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B H Wainer

Publications and source records attributed to B H Wainer.

At least 91 records · Page 5Linked to original sources

A correlated light and electron microscopic study of identified cholinergic basal forebrain neurons that project to the cortex in the rat.

Cholinergic neurons in the basal forebrain which project to the frontal cortex were studied by combining the retrograde transport of a conjugate of horseradish peroxidase and wheat germ agglutinin with choline acetyltransferase immunohistochemistry. Neurons that were both retrogradely labelled and immunoreactive were found on the medial, lateral, and ventral borders of the globus pallidus, within the globus pallidus, as well as in the substantia innominata and ventral pallidum region. The cell bodies averaged 31 by 19 micron in size and had sparsely branching dendrites. Cells which were labelled by both techniques were first characterised in the light microscope and then studied in the electron microscope. The perikarya had large amounts of cytoplasm with abundant organelles. The nuclei were indented, were usually eccentrically placed, and contained prominent nucleoli. The synaptic input onto the cell bodies and their dendrites was studied in serial sections. The synaptic input onto the perikarya and proximal dendrites was sparse but the density increased on more distal regions of the dendrites. Subjunctional bodies were associated with the postsynaptic membrane in 20-30% of the synaptic contacts and these were classified as asymmetrical; the remaining contacts could not be classified because of an association of the immunoreaction product with the postsynaptic membrane. The synaptic input to these cells was distinctly different from that onto typical globus pallidus cells, the perikarya and dendrites of which were characteristically ensheathed in synaptic boutons.

Animals↗

Cholinergic neurons in the telencephalon of the reptile Caiman crocodilus.

Choline acetyltransferase (ChAT) immunohistochemistry was used to characterize the distribution of cholinergic neurons in the telencephalon of the reptile, Caiman crocodilus. ChAT-positive cell bodies were observed within the small-celled portion of the ventrolateral area of the telencephalon (VLA s.c.), a region previously considered comparable to the mammalian caudate nucleus and putamen. A large field of cholinergic neurons was observed within the ventral paleostriatum (VP), a region comparable to the substantia innominata and ventral pallidum of mammals. Cholinergic neurons were also observed within cranial motor nuclei of the brainstem, within the isthmic nucleus and within portions of the lateral reticular formation of the pons and medulla. A rich plexus of cholinergic fibers was observed within the intermediate and deep layers of the optic tectum. The results of this study indicate that many aspects of cholinergic system organization are similar in caiman and mammals, and suggest a common derivation of these systems from ancestral forms.

Acetylcholinesterase↗

Evidence for the coexistence of acetylcholine and enkephalin in the sympathetic preganglionic neurons of rats.

The localization of the cholinergic neurons in the lower thoracic segments of the spinal cord of rats was examined by a monoclonal antibody against choline acetyltransferase (ChAT). The ChAT-immunoreactive neurons were located in the intermediate as well as anterior gray matters. In the intermediate gray the highest incidence of the immunoreactive neurons was in the nucleus intermediolateralis, followed by the nucleus intercalatus pars paraependymalis and a few immunoreactive neurons were seen in the nucleus intercalatus proprius. In the sequential immunostaining of one and the same section of the spinal cord pretreated with colchicine using the ChAT antibody and a polyclonal antibody against methionine-enkephalin-argynine-glycine-leucine (Met-Enk-Arg-Gly-Leu), substantial numbers of neurons were immunostained simultaneously by the two antibodies in the intermediate gray matter. The present finding gives strong evidence for the coexistence of acetylcholine and enkephalins in, at least, some of the preganglionic neurons projecting their axons to the periphery.

Animals↗

Discrete distribution of cholinergic and vasoactive intestinal polypeptidergic amacrine cells in the rat retina.

The distribution and fine structure of cholinergic amacrine cells of the rat retina were observed using monoclonal anti-choline acetyltransferase (ChAT) antibody. The ChAT-immunoreactive cholinergic cells were located in the innermost zone of the inner nuclear layer and in the outer zone of the ganglion cell layer, and they formed two distinct bands in the inner plexiform layer. The ChAT-immunoreactive materials were diffusely localized throughout the cytoplasm except for the nucleus, mitochondria, Golgi apparatus and the interior of the endoplasmic reticulum and vesicular components. The sequential immunostaining of one and the same section with both the ChAT antibody and the antiserum against vasoactive intestinal polypeptide (VIP) revealed clearly that the cholinergic amacrine cells are distinct from the VIP-immunoreactive amacrine cells.

Animals↗

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.

Acetylcholinesterase↗

Stabilization of TMB reaction product for electron microscopic retrograde and anterograde fiber tracing.

Use of the highly sensitive tetramethylbenzidine (TMB) method of horseradish peroxidase histochemistry for electron microscopy has been limited by the solubility of the reaction product in aqueous and alcoholic solutions. We have found that following the TMB reaction with a diaminobenzidine-cobalt (DAB-Co) step causes the TMB crystals to become coated with DAB-Co. The resultant reaction complex is insoluble, and easily localized using electron microscopy. By systematically varying the pH at which the TMB reaction is run, the size and shape of the reaction complex can be controlled. The pH 4.0 reaction complex was the most suitable for electron microscopic identification of labeled structures less than 1.0 micron in diameter (e.g., axon terminals).

