Improvement of the method of Karnovsky and Roots for the histochemical demonstration of acetylcholinesterase

The aqueous reaction medium of Karnovsky and Roots for the demonstration of acetylcholinesterase (AChE) activity was modified to obtain a gel-incubation medium that prevents diffusion of the reaction product and the soluble part of AChE into the incubation medium and along the plane of the section. The incubation medium contained 27% polyvinyl alcohol and suitable concentrations of the components of the medium of Karnovsky and Roots. The application of this incubation medium to sections of rat hippocampus resulted in intense staining, with the reaction products being mainly localized in fibrous structures.     

A histochemical method for the demonstration of acetylcholinesterase activity using semipermeable membranes

The "direct coloring" thiocholine method of Karnovsky and Roots (1964) for the demonstration of acetylcholinesterase (AChE) activity was modified and adapted to the technique of semipermeable membranes. In this way it is possible to demonstrate histochemically both the bound as well as the soluble part of AChE activity. The localization of the reaction product is very distinct. Microdensitometric investigations of results of this method showed a linear increase of the amount of reaction product up to an incubation time of 180 min and section thickness up to 24 micron. The medium supplemented with buffer (instead of agar) can be used for the demonstration of AChE activity in cryostat sections adherent to slides and is also very suitable for the detection of multiple forms of AChE in polyacrylamide or agarose gels.     

A histochemical method for the demonstration of acetylcholinesterase activity using semipermeable membranes

Summary The direct coloring thiocholine method of Karnovsky and Roots (1964) for the demonstration of acetylcholinesterase (AChE) activity was modified and adapted to the technique of semipermeable membranes. In this way it is possible to demonstrate histochemically both the bound as well as the soluble part of AChE activity. The localization of the reaction product is very distinct. Microdensitometric investigations of results of this method showed a linear increase of the amount of reaction product up to an incubation time of 180 min and section thickness up to 24 m. The medium supplemented with buffer (instead of agar) can be used for the demonstration of AChE activity in cryostat sections adherent to slides and is also very suitable for the defection of multiple forms of AChE in polyacrylamide or agarose gels.In honour of Prof. P. van Duijn     

Quantitative aspects of cytochemical methods for acetylcholinesterase studied with a cytochemical model system

A model system of polyacrylamide films containing the Triton extract of rat brain homogenate was applied to investigate quantitatively some aspects of three methods for the cytochemical demonstration of acetylcholinesterase activity (Lewis 1961; Karnovsky and Roots 1964; Tsuji 1974). Biochemical determinations showed that about 90% of the acetylcholinesterase activity originally present in the Triton extract were still detectable in the films. The relationship of the formation of cuprous thiocholine iodide in the case of the methods of Lewis (1961) or Tsuji (1974) and of cupric ferrocyanide at the reaction of Karnovsky and Roots (1964) to either enzyme concentration or incubation time were tested in detail. The results showed that for the method of Tsuji and, with some restrictions, also for the method of Karnovsky and Roots a linearity exists in these two respects. In the case of the Lewis technique, an approximate linearity between the amount of reaction product and incubation time could only be found from 90 min onward, but no linearity was detected in relation to the enzyme concentration. At low enzyme concentrations, too little white precipitate was formed in comparison to higher ones. Therefore it is suggested that this technique, as compared to the methods of Tsuji and Karnovsky and Roots, probably is less suitable as a quantitative cytochemical method.     

Quantitative aspects of cytochemical methods for acetylcholinesterase studied with a cytochemical model system

Summary A model system of polyacrylamide films containing the Triton extract of rat brain homogenate was applied to investigate quantitatively some aspects of three methods for the cytochemical demonstration of acetylcholinesterase activity (Lewis 1961; Karnovsky and Roots 1964; Tsuji 1974).Biochemical determinations showed that about 90% of the acetylcholinesterase activity originally present in the Triton extract were still detectable in the films. The relationship of the formation of cuprous thiocholine iodide in the case of the methods of Lewis (1961) or Tsuji (1974) and of cupric ferrocyanide at the reaction of Karnovsky and Roots (1964) to either enzyme concentration or incubation time were tested in detail. The results showed that for the method of Tsuji and, with some restrictions, also for the method of Karnovsky and Roots a linearity exists in these two respects. In the case of the Lewis technique, an approximate linearity between the amount of reaction product and incubation time could only be found from 90 min onward, but no linearity was detected in relation to the enzyme concentration. At low enzyme concentrations, too little white precipitate was formed in comparison to higher ones. Therefore it is suggested that this technique, as compared to the methods of Tsuji and Karnovsky and Roots, probably is less suitable as a quantitative cytochemical method.This word was performed while one of us (Dr. Andrä) was in receipt of a visitor grant from the Netherlands Organization for the Advancement of Pure Research (ZWO)     

