High Acetylcholinesterase Activity Research Articles

Extraocular muscles in the lamprey, Lampetra fluviatils L. II. Motor end plates

Motor innervation and particularly the structure of motor end plates (MEPs) was studied in the extraocular muscles of the lamprey, Lampetra fluviatilis L., by light and electron microscopy. Each muscle is supplied with numerous thin motor nerve fibres. Motor end plates are located at their ends or along their course. Two motor end plate types were distinguished: en grappe‐like plates with a low acetylcholinesterase (AChE) activity were observed on thin muscle fibres, whilst en plaque‐like plates with a high AChE activity were found on thick mitochondria‐rich and thick multifibrillar muscle fibres. The postsynaptic membrane of the former MEP type does not show the presence of infoldings, MEPs located on thick mitochondria‐rich fibres show occasional infoldings, whereas the postsynaptic membrane of MEPs present on thick multifibrillar fibres reveals numerous infoldings. Motor end plates present in the extraocular muscles in the lamprey possess features typical for higher vertebrates and elasmobranch fishes, as well as for Tunicata.

Convergence of afferents from frontal cortex and substantia nigra onto acetylcholinesterase-rich patches of the cat's superior colliculus

The patterns of distribution of frontotectal and nigrotectal fibers were studied with the anterograde horseradish peroxidase method in the cat. Direct serial-section comparisons were made between the afferent-fiber patterns and the compartmentalized arrangements of acetylcholinesterase staining within the intermediate and deep collicular layers. Many of the patches of high acetylcholinesterase activity in the intermediate gray layer proved to be zones in which labeled frontotectal and nigrotectal fibers converged. These acetylcholinesterase-rich patches may thus represent sites at which functional influences from the basal ganglia and frontal cortex are coordinated. In the deeper tiers of the intermediate gray layer and layers ventral to it, there were also zones of heightened and diminished acetylcholinesterase staining. Much of this histochemical patterning was reflected in the arrangement of fibers labeled by large rostromedial frontal injections, but these deeper tiers were not strongly labeled after more lateral frontal injections or after injections placed in the substantia nigra. The deeper parts of the acetylcholinesterase-positive gridwork in the superior colliculus are thus distinct from its upper tier of acetylcholinesterase-positive patches. We conclude that the compartmentalized patterning of dense acetylcholinesterase staining in the intermediate and deep collicular layers represents a mosaic architecture to which collicular afferent circuitry is tightly related. This gridwork may serve to set up functional domains within which different aspects of collicular processing are accommodated.

The neostriatal mosaic: compartmentalization of corticostriatal input and striatonigral output systems.

The striatum (caudate-putamen) of the basal ganglia in the mammalian forebrain is a mosaic of two interdigitating, neurochemically distinct compartments. One type, the 'patch' compartment, is identified by patches of dense opiate receptor binding, and is enriched in enkephalin- and substance P-like immunoreactivity. The other compartment, the 'matrix', has a high acetylcholinesterase activity, and is shown here to have a dense plexus of fibres displaying somatostatin-like immunoreactivity. The present study demonstrates the two compartments have distinct connections, using a method that concurrently reveals striatal input, output and neurochemical systems in the rat. Patches receive inputs from the prelimbic cortex (a medial frontal cortical area with direct 'limbic' inputs from the amygdala and hippocampus); they also project to the substantia nigra pars compacta (the source of the nigrostriatal dopaminergic system). Conversely, the matrix receives inputs from sensory and motor cortical areas; here it is shown to project to the substantia nigra pars reticulata (the source of the non-dopaminergic nigrothalamic and nigrotectal system). Also, an intrinsic striatal somatostatin-immunoreactive system is described that may provide a link between the two compartments. The striatal patch and matrix compartments thus appear to be functionally distinct and interactive parallel input-output processing channels.

