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)     

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.     

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 die visualization of motor end plate patterns in superficial muscles or deeper muscles exposed by dissection.     

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

Summary 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.Supported by the Deutsche Forschungsgemeinschaft (Ku 541/2-1)     

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.     

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.     

Acetylcholinesterase distribution in chick spinal cord cultures

Summary Explants of 10–12 day chick embryo spinal cord were cultured by coverslip-roller tube method for 3–80 days. The cellular and subcellular localization of acetylcholinesterase activity in cultured neurons was studied by the thiocholine techniques of Karnovsky and Roots and Lewis and Shute.At the light microscopic level, acetylcholinesterase was demonstrated in the neurons of both ventral and dorsal horn regions. Occasionally neurons migrated in the outgrowth zone exhibited strong intracellular activity.At the electron microscopic level, acetylcholinesterase activity was found in the nuclear envelope, granular endoplasmic reticulum and the Golgi apparatus of the neurons. No enzyme reaction was detected in the glial cell cytoplasm.     

Extensive Distribution of Acetylcholinesterase in Guard Cells of Vicia faba

Acetylcholinesterase, an enzyme responsible for hydrolyzing of acetylcholine to choline and acetic acid residues, is detected in the guard cell protoplasts. Extensive acetylcholinesterase activity has been found in the guard cell protoplasts as compared with the mesophyll cell protoplasts. Moreover, light could stimulate the enzyme activity. Localization of acetylcholinesterase in the stomata of Vicia faba L. was undertaken using Karnovsky and Roots cytochemical method. It was found that in the stomata of this plant products of acetylcholinesterase enzymatic reaction mainly appeared in the outer side of the guard cell ventral wall and inner wall. When the staining time was prolonged, products of acetylcholinesterase enzymatic reaction could also be found in the ventral and inner wall of the guard cells. In addition, more extensive product of enzymatic reaction was observed in the opened stomata than in the closed stomata. It was assumed that acetylcholineaterase may participate in the regulation of stomatal movement by hydrolyzing acetylcholine around the stomata.     

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.     

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.     

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.     

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     

Density of the neurovegetative innervation of the uterus of rats

Using the Karnovsky and Roots modified by E1 Badawi and Schenk's technic for the cholinergic innervation and with the method of Lindvall and Bj?rklund modified by de la Torre and Surgeon for the adrenergic ones, we demonstrate the innervation of each tissular layer, in cervix, corpus and cornua of the uterus of the she-rat in pro-oestrus state. A statistic evaluation is established for the different areas. The density of cholinergic innervation is richer than the adrenergic one. The number of cholinergic fibers is maximum in the cervix. It decreases in corpus and cornua especially in the mucosal and sub-mucosal layers. The density of the muscular nervous network is predominant in the corpus versus the cervix. The number of adrenergic nerves is maximum in corpus it diminishes in the cervix and the cornua. In each region, the muscular adrenergic network domines over the other tissular layers. We confirm statistically morphometric non parametric observations of these other authors.     

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.     

Influence of glutaraldehyde fixation of cells adherent to solid substrata on their detachment during exposure to shear stress.

In order to determine the response of fixed and nonfixed cells adherent to a solid substratum to shear stress, human fibroblasts were allowed to adhere and spread on either hydrophilic glass or hydrophobic Fluoroethylene-propylene (FEP-Teflon) and fixed with glutaraldehyde. Then, the cells were exposed to an incrementally loaded shear stress in a parallel plate flow chamber up to shear stresses of about 500 dynes/cm2, followed by exposure to a liquid-air interface passage. The cellular detachment was compared with the one of nonfixed cells. In case of fixed cells, 50% of the adhering cells detached from FEP-Teflon at a shear stress of 350 dynes/cm2, whereas 50% of the adhering, nonfixed cells detached already at a shear stress of 20 dynes/cm2. No fixed cells detached from glass for shear stresses up to at least 500 dynes/cm2. More than 50% of the nonfixed cells were detached from glass at a shear stress of 350 dynes/cm2. Furthermore, the shape and morphology of fixed cells did not change during the incrementally loaded flow, in contrast to the ones of nonfixed cells, which clearly rounded up prior to detachment.     

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.     

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.     

Cytophotometric analysis of the apoprotein B content of sections of the human aorta

A cytophotometric technique to quantitate the content of apolipoprotein B (apo B) in cryostate sections of human aorta is described. The method is based on the comparison of different regions of the sections stained by indirect immunoperoxidase technique, using antibody to apo B. It has been found that the optical density of stained intimal layer was 8-22 times higher than that of the stained aortic media, which in its turn, differed from the control values. The intensity of specific staining of fibrous plaques was 58-87% of the staining intimal tissue of the apparently unaffected intima. About 25-42% of apo B content was extracted from the sections of the normal part of intima and fibrous plaque tissue during incubation of nonfixed sections in a physiologic saline.