Visualization of detailed acetylcholinesterase fiber and neuron staining in rat brain by a sensitive histochemical procedure

A sensitive method for acetylcholinesterase (AChE) histochemistry has been developed which permits simultaneous observation of fine fiber processes and neuron cell bodies. In rat brain, distinctive configurations can be observed which have been difficult to see by other techniques. The staining procedure involves two steps. Tissue sections are incubated first in Karnovsky and Roots medium diluted one-hundredfold; and then with a mixture containing diaminobenzidine (DAB) and H2O2. The reaction product of the first step induces cleavage of hydrogen peroxide in the second step, with a resulting oxidation of DAB to yield a fine precipitate. Addition of metal ions, such as nickel, to the DAB-H2O2 mixture produces high-contrast, Golgi-like images of neuron structures. The technique is much more sensitive than previous methods and greatly reduces background staining caused by crystallization of reaction products. Many potential applications exist for this new technique, in addition to the initial results described here.     

Acetylcholinesterase activity in spiral ganglion cells in rats]

Acetylcholinesterase has been demonstrated in the spiral ganglion cells by the method of Karnovsky and Roots. After incubation for 24 h the content of AchE was measured with the help of a three-stage scale. 22% of the cells showed strong, 50% medium and 28% weak AchE activity. Within the spiral ganglion perikarya, the enzyme was equally distributed.     

A morphological and histochemical study of the developing tongue musculature in the mouse: its relationship to palatal closure.

Some question exists concerning the ability of the embryonic tongue to undergo reflex movements at the time of palatal closure (15.5 days of development). Functional motor endplates are prerequisite for such movements to occur. Light and ultrastructural cytochemical methods were employed to elucidate the morphology of neuromuscular relationships in the developing mouse tongue. The A/Jax mice used in the experiments demonstrated a 12-20% incidence (seasonal variation) of spontaneous cleft palate, allowing a correlation between normal and teratological processes. Organized myofibrils were first seen in tongues of normal and spontaneous cleft lip-cleft palate (SCL-CP) specimens at 14.5 days of development. The thiocholine technique of Karnovsky and Roots was used to demonstrate acetylcholinesterase (AChE) activity at the light microscope level. The Lewis and Schute method was used for ultrastructural localization of this enzyme. Tissues from normal and SCL-CP specimens from 12.5 to 20.5 days of gestation failed to show differences in amounts or distribution of AChE activity. AChE activity was seen as early as 14 day's gestation. Electron microscopic studies demonstrated reaction product in the endoplasmic reticulum and nuclear envelope of developing myoblasts. AChE activity at the developing neuromuscular junction and the occurrence of myofilaments preceded palatal closure by several days. Based on these morphological and histochemical findings the tongue of normal and SCL-CP embryos appears capable of responding to a neurogenic stimulus at the time of palatal closure. The findings suggest that the tongue of animals exhibiting a spontaneous cleft palate is not actively involved in the etiology of this condition.     

Biochemical and cytochemical evidence indicates that coated vesicles in chick embryo myotubes contain newly synthesized acetylcholinesterase

We have isolated highly purified coated vesicles from 17-d-old chick embryo skeletal muscle. These isolated coated vesicles contain acetylcholinesterase (AChE) in a latent, membrane-protected form as demonstrated enzymatically and morphologically using the Karnovsky and Roots histochemical procedure (J. Histochem. Cytochem., 1964, 12:219- 221). By the use of appropriate inhibitors the cholinesterase activity can be shown to be specific for acetylcholine. It also can be concluded that most of the AChE represents soluble enzyme since it is rendered soluble by repeated freeze-thaw cycles. To determine the origin of the coated vesicle-associated AChE, we have isolated coated vesicles from cultured chick embryo myotubes which have been treated with diisopropylfluorophosphate, an essentially irreversible inhibitor of both intra- and extracellular AChE, and have been allowed to recover for 3 h. This time is not enough to allow any newly synthesized AChE to be secreted. These coated vesicles also contain predominantly soluble AChE. These data are compatible with the hypothesis that coated vesicles are important intermediates in the intracellular transport of newly synthesized AChE.     