Acetylcholine Receptors on the Cell Body Membrane of Giant Interneurone 2 in the Cockroach, Periplaneta Americana

ABSTRACT lonophoresis of acetylcholine (ACh) onto the cell body membrane of an identified giant interneurone (GI2) in the central nervous system of the cockroach Periplaneta americana induced a depolarizing response at resting potential which was attributed to a population of extrasynaptic ACh receptors. The sensitivity of the cell body membrane of GI 2 to ionophoresis of ACh was determined. Perfusion of 1·0 × 10−6M neostigmine, an inhibitor of acetylcholine esterase, potentiated the ACh sensitivity of the cell body membrane of GI 2. This indicated that a high acetylcholinesterase activity was present in the periphery of the sixth abdominal ganglion (A6). The nicotinic antagonist, a-bungarotoxin (at a concentration of 1·0 × 10−7M) was found to block the ACh response of the cell body membrane of GI 2. By contrast, the muscarinic antagonist, quinuclidinyl benzilate, (at concentrations up to 1·0 × 10−5 M) had no detectable effect on the ACh response. It is suggested that an extrasynaptic nicotinic type of ACh receptor is present on the cell body membrane of GI 2.

Association of acetylcholinesterase with the external surface of the presynaptic plasma membrane in Torpedo electric organ

A high acetylcholinesterase (AChE) activity was found associated with pure cholinergic synaptosomes prepared from Torpedo electric organ. This activity was bound to the presynaptic plasma membrane upon subfractionation on sucrose density gradients. It was not solubilized in the presence of 2 M MgCl 2 but in the presence of Triton X 100. This presynaptic AChE activity corresponded to a hydrophobic form of the enzyme with a sedimentation coefficient of 5.5 S in our conditions. More than 80% of the AChE activity of intact synaptosomes was externally oriented. The presynaptic AChE activity could represent as much as 25% of the total activity in Torpedo electric organ.

Axon/Schwann-cell relationships in the giant nerve fibre of the squid.

This communication summarizes the experimental evidence obtained in the giant nerve fibre of the tropical squid Sepioteuthis sepioidea, on the nature of the mechanism responsible for the long-lasting effects of axonal excitation on the membrane potential of the Schwann cell. In these nerve fibres the propagation of a train of nerve impulses by the axon is followed by a prolonged hyperpolarization of the Schwann cell which can be reproduced, or modified, by the external application of cholinergic compounds. The presence and exact localization of the different components of the acetylcholine system directly involved in such Schwann-cell responses was detected by means of pharmacological, histochemical and chemical procedures. Thus, the results of the experiments herein discussed revealed that, under physiological conditions: the Schwann cell is able to synthesize, store and release acetylcholine; and that it has acetylcholine receptors of the nicotinic type, and acetylcholinesterase enzyme activity in its plasma membrane. On the other hand, the axon has low acetyltransferase activity and acetylcholine concentration in its axoplasm, and a high acetylcholinesterase activity in its axolemma. It was also found that acetylcholine hyperpolarizes the Schwann cell by increasing its relative permeability to the potassium ion. The distribution pattern of the acetylcholine system indicates that it operates as a feedback mechanism for the regulation of the Schwann-cell membrane potential and ionic permeability following axonal excitation.

Partial purification and characterization of a nerve trophic factor regulating muscle acetylcholinesterase activity

A protein influencing acetylcholinesterase activity of newt triceps muscle in organ culture has been identified and partially purified from rat brain homogenates by gel filtration, ion-exchange chromatography, and isoelectric focusing. Muscles treated with fractions containing the active factor had higher acetylcholinesterase activity than paired untreated muscles after 1 week in organ culture. Analysis of the molecular forms of acetylcholinesterase by velocity sedimentation showed that the factor had the greatest effect on the 16.5 S acetylcholinesterase, increasing it more than 100% of untreated controls. With isoelectric focusing, activity was present in a fraction focusing at pH 8.3. On SDS-polyacrylamide gel electrophoresis, a protein with a molecular weight of 34,000 was present in active fractions and was the major component of the isoelectric focusing pH 8.3 fraction. These findings indicate that a small basic protein, which we have termed neurotrophin, mediates the neuronal trophic effect on skeletal muscle acetylcholinesterase.

