsal tegmental gray of the periventricular area (Ch6). These sectors provide the major cholinergic innervation of the thalamus. The Ch5-Ch6 neurons also provide a minor component of the corticopetal cholinergic innervation. These central cholinergic pathways have been implicated in a variety of behaviors and especially in memory function. It appears that the age-related changes of memory function as well as some of the behavioral disturbances seen in the dementia of Alzheimers Disease may be related to pathological alterations along central cholinergic pathways.

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.

Co-localization of acetylcholinesterase and choline acetyltransferase in the rat cerebrum

Acetylcholinesterase-histochemistry has been widely used for localizing cholinergic neurons despite specificity problems. The distribution of cells stained with this method has never been directly compared on a histochemical level with the specific cholinergic marker, choline acetyltransferase. We recently reported the immunohistochemical localization of choline acetyltransferase using monoclonal antibodies [Levey A. I., Armstrong D., Atweh S. F., Terry R. D. & Wainer B. H. (1983) J. Neurosci 3, 1-9]. Here we report the development of a combined histochemical and immunohistochemical method for the co-localization of the 2 cholinergic markers, and their comparison in the rat cerebrum. Although the precise relationship between the markers was complex, the important results were: (1) all neurons which contained choline acetyltransferase also contained some acetylcholinesterase; (2) many acetylcholinesterase-containing neurons did not contain any demonstrable choline acetyltransferase; (3) all neurons which stained intensely for acetylcholinesterase in the neostriatum and basal forebrain also contained choline acetyltransferase; and (4) many choline acetyltransferase-containing neurons did not stain intensely for acetylcholinesterase. The results corroborate the assumption that choline acetyltransferase is a more specific marker for cholinergic neurons than acetylcholinesterase. Intense staining for acetylcholinesterase can be reliably used in some regions of the cerebrum for identifying cholinergic neurons, however, it should be recognized that this criterion s not essential for all cholinergic neurons.

Cholinergic systems in mammalian brain identified with antibodies against choline acetyltransferase

Publisher Summary This chapter presents a summary of recent advances in choline acetyltransferase (ChAT) antibody development and immunohistochemical applications and discusses areas where there is general agreement and areas that will require further investigation. It focuses on studies describing localization of ChAT immunoreactivity. The accumulation of new knowledge about the function of acetylcholine in brain has been hampered by the lack of reliable methods for visualizing cholineric structures. There has been general agreement that ChAT is potentially the best marker to use for these purposes. Although successful purification and antibody development has proven to be a real struggle, it is apparent that there are now several highly specific antibody reagents against ChAT enzyme derived from a number of different species. The chapter presents information concerning the development of these reagents and discusses the results thus far on their usefulness in localizing cholinergic structures in mammalian brain. Of the monoclonal antibodies developed in the laboratory, one cross-reacts with enzyme derived from all mammalian species tested and has proven to be a very useful reagent for localization of ChAT immunoreactivity. This antibody has been employed to localize ChAT in rat, guinea pig, monkey, ferret, and mouse brain.

Choline acetyltransferase-immunoreactive neurons intrinsic to rodent cortex and distinction from acetylcholinesterase-positive neurons

Cholinergic neurons intrinsic to rat cortex were studied using a sensitive method for the localization of choline acetyltransferase immunoreactivity, acetylcholinesterase histochemistry, combined localization of choline acetyltransferase and acetylcholinesterase, and combined localization of choline acetyltransferase and retrogradely transported horseradish peroxidase-wheat germ agglutinin. Choline acetyltransferase immunoreactivity was localized predominantly in small bipolar cortical neurons within the upper layers of isocortex, while small multipolar neurons were the predominantly stained cell type in allocortical regions. Acetylcholinesterase histochemistry demonstrated mainly small polymorphic cells scattered throughout all cellular layers in all cortices. Combined staining for choline acetyltransferase and acetylcholinesterase resulted in localization of the markers in different cell populations; choline acetyltransferase-immunoreactive neurons did not contain detectable acetylcholinesterase and acetylcholinesterase-positive neurons did not contain detectable immunoreactivity to choline acetyltransferase. Some possible connections of the cortical choline acetyltransferase-immunoreactive cells were studied in rats which had received injections of horseradish peroxidase-wheat germ agglutinin into either cortex or brainstem. The choline acetyltransferase-immunoreactive cells were frequently admixed with cells labeled with the retrograde marker; however, no double-labeled cells were observed. It was concluded that cortical cholinergic cells are not visualized by acetylcholinesterase histochemistry, and are likely to be involved in local circuitry.