Cholinergic innervation of the cornea and iris of the guinea pig

Cholinergic innervation of the cornea and iris of the newborn and adult guinea pig was studied by the technique of Karnovsky and Roots (1964). The given structures are both richly innervated. The cholinesterase reaction of the cornea is more strongly positive in adult animals, whereas the intensity of the reaction of the iris in newborn and adult guinea pigs is almost identical.     

Dot-blot test for identification of insecticide-resistant acetylcholinesterase in single insects

A test was developed to detect the presence of insecticide-resistant acetylcholinesterase (AChE) in single insects based on the quasipermanent binding of proteins onto blotting membranes. The method is simple, sensitive, requires inexpensive equipment, and produces a permanent record of results. AChE activity is revealed by the Karnovsky & Roots staining technique in the presence of propoxur, or after exposure of the membrane to paraoxon and rinsing with water. We chose insecticide concentrations that inhibited the sensitive AChE while allowing detectable residual activity of the resistant AChE to remain. By comparing the staining of insecticide-treated and control membranes, susceptible and resistant genotypes for the AChE gene could be distinguished in laboratory strains of mosquitoes (Culex spp. and Anopheles albimanus Wiedemann) and the house fly (Musca domestica L.). Resistant AChE from mosquitoes was less susceptible both to propoxur and paraoxon than the corresponding sensitive AChE, whereas resistant AChE from house fly was less susceptible mainly to paraoxon. The technique worked well for mosquito adults and house fly heads but not for mosquito larvae. Blotted AChE did not show detectable loss of activity during storage of the membranes for 3 wk at 25 degrees C. Storage is an important asset of the technique because transportation of live insect material to the laboratory may not be necessary.     

Distribution of acetyl cholinesterase in the hippocampal region of the guinea pig

Summary 1. The distribution of acetyl cholinesterase (AChE) has been described in the dentate area, a part of the hippocampal region, in the adult guinea pig. The enzyme was demonstrated histochemically with a modification of the Koelle thiocholine method applied to formaldehyde-fixed frozen sections and unfixed cryostat sections. Non-specific cholinesterase was suppressed by ethopropazine, while the staining reaction for AChE was controlled by complete specific inhibition with BW 284c51. A single brain was stained according to the method of Karnovsky and Roots.2. The abundant AChE found in the dentate area exhibited a distinctly stratified distribution pattern. In the molecular layer, strong reaction was present in the outer third and immediately above the granular cell layer, the intermediate zone being light. The granular cell bodies were unstained. In the hilus, five layers showing alternating stronger and weaker reaction for AChE were recognizable.3. In view of the opinions of Cajal, Lorente de Nó, and Blackstad criteria for the definition of the dentate area are discussed. The present results fit into a concept of a layered guinea pig hilus representative of one group of mammals (other members being rabbit, monkey, and man) differing morphologically from the non-layered hilus of rat and mouse. The distribution of metal in the guinea pig hilus supports the concept.4. Possible structural correlates to the AChE are considered and a comparison with the distribution of AChE in the rat, reported earlier, has been made. In the molecular layer, the most striking difference was the heavy activity observed in the outer third in the guinea pig, where the content is moderate in the rat. The granular cell layer appeared virtually identical in both species. In the hilus the stratified pattern in the guinea pig, contrasting with the more diffuse distribution in the rat, essentially reflects the differing structural architectonics in the hilus of the two species.I am indebted to Mrs. L. Knudsen, Mr. A. Meier, Mr. Th. Nielsen, Mrs. K. Sørensen, Miss M. Sørensen, and Miss B. Ørum for skillful technical assistance.This study was supported in part by U.S.P.H.S. Grant NS 07998.     