The significance of type 2C muscle fibers in Duchenne muscular dystrophy.

Undifferentiated type 2C fibers, which are dark on ATPase staining with both alkaline and acid preincubation, comprised on average 16.1% of the muscle fibers in 12 patients with Duchenne muscular dystrophy (DMD). Many of type 2C fibers had characteristics of regenerating fibers: basophilic cytoplasm, vesicular nuclei with occasional prominent nucleoli, high alkaline phosphatase and nonspecific esterase activity, and also high oxidative enzyme activity at the periphery of the fiber. A localized high acetylcholinesterase activity suggested the presence of a neuromuscular junction in some of the type 2C fibers. In serial sections, histochemical reactions characteristic of the type 2C fiber were present in segments of a single fiber, which in other parts was either a type 1 or a type 2 fiber. Since most of the opaque (hyaline, dark) fibers, which previously have been thought to be precursors of necrotic fibers, behaved as differentiated type 1 or type 2 fibers, the presence of type 2C fibers in DMD may not reflect "dedifferentiation" of fiber type, but rather indicate an active regenerating process. It remains unknown whether the type 2C fiber segments in DMD develop into fully differentiated functional fibers or remain as incompletely regenerated fibers.

Phenytoin, methysergide, and penicillamine in hereditary muscular dystrophy of the chicken

Chickens with an inherited muscular dystrophy (line 413) were treated from days 1 through 90 ex ovo with either phenytoin, methysergide, or d-penicillamine. Under the conditions of the study, phenytoin and methysergide were equally effective in improving the righting ability of dystrophic birds, but penicillamine had little or no effect. Phenytoin and penicillamine decreased the abnormally high creatine kinase activity in dystrophic plasma, but only phenytoin reduced the high acetylcholinesterase activity in plasma and fast-twitch muscles of dystrophic birds. Beneficial effects of methysergide and phenytoin were also shown in trials as short as 25 days. None of the treatments, improved the abnormal histological appearance of dystrophic muscles.

Effects of later isolation housing on both scent marking behavior and brain cholinergic activites in Mongolian gerbils

Significant difference in the frequency of marking was found between aggregated and isolated gerbils. As compared with the aggregated gerbils, isolated gerbils showed high acetylcholinesterase activity in the hippocampus and high choline acetyltransferase activity in the hypothalamus.

The nucleus basalis magnocellularis: The origin of a cholinergic projection to the neocortex of the rat

The cells of origin of a neocortical cholinergic afferent projection have been identified by anterograde and retrograde methods in the rat. Horseradish peroxidase injected into neocortex labelled large, acetylcholinesterase-rich neurons in the ventromedial extremity of the globus pallidus. This same group of neurons underwent retrograde degeneration following cortical ablations. The region in which cell depletion occurred also showed significant decreases in the activities of choline acetyltransferase and acetylcholinesterase. Discrete electrolytic and kainic acid lesions restricted to the medial part of the globus pallidus each resulted in significant depletions of neocortical choline acetyltransferase and acetylcholinesterase. Hemitransections caudal to this cell group did not result in such depletions. Taken together these observations suggest that the acetylcholinesterase-rich neurons lying in the ventromedial extremity of the globus pallidus, as mapped in this study, constitute the origin of a major subcortical cholinergic projection to the neocortex. The utility of acetylcholinesterase histochemistry in animals pretreated with di-isopropylphosphorofluoridate in identifying cholinergic neurons is discussed in the light of this example; specifically, it is proposed that high acetylcholinesterase activity 4–8 h after this pretreatment is a necessary, but not sufficient, criterion for the identification of cholinergic perikarya. The neurons in question appear to be homologous to the nucleus basalis of the substantia innominata of primates, and are thus termed ‘nucleus basalis magnocellularis’ in the rat. No evidence was obtained to support the hypothesis that nucleus of the diagonal band projects to neocortex. However, striking similarities in size and acetylcholinesterase activity were observed among the putative cholinergic perikarya of the nucleus basalis magnocellularis, the nucleus of the diagonal band, and the medial septal nucleus. Kainic acid lesions of the neocortex produced uniform and complete destruction of neuronal perikarya. These lesions decreased neocortical glutamic acid decar☐ylase activity, suggesting that there are GABAergic perikarya in the neocortex. However, the same lesions did not affect neocortical choline acetyltransferase. This observation suggests that there are no cholinergic perikarya in the neocortex, a conclusion that is consistent with the absence of intensely acetylcholinesterase-reactive neurons in the neocortex.