Cholinergic and non-cholinergic septohippocampal pathways **

Cholinergic innervation of the hippocampus was examined in the rat by immunocytochemical localization of choline acetyltransferase immunoreactivity combined with retrograde transport of horseradish peroxidase-conjugated wheatgerm agglutinin. It was found that at least 50% of hippocampal afferents arising in the septal-diagonal band region consisted of non-cholinergic projection neurons. In addition, scattered choline acetyltransferase-immunoreactive neurons were localized to the hippocampal formation. These results indicate that: (1) the septohippocampal pathway is neither uniformly nor predominantly cholinergic; and (2) confirm that cholinergic innervation of the hippocampal formation of the rat is derived in part from intrinsic neurons.

Substance P-Containing terminals in synaptic contact with cholinergic neurons in the neostriatum and basal forebrain: a double immunocytochemical study in the rat

Antibodies against substance P and choline acetyltransferase (ChAT) have been used in a sequential double-immunocytochemical ultrastructural study of the rat forebrain. The peroxidase-anti-peroxidase procedure was used for both antigens, however, two different substrates for the peroxidase reactions were used. The substance P-immunoreactive sites were first localized using 3,3-diaminobenzidine as the substrate, then the ChAT-immunoreactive sites were localized using benzidine dihydrochloride. The reaction product formed by the two substrates was distinguishable in both the light and electron microscopes. Using this procedure, the cell bodies and proximal dendrites of identified cholinergic neurons in the neostriatum were found to receive symmetrical synaptic input from substance P-immunoreactive boutons. A similar pattern of substance P-immunoreactive synaptic input was observed onto magnocellular basal forebrain cholinergic neurons in the ventral pallidum and ventromedial globus pallidus. In both the striatum and basal forebrain substance P-immunoreactive boutons were also seen in contact with structures that did not display ChAT immunoreactivity.

A light and electron microscopic procedure for sequential double antigen localization using diaminobenzidine and benzidine dihydrochloride.

Very few double-antigen staining methods are available that are applicable to both light and electron microscopy. The objective of this study was to develop for localization of two neural antigens simultaneously a procedure which would be sensitive, simple to perform, offer permanent reaction products, and permit correlated light and ultrastructural analysis. The method employs sequential immunoperoxidase staining without antibody elution, in which the first sequence of antibodies is visualized with 3,3-diaminobenzidine (DAB) and the second with benzidine dihydrochloride (BDHC). The DAB reaction product (brown and diffuse) was easily distinguishable from the BDHC deposit (blue, granular, and more electron-dense) by both light and electron microscopy. The procedure was used to simultaneously localize choline acetyltransferase-and either substance P or tyrosine hydroxylase in rat brain at both light and ultrastructural levels. Control experiments demonstrated the absence of both color mixing and antibody crossreactions, even when both primary antibodies were from the same species. This study demonstrates the usefulness of BDHC as a chromogen for immunoperoxidase staining either alone or in combination with DAB, and describes a double method which should have wide applicability for detailed studies of most pairs of antigens at both light and ultrastructural levels.

Cholinergic nucleus basalis neurons may influence the cortex via the thalamus

The cholinergic neurons of the nucleus basalis of Meynert have been shown to provide the major cholinergic innervation of the cerebral cortex through which cholinergic transmission may modulate cortical activity. This study describes a projection from the cholinergic and non-cholinergic neurons of the nucleus basalis to the reticular nucleus of the thalamus, and a projection from the brainstem cholinergic neurons to the reticular nucleus as well as to other thalamic nuclei. The projection from the nucleus basalis to the reticular nucleus, which itself is synaptically interconnected with other thalamic nuclei, may provide an additional pathway for the modulation of cortical activity by the cholinergic basal forebrain and brainstem groups.

Choline acetyltransferase-like immunoreactivity in the forebrain of the red-eared pond turtle (Pseudemys scripta elegans)

Choline acetyltransferase (ChAT) immunohistochemistry was used to map the cholinergic neurons in the forebrain of Pseudemys turtles. Cell bodies with ChAT-like immunoreactivity were seen in the septum, the nucleus of the diagonal band, and embedded within the medial and lateral forebrain bundles. The region of the medial and lateral forebrain bundles contained the greatest concentration of ChAT-positive neurons. Virtually no ChAT-like immunoreactivity was seen in the areas composing the reptilian homologue of the mammalian striatum. It is suggested that the turtle basal forebrain cholinergic neurons may represent the evolutionary precursors to the mammalian cholinergic neurons of the basal forebrain and even the striatum.