On the intrinsic innervation of normal rat liver

Summary With the Bodian method stained fibers were observed in the lobules of the rat liver and with the modified Karnovsky and Roots thiocholine method cholinesterase (presumably acetylcholinesterase (AChE))-positive nerve fibers were found in a pattern similar to that of the Bodian-stained fibers. The AChE positive nerve fibers form a network in the liver lobules in close relation to hepatocytes and sinusoids. Fluorescent varicose nerve fibers demonstrated by the glyoxylic acid and Falck-Hillarp fluorescence methods were found only in the interlobular spaces associated with vessels. As no overlapping of distribution patterns of AChE-positive nerve fibers and fluorescent nerve fibers occurs, the AChE activity of the nerves of the liver lobules probably reflects the associated presence of acetylcholine in the nerve fibers. In consequence we suggest that nerves of the liver lobules belong to the autonomic parasympathetic nervous system.SEM of liver tissue revealed light cords apparently situated in smooth surfaced channels between adjacent hepatocytes and in the space of Disse, where fibers also cross sinusoids. We tentatively suggest that the cords of the SEM represent the AChE-positive nerve fibers of our LM observations'.The skilled assistance of Dr. Esther Hage, Department of Pathology, Odense Sygehus, Denmark, in the Falck-Hillarp fluorescence work is gratefully acknowledged     

Use of potassium ferricyanide in histochemistry

Karnovsky and Roots offer to use potassium ferricianide for coloured detecting the products of acetylcholine hydrolysis by cholinesterase. The method is based on the reduction of ferricianide to ferrocianide which forms with copper ions, present in the solution, unsoluble ferrocianide. Some properties of ferricianide ion, however, (stability, large size and great hydratation) make it difficult for the substance to penetrate the native cell membranes. The method by Karnovsky and Roots applied to laminated muscular tissue and to the rat nonfixed whole diaphragm, and to the sections from nonfixed tissue of the cat skeletal muscle verifies space isolation of ferricianide from the enzyme localized at the other side of cell membrane.     

Ultrastructure of cholinergic innervation in the cirrhotic liver in guinea pigs. Neurohistochemical and ultrastructural study

Hepatic cirrhosis was induced in guinea pigs by ligation of the common bile duct and innervation of the liver was studied by fluorescence histochemistry (glyoxylic acid method), acetylcholinesterase (AChE) neurohistochemistry (modified Karnovsky and Roots method), and transmission electron microscopy. In control animals the adrenergic terminals showed connections with endothelial cells, hepatocytes and fat-storing cells, but no cholinergic terminals were evident. Cirrhosis was present 6 weeks after the bile duct ligation and marked fibrosis, accompanied by bile duct proliferation, was evident in the portal areas. Numerous AChE-positive nerve fibers traversed the collagenous bundles in the fibrotic areas, and cholinergic terminals formed close contacts with fibroblasts. Each axon terminal was found to contain numerous small coreless vesicles and AChE-reaction products were confirmed in the space between a nerve terminal and a fibroblast. In contrast, fluorescence adrenergic nerve fibers and their terminals remained unchanged. This study demonstrates that parasympathetic cholinergic innervation participates in some stages in the development of hepatic cirrhosis.     

Graphic representation of the distribution of acetylcholinesterase in cat dorsal root ganglion neurons

Synopsis Acetylcholinesterase (AChE) activity of primary sensory neurons of the cat has been quantitated and correlated with cell size. Dorsal root ganglia of the fourth and fifth thoracic spinal levels were studied. Frozen longitudinal and cross-sections were collected serially and stained with Cresyl Violet for total cell counts of the ganglia on the left; the average count was 3375 cells. Ganglia from the right were stained for AChE after the method of Karnovsky & Roots (1964) as modified by El Badawi & Schenk (1967), and counterstained with Haematoxylin. Cells were counted in every fourth section and the diameter of each was recorded. AChE-positive cells were classified as brown (B₁, B₂, B₃) and AChE-negative ones as blue (BL).An inverse correlation exists between cell size and AChE activity. High activity was demonstrated in 29% of the cells (B₁), moderate activity in 52% (B₂), minimal activity in 15% (B₃) and 4% were classified as AChE-negative (BL). Small cells with high activity were centrally located in the ganglia whereas large AChE-negative cells were peripherally distributed. Chi-Square analysis revealed that the size of the cell was not independent of the enzyme colour category.     