Acetylcholinesterase in the ocellus of Apis mellifica

The ultrastructural localization of the enzyme acetylcholinesterase (AChE) in the ocellus of the honey bee ( Apis mellifica) was studied by electron microscopy. High AChE activity was found both in the receptor-cell axons and in the surrounding glial cells. Second order neurones exhibited a remarkably lower anzyme activity. AChE was also detected in the intercellular spaces between the receptor-cell axons and the second order neurones. These results provide additional support to the assumed cholinergic nature of the photoreceptor cells in the insect ocellus.

Sensitivity of the central nuclei of the vagus nerve to insulin deficiency in rats (Enzyme-histochemical study)

The distribution and activity of acetylcholinesterase (AChE) in the central nuclei of the vagus nerve (nucleus dorsalis and nucleus ambiguus) were investigated by Gomori's (1952) histochemical method in sexually mature male rats, intact or with experimental alloxan diabetes. The number of cells with high AChE activity in nucleus dorsalis was increased by 6% in the rats with alloxan diabetes. The results suggest a role of the dorsal nucleus of the vagus nerve in the regulation of pancreatic endocrine function.

Acetylcholinesterase activity of synaptic structures in the spinal trigeminal nucleus

The electron microscope has been used to study the localization of acetylcholinesterase (AChE) activity in the spinal trigeminal nucleus of normal cats with special emphasis on the distribution near synaptic structures. Reaction product is found around both round and flattened synaptic vesicle-containing axon terminals, particularly in synaptic clefts and often specifically associated with the presynaptic, or less frequently the postsynaptic membrane. The presence of reaction product at these specific sites suggests that these are areas of high AChE activity and that acetylcholine may be important in neurotransmission in these regions.

Distribution of acetylcholinesterase activity in normal, dystrophic, and denervated muscles of the chicken

One distinctive property of denervated and genetically dystrophic muscles of chickens is high acetylcholinesterase activity found in the fibers. The distribution of AChE activity in single fibers from these muscles was studied by using fresh frozen serial sections, a specific histochemical stain, and photodensitometry. The results confirm the findings of a previous report (22) that high AChE activity occurs only around the neuromuscular junction region in fibers from biceps muscles of 6-week-old dystrophic chickens. In contrast, both normal and dystrophic biceps muscle of 6-week old chickens that had been denervated for 5 days exhibited high AChE activity throughout the length of the fibers. These results suggest that changes in AChE activity due to denervation are superimposed on the activity already present in dystrophy. The data support the idea that inherited muscular dystrophy in the chicken involves a specific defect in the regulation of AChE and perhaps other molecules around the neuromuscular junction.

