AChE Staining Research Articles

Effects of vagotomy on the distribution of cholinesterases in the cat medulla oblongata

Histochemical study of the distribution of cholinesterases in the cat medulla oblongata reveals that all neurones in the dorsal motor nucleus of the vagus (DMV) contain true cholinesterase (AChE) but, while most contain this enzyme alone, a small proportion of cells contain pseudocholinesterase (BuChE) as well. Cervical vagotomy affects the two types of cell to different degrees of severity. The BuChE-containing neurones lose their enzyme completely within 2–3 weeks and they atrophy and disappear as a result of the operation. On the other hand, the reaction is less severe and is reversible in those cells containing AChE only. Vagotomy also causes reduction of AChE and BuChE staining in the nearby area subpostrema; the depletion here is pronounced at 2–3 weeks and recovery occurs within the year. These findings suggest that some cells in the area subpostrema project peripherally via the vagus and that the area is part of the vagal nuclear complex. Moreover, capillaries in the area contain AChE and BuChE in the endothelial lining and this is one of the few areas of the cat hindbrain to exhibit such vascular enzyme activity. The ependyma of the area postrema, which overlies the area subpostrema, is heavily stained for BuChE but this is unaffected by vagotomy.

Some morphological features of the myoneural junctions in certain normal muscles of the rat.

AbstractIn frozen sections and whole mount preparations of the normal soleus, diaphragm, or intercostal muscles of the rat, myoneural junctions of different AChE and silver staining intensities were regularly observed. Muscle fibers of different diameters showed end‐plates of varying staining intensities. No correlation was found between a certain staining intensity and diameter of the end‐plates or between muscle fiber diameters and the ratio of pale to strongly stained end‐plates. There was, however, a tendency for the smaller muscle fibers to have end‐plates of smaller mean diameter than those in the larger muscle fibers.

The Schwartz-Jampel syndrome: Its clinical, physiological and histological expressions

Patients with the Schwartz-Jampel syndrome represent an example of continuous muscle fiber activity at rest. Abolition of the continuous repetitive discharges with curare showed that this was not myotonia as previously reported. The likelihood that the abnormal discharges originated from the muscle component of the neuromuscular junction rather than the nerve was suggested by the observation that spontaneous electrical activity persisted long after peripheral nerve injury in 1 of the cases. Electron-microscopic examination of biopsy specimens showed marked dilatation of the endoplasmic reticulum of skin fibroblasts and moderate dilatation of the sarcoplasmic reticulum of muscle fibres. Tissue culture studies of the fibroblasts revealed a low level of lipid label incorporation representing a non-specific depression of the synthetic machinery of these cells. These studies failed to demonstrate any mucopolysaccharide abnormality, even though the radiologic findings in 1 of the cases were consistent with those found in the Morquio-Brailsford syndrome. Histochemical evaluation showed hypertrophy of both Type I and II fibers with SDH activity uniformly high in both fiber types. AChE staining revealed marked diffuse activity throughout the sarcoplasm of the muscle similar to that reported in embryonic muscle. These findings suggest the possibility that the abnormality in this syndrome occurs during fetal development.

Cholinesterase activity in rough and smooth brain microsomal membranes.

AbstractRat brain cortex was homogenized in 0.25 M or 0.32 M sucrose and sedimented at 10,000 Xg for 20 min. The supernatant was further separated into two subtractions by density gradient centrifugation in Cs+‐containing sucrose. Electron microscopy, RNA/protein and phospholipid/ protein indices showed that one of the subfractions was similar to the rough‐surfaced (R) and the other to the smooth‐surfaced (S) microsomal fractions separated from liver tissue by Dallner (1963). Cholinesterase (ChE) activity was studied by titration. Only 8–13 % of the total ChE activity was recovered in the 10,000 × g supernatant and about 3–6 % in the subfractions. Both the R and S microsomal subfractions displayed ChE activity; however, the S sub‐fraction had about three times as much activity per mg protein as the R subfraction. Activity due to nonspecific cholinesterase was 13 % of the total ChE activity in the R fraction and 7 % in the S fraction when the homogenate was prepared by homogenization with a clearance of 250 urn at 840 rpm. More vigorous homogenization increased the activities. Histochemical staining for AChE and ns. ChE activities revealed reaction product adjacent to both R and S vesicles.

The development of neuromuscular junctions in cultures of chick embryo tissues.

AbstractNeuromuscular junctions formed in vitro have been obtained previously only in cultures of cross‐sections of embryos. These have represented simply the further development in vitro of the anlage of neuromuscular junctions previously formed in vivo. The present experiments were designed to determine whether such junctions could form de novo in tissue culture and in organ culture from spatially separated spinal cord and skeletal muscle. Cultures of dissociated spinal cord and muscle cells could not be maintained sufficiently long to lead to differentiated neuromuscular junctions. However, when fragments of spinal cord and muscle from chick embryos of varying developmental stages were cultivated for several weeks neuromuscular junctions could be identified by phase contrast microscopy, silver impregnation, and AChE staining. The neuromuscular junctions were similar histologically and histochemically to those developing in vivo.

The effect of axotomy on the acetylcholinesterase of the superior cervical ganglion of the cat

The effects of chronic axotomy of postganglionic fibers on the AChE of sympathetic ganglia of the cat were investigated histochemically and quantitatively. Partial axotomy of the superior cervical ganglion was performed by sectioning the main postganglionic (internal carotid) branch or the external carotid branch, or by enucleation of the anterior contents of the orbit; the inferior mesenteric ganglion was partially axotomized by sectioning both hypogastric nerves. During the postoperative periods of 8 to 44 days, at the end of which the ganglia were studied, there was a marked but reversible loss of AChE from the perikarya of most of the axotomized, heavily and moderately stained sympathetic neurons; in the normal superior cervical ganglion of the cat, these groups together comprise less than 20% of the total ganglion cells, while in the inferior mesenteric ganglion the corresponding figure is even smaller; the remaining sympathetic neurons of both ganglia show only faint AChE staining. Although there was an initial impression of a concomitant loss of AChE from the preganglionic fibers, this was not borne out by more careful examination. Manometric determinations indicated that there was a loss of only 6% of the ganglionic AChE following chronic axotomy of the internal carotid branch of the superior cervical ganglion; after preganglionic denervation, there was a loss of approximately 75 %. It was concluded that the fall in ganglionic AChE after postganglionic axotomy can be accounted for entirely on the basis of the loss of the enzyme from the axotomized ganglion cells themselves, and that the ensuing changes in synaptic transmission and drug sensitivity that have been reported for such ganglia are not due to this factor.

Localization of cholinesterases in adult mammalian brain cell cultures

1. AChE was localized in all neuronal elements and BuChE in glial elements in the explants and migratory zones of whole mount adult rabbit and human cortex cultures maintained up to 6 months. Koelle's technique ( Koelle, 1955) with AChTh and BuTh as substrates and suitable inhibitors was used. Eserine (10 −5 to 10 −3 M) inhibited cholinesterases. DFP (10 −8 to 10 −6 M) and RO2-0682 (6 × 10 −8M) inhibited BuChE. 2. AChE was present in the perikarya and fibers and was found to highly concentrated in axonal endings, especially presynaptic endings. 3. Cultures from human subcortical white matter after 2 months in culture showed an absence of AChE but the presence of BuChE in glial elements. AChE and BuChE could be localized in corresponding gray matter cultures. 4. Sodium barbital decreased the intensity of staining for AChE and increased the staining of glial elements for BuChE. Epinephrine reduced the stain intensity of AChE in the perikarya of neurons while at the same time the fibers and boutons stained intensely.