Bruce H. Wainer

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.

Cortical projections arising from the basal forebrain: a study of cholinergic and noncholinergic components employing combined retrograde tracing and immunohistochemical localization of choline acetyltransferase

The neurochemical identity of ascending putative cholinergic pathways from the rat basal forebrain was investigated employing a method for simultaneously visualizing choline acetyltransferase immunoreactivity and retrogradely transported horseradish peroxidase-conjugated wheatgerm agglutinin. This histochemical procedure revealed three distinct populations of neurons: (1) cells which stained only for choline acetyltransferase immunoreactivity; (2) cells which stained only for retrograde tracer and (3) cells which stained simultaneously for choline acetyltransferase immunoreactivity and retrograde tracer. The results demonstrated that this projection is topographically organized and consists of both cholinergic and noncholinergic components. The relative contribution of each component varied with the telencephalic target area as follows: the olfactory bulb receives a projection from cells of the horizontal limb nucleus, 10-20% of which are cholinergic (Ch3); the hippocampal formation receives afferents from cells of the medial septal and vertical limb nuclei, 35-45% of which are cholinergic (Ch1 and Ch2); and the cortical mantle receives afferents primarily from cells within the substantia innominata-nucleus basalis complex, 80-90% of which are cholinergic (Ch4). The topographical organization of Ch4 projections is not as completely differentiated as we have previously observed in the primate.

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.

Stabilization of the tetramethylbenzidine (TMB) reaction product: application for retrograde and anterograde tracing, and combination with immunohistochemistry.

Tetramethylbenzidine (TMB) as a substrate for horseradish peroxidase (HRP) histochemistry is more sensitive than other chromogens. Its instability in aqueous solutions and ethanol, however, has limited its application. We now report a method for stabilizing TMB by incubation in combinations of diaminobenzidine (DAB)/cobalt (Co2+)/H2O2. The stabilized TMB product was unaffected by long-term exposures to ethanol, neutral buffers, and subsequent immunohistochemical staining procedures. A procedure is recommended for optimal stabilization of TMB that affords a sensitivity for demonstrating retrogradely labeled perikarya comparable to standard TMB histochemistry. The physical characteristics of the reaction product make it suitable for combination with the unlabeled antibody, peroxidase-antiperoxidase (PAP) immunohistochemical staining procedure. This was established by staining retrogradely labeled neurons in the basal forebrain with a monoclonal antibody against choline acetyltransferase. Because the stabilized TMB product exhibited a superior sensitivity over cobalt ion intensification of the DAB-based reaction product (DAB-Co), it offers a distinct advantage over previously described combination procedures.

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.

Cholinergic projections from the laterodorsal and pedunculopontine tegmental nuclei to the pontine gigantocellular tegmental field in the cat

This study demonstrates that the laterodorsal tegmental nucleus (LDT) and pedunculopontine tegmental nucleus (PPT) are sources of cholinergic projections to the cat pontine reticular formation gigantocellular tegmental field (PFTG). Neurons of the LDT and PPT were double-labeled utilizing choline acetyltransferase immunohistochemistry combined with retrograde transport of horseradish peroxidase conjugated with wheat germ agglutinin (WGA-HRP). In the LDT the percentage of cholinergic neurons retrogradely labeled from PFTG was 10.2% ipsilaterally and 3.7% contralaterally, while in the PPT the percentages were 5.2% ipsilaterally and 1.3% contralaterally. These projections from the LDT and PPT to the PFTG were confirmed and their course delineated with anterograde labeling utilizing Phaseolus vulgaris leucoagglutinin (PHA-L) anterograde transport.

Cholinergic synapses in the rat brain: a correlated light and electron microscopic immunohistochemical study employing a monoclonal antibody against choline acetyltransferase

Using a monoclonal antibody to choline acetyltransferase, immunoreactive synaptic boutons were identified in the neostriatum, cingulate cortex, basolateral nucleus of the amygdala, hippocampus and interpeduncular nucleus of the rat. The synapses were generally symmetrical although some asymmetrical membrane specializations were observed. Postsynaptic targets included perikarya, dendritic shafts and dendritic spines.