Fiber analysis of human ventral and dorsal roots on the basis of different acetylcholinesterase activity]

Marked differences in the AChE activity of myelinated nerve fibers of ventral and dorsal roots could be established in human post mortem material. After a fixation time of 3 h and a critical incubation period of 24 h, in the mean 96% of the myelinated ventral root but only 4% of dorsal root fibers showed reaction product, detectable by the light microscope. The percentage of stained fibres varies, to some extent, in the different segments. Groups of very thin myelinated fibres within the ventral roots between the segments C-8 and L-3, showing a conspicuous high enzyme activity, are interpreted as pre-ganglionic sympathetic fibres; similar elements in the sacral ventral roots may represent parasympathetic fibres. The method of Karnovsky, applied under conditions established in this study, can be used for analysis of fibre types in a given human peripheral nerve.     

Opsin-immunoreactive outer segments and acetylcholinesterase-positive neurons in the pineal complex of Phoxinus phoxinus (Teleostei,Cyprinidae)

Summary The pineal complex of the teleost, Phoxinus phoxinus L., was studied light-microscopically by the use of the indirect immunocytochemical antiopsin reaction and the histochemical acetylcholinersterase (AChE) method.Opsin-immunoreactive outer segments of photoreceptor cells were demonstrated in large numbers in all divisions of the pineal end-vesicle and in the pineal stalk. Moreover, they were found in the roof of the third ventricle, adjacent to the orifice of the pineal recess as well as scattered in the parapineal organ. These immunocytochemical observations provide direct evidence of the presence of an opsin associated with a photopigment in the photosensory cells of the pineal and parapineal organs of Phoxinus. By means of the AChE reaction (Karnovsky and Roots 1964) inner segments of pineal photoreceptors, intrinsic nerve cells, several intrapineal bundles of nerve fibers, and a prominent pineal tract were specifically marked. The pineal neurons can be divided into two types: one is located near the pineal lumen, the other near the basal lamina. The latter perikarya bear stained processes directed toward the photoreceptor layer. A rostral aggregation of two types of AChE-positive nerve cells occurs in the ventral wall of the pineal end-vesicle. The main portion of the AChE-positive pineal tract, which lies within the dorsal wall of the pineal stalk, can be followed to the posterior commissure where some of the nerve fibers course laterally. A few AChE-positive pineal nerve fibers run toward the lateral habenular nucleus via the habenular commissure. In the region of the subcommissural organ single AChE-positive neurons accompany the pineal tract. The nerve cells of the parapineal organ exhibit a moderate AChE activity.These findings extend the structural basis for the remarkable light-dependent activity of the pineal organ of Phoxinus phoxinus. To the memory of Professor Karl von Frisch, pioneer and master in the field of photoneuroendocrine systemsThis investigation was supported by grants from the Deutsche Forschungsgemeinschaft to A.O. (Ok 1/24; 1/25: Mechanismen biologischer Uhren) and to H.-W. K. (Ko 758/1; 758–2)On leave from the 2nd Department of Anatomy, SOTE, Budapest, Hungary     

A histochemical method for gross demonstration of motor end plates.

The thiocholine-ferricyanide method of Karnovsky and Roots for histochemical demonstration of cholinesterases has been applied to whole fetal and neonatal mice and chicks for the visualization of motor end plate patterns in superficial muscles or deeper muscles exposed by dissection.     

Vitellogenin synthesis by the fat body of the mosquito Aedes aegypti: evidence of transcriptional control

The acetylcholinesterase (AChE) activity of cultures from 11-day-old chick embryo muscle cells was studied for up to 4 weeks in vitro. AChE activity was found in mononucleated cells and multinucleated myotubes. The activity increased greatly after fusion. Maximum AChE levels were reached after 7–10 days of incubation and tended to decline thereafter. Multiple forms of AChE found in embryo muscle in situ were present in cultures before and after fusion. Selective inhibitors and substrates were used to show that AChE was released by the cells into their medium. Within a 2-day period the AChE that accumulated in the medium averaged over 6 times that remaining in the cells. Release of AChE from the cells was inhibited by cycloheximide, and AChE levels in cells and medium were much reduced when differentiation was inhibited by bromodeoxyuridine. Little AChE was present in subcultures of fibroblasts from muscle cultures. Acetyl-β-methylcholine and, to a lesser degree, choline itself, prevented the decrease in AChE levels of 2- to 3-week-old muscle cultures.