Animals↗

Immunoaffinity purification of human choline acetyltransferase: comparison of the brain and placental enzymes.

A rapid and efficient immunoaffinity purification procedure has been developed for human placental choline acetyltransferase (ChAT). Using this procedure, human placental ChAT was purified to homogeneity with high recovery of enzyme activity (50-60%). Purified ChAT was used to raise a monospecific anti-human ChAT polyclonal antibody in rabbits. A comparison of the physical properties of ChAT was made between the enzymes purified from human brain and human placenta. Only one form of the enzyme exists in either tissue, having identical molecular weights of 68,000 and a single apparent pI of 8.1. A more detailed comparison of the two enzymes using peptide mapping and epitope mapping indicates identity between the brain and placental enzymes.

Amino Acids↗

Evidence for cholinergic neurites in senile plaques.

In the neocortices and amygdalae of young and aged macaques, cholinergic axons were identified by means of a monoclonal antibody to bovine choline acetyltransferase. Many fine, linear, immunoreactive profiles were seen in these animals. In the older animals, some cholinergic axons showed multifocal enlargements along their course. In some instances, neurites with choline acetyltransferase immunoreactivity were associated with deposits of amyloid (visualized with thioflavin T fluorescence). The appearance of these amyloid-associated abnormal cholinergic processes was similar to that of neurites in senile plaques, as shown by conventional silver impregnation techniques. Cholinergic systems thus give rise to some of the neurites within senile plaques.

Aging↗

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.

Acetylcholinesterase↗

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.

Acetylcholine↗

Multiple isoelectric and molecular weight variants of choline acetyltransferase. Artifact or real?

The existence of isoelectric variants of bovine brain and human placental choline acetyltransferase was confirmed by chromatofocusing. The identification of molecular weight variants (bovine brain, Mr = 68,000 and 63,000; human placental, Mr = 66,000 and 64,000) was also demonstrated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by "Western blotting" and immunochemical visualization of choline acetyltransferase with monoclonal antibodies. No correlation between the isoelectric variants and molecular weight variants could be observed; however, in the case of the bovine brain enzyme the more alkaline isoelectric variant was enriched in the higher molecular weight form of the enzyme. Treatment of the bovine brain enzyme with Staphylococcus aureus V8 protease resulted in the conversion of the Mr = 68,000 form of the enzyme to the Mr = 63,000 form. During this conversion no change in the enzyme activity was observed demonstrating that the Mr = 63,000 form of the enzyme and probably also the Mr = 68,000 form of the enzyme are active. Preparation of the bovine brain enzyme in the presence of proteolytic enzyme inhibitors yielded a new higher molecular weight form of the enzyme, Mr = 73,000, which was enzymatically active. The Mr = 73,000 form of the enzyme exhibited a single isoelectric form when analyzed by chromatofocusing. These results suggest that the Mr = 73,000 form of the enzyme represents the native form, and that other molecular weight and isoelectric variants may arise by proteolysis.

Animals↗

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.

Animals↗

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.

Acetylcholinesterase↗

Characterization of cholinergic neurons in the rat neostriatum. A combination of choline acetyltransferase immunocytochemistry, Golgi-impregnation and electron microscopy.

Immunocytochemistry with a monoclonal antibody against choline acetyltransferase has been used to characterise cholinergic neurons in the rat neostriatum. The light microscopic morphology, ultrastructure and synaptic input of these neurons was compared to that of the three types of large neuron found in Golgi preparations of the striatum. The cholinergic neurons are large and have long infrequently branching dendrites. Two of the immunoreactive neurons were also Golgi-impregnated and showed characteristics of the "classical" large neurons of the striatum. Examination in the electron microscope revealed that the synaptic input to perikarya and proximal dendrites is sparse, thus distinguishing them from another large type of neuron, found in the ventral regions of the striatum, whose dendrites and perikarya are ensheathed in synaptic boutons. It is concluded that one of the three morphologically distinguishable classes of large neuron in the striatum is a cholinergic neuron.

Animals↗

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.

Acetylcholinesterase↗

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.

Animals↗

Retrograde horseradish peroxidase tracing combined with localization of choline acetyltransferase immunoreactivity.

Localization of choline acetyltransferase (ChAT) immunoreactivity in rodent brain (AI Levey, DM Armstrong, SF Atweh, RD Terry, BH Wainer: J Neurosci 3 1, 1983) with a monoclonal antibody (Ab8) has been previously reported. Now a procedure for combining ChAT immunohistochemistry with retrograde tracing for the purpose of mapping cholinergic pathways is presented. Rats were injected with horseradish peroxidase-wheat germ agglutinin in the tongue and cerebral cortex. Sections from their perfusion-fixed brains were reacted with 3,3'-diaminobenzidine (DAB)/H2O2/cobalt acetate followed by ChAT localization with monoclonal antibody Ab8 using the peroxidase-antiperoxidase method and visualization using DAB/H2O2. Double-labeled cells were visualized with black punctate staining (retrograde tracer) on a diffuse brown cytoplasmic background (ChAT immunoreactivity) in the hypoglossal nucleus and ventral telencephalon (substantia innominata-nucleus basalis). Conditions of fixation, histochemistry, and immunohistochemistry that contribute to optimal resolution for this procedure are discussed.

Animals↗

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.

Animals↗