Biomedical subjects

D B Rye

Publications and source records attributed to D B Rye.

47 records · Page 3Linked to original sources

PubMed1986

Cholinergic innervation displays strikingly different laminar preferences in several cortical areas.

A new rabbit polyclonal antiserum against choline acetyltransferase (ChAT) reveals that cholinergic innervation of the cortex varies strikingly among different cytoarchitecturally defined areas in the rat neocortex. These findings suggest that cholinergic transmission may be integrated differently into the local circuitries of various regions of the cerebral cortex. In addition, the pattern of staining observed with acetylcholinesterase histochemistry, which has been used for many years to demonstrate putative cholinergic fibers, only partially matches the staining pattern obtained with the more specific cholinergic marker, ChAT.

Acetylcholinesterase↗

PubMed1986

Colocalization of gamma-aminobutyric acid and acetylcholinesterase in rodent cortical neurons.

We have previously demonstrated that neurons of the rat cerebral cortex which stain positively for acetylcholinesterase are not likely to be cholinergic since they do not colocalize with choline acetyltransferase immunoreactivity [Levey, Rye, Wainer, Mufson and Mesulam (1984) Neuroscience 9, 9-22]. These noncholinergic acetylcholinesterase-positive cells were similar in morphology to cortical neurons which localize gamma-aminobutyric acid or glutamate decarboxylase immunoreactivity. In order to investigate the possibility that the two substances may be colocalized to the same cortical neurons, gamma-aminobutyric acid immunohistochemistry and acetylcholinesterase histochemistry were combined in single sections of rat cerebral cortex. We found that 18% of gamma-aminobutyric acid-immunoreactive cortical neurons are also acetylcholinesterase-positive, and about 36% of acetylcholinesterase-positive cells are gamma-aminobutyric acid-immunoreactive. Neurons which colocalized both substances were multipolar and bipolar neurons in cortical laminae II-VI and were observed in every cortical area examined. The possibility that gamma-aminobutyric acid-immunoreactive/acetylcholinesterase-positive cortical neurons may be postsynaptic targets of cholinergic afferents to the cerebral cortex is discussed.

Acetylcholinesterase↗

PubMed1986

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.

3,3'-Diaminobenzidine↗

PubMed1985

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↗

PubMed1985

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↗

PubMed1984

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↗

PubMed1984

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↗

PubMed1984

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↗

PubMed1984

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↗

PubMed1982

Immunochemical studies of bovine and human choline-O-acetyltransferase using monoclonal antibodies.

Immunochemical properties of bovine and human choline acetyltransferase (ChAT, EC 2.3.1.6, acetyl-CoA:choline-O-acetyltransferase) were studied using six monoclonal antibodies (AB1, AB5, AB6, AB7, AB8, and AB9) reactive with the enzyme. All antibodies except AB1 bound specifically to two proteins of 68,000 and 70,000 MW on "Western" blots of sodium dodecyl sulfate-polyacrylamide gels containing human or bovine ChAT. The enzyme was specifically absorbed to immobilized antibody and could not be eluted by low pH and/or high salt concentrations although the enzyme retained activity on the immunoabsorbent. Pure bovine enzyme consisting of the same two proteins as seen in the Western blotting studies was eluted from immobilized AB1 in the presence of sodium dodecyl sulfate. Although active enzyme could not be eluted from immobilized antibodies by standard conditions, various combinations of free and immobilized antibodies were effective in competing off bound enzyme. Free antibody AB1 quantitatively eluted the active enzyme from immobilized AB1. The different capacities of the antibodies to elute enzyme from various immunoabsorbents reflect interesting properties of both the enzyme and the antibodies.

Animals↗

PubMedDate not supplied

Hemodialysis disrupts basic sleep regulatory mechanisms: building hypotheses.

BACKGROUND: The proposition that hemodialysis may induce alterations in the sleep-wake cycle is based on two clinical observations: (a) Patients on dialysis frequently sleep during and after dialysis, and (b) the procedure often is associated with temperature elevations. A review of pertinent literature showed that sleepiness and temperature are physiologically related, and that these two variables are important indicators of sleep and circadian regulatory processes. OBJECTIVES: To describe possible associations among hemodialysis, body temperature, and sleepiness; to assist in building hypotheses; and to provide direction for future research. METHODS: Three exploratory studies were performed to investigate (a) dialysis-associated elevations in body temperature, (b) changes in body temperature patterns across the day, and (c) alterations in daytime sleepiness. RESULTS: The current study demonstrated that many patients manifest dialysis-associated episodic elevations of body temperature, changes in the circadian pattern of body temperature, and increases in daytime sleep propensity. In the context of the Two-Process Model of Sleep Regulation, these alterations provide a parsimonious explanation for the poor subjective sleep quality, prolonged sleep latency, and daytime sleepiness reported by these patients. CONCLUSIONS: These preliminary studies lead to the suggestion that hemodialysis may disrupt basic sleep regulatory mechanisms, and that further investigation in this area is warranted.

Adult↗