Tissue acetylcholinesterase in plasma of chick embryos and dystrophic chickens

The plasma cholinesterases of chickens (line 304) with inherited muscular dystrophy and 2 normal lines were examined from 12 days of incubation to 14 weeks after hatching. Acetylcholinesterase (AChE) and non-specific cholinesterase (BChE) activities were distinguished by spectrophotometric, electrophoretic and titrimetric analyses using acetylcholine, butyrylcholine, and their thioester analogs acetylthiocholine and butyrylthiocholine as substrates and 284C51 and iso-OMPA as selective inhibitors. Plasma from normal and dystrophic embryos and from dystrophic chicks had high AChE activity. For example, by 12 weeks of age, 37% of plasma acetylthiocholine hydrolysis was inhibited by the anti-AChE agent 284C51. The results support the view that embryo plasma contains AChE and BChE forms. After hatching the AChE forms decrease greatly in normal plasma and a shift in BChE forms occurs. Later, the AChE forms return in dystrophic line plasma. AChE activity in plasma was circumstantially associated with multiple molecular forms of AChE in the sarcoplasm of embryo and dystrophic muscles and it is likely that these muscles are sources of the plasma AChE activity. The results also confirmed that acetyl-β-methylcholine, unlike the situation which exists in mammals, is hydrolyzed by both AChE and BChE forms in the chicken, and cannot be used to distinguish these cholinesterases.

Dual vasoconstrictor and vasodilator innervation of the uterine arterial supply in the guinea pig.

The innervation of the extrinsic uterine arterial supply of the guinea pig has been studied using an isolated perfused preparation. The preparation was normally at minimal tone and responded to periarterial stimulation by a vaso-constriction which was mimicked by norepinephrine and abolished by bretylium. Constrictor responses both to stimulation and to norepinephrine were similar in both pregnant and non-pregnant states. In pregnant preparations, raising the tone of the vessels with norepinephrine revealed a powerful dilator response to periarterial stimulation. This response was mimicked by acetylcholine, potentiated by anticholinesterases, reduced by hyoscine, and abolished by-local anesthetic treatment. In contrast, virgin preparations showed only a weak dilator response to stimulation, while the response to acetylcholine even at high concentrations was usually negligible. Histochemical examination of the vessels revealed a dense plexus of fine nerve fibers exhibiting high acetylcholinesterase activity along the main uterine artery. The fibers lay in close apposition to the vascular muscle layer. Nerve fibers exhibited fluorescence for catecholamines were also abundant, but were distributed along the secondary as well as the main arteries. It is concluded that the uterine arterial supply of the guinea pig is innervated by both adrenergic constrictor and cholinergic dilator fibers. The dilator fibers appear to be functional only during pregnancy. The possibility of a noncholinergic dilator innervation is also discussed.

CHOLINESTERASES IN THE HYPOTHALAMO-HYPOPHYSIAL NEUROSECRETORY SYSTEM OF THE WHITE-CROWNED SPARROW, ZONOTRICHIA LEUCOPHRYS GAMBELII.

The distribution of cholinesterases in hypothalamo-hypophysial neurosecretory system of the White-crowned Sparrow has been examined histochemically. The perikarya of the neurosecretory cells of the paraventricular and supraoptic nuclei have a high acetylcholinesterase activity. Acetylcholinesterase activity also occurs in the cells of the infundibular nucleus. The proximal parts of the axons of the cells of the neurosecretory and infundibular nuclei have strong acetylcholinesterase activity and weak non-specific cholinesterase activity. In the median eminence, the activity of acetylcholinesterase is strongest in the palisade layer. In the pars nervosa, there is definite, although weak, acetylcholinesterase activity.

Attivit� acetilcolinesterasica nel ganglio ciliare di cane

The authors have studied the histochemical localization of acetylcholinesterase activity in the dog's ciliary ganglion by using the modified Koelle and Friedenwald technique. For better localisation of the structures stained by the histochemical method, some sections were in addition submitted to the Feulgen reaction, stained with Neutral red or impregnated according to the method of Bielschowsky. The results showed that if the pH of the incubating medium was 6,4, the pre- and postganglionic nerve branches as well as the nerve cell bodies react with a mean intensity only; the pericellular capsules, the sheathes formed by the satellite cells round the short processes and round the proximal part of the axone as well as the neurilemma of the nerve fibers between the ganglionic cells react as if they were the site of a very high enzymatic activity. At pH 5,2 of the incubating medium the authors observed the copper sulphide precipitate due to enzymatic activity to form a pattern resembling fibrillar woofs, sheathes, cords and rings of various dimensions; it now seems to be situated close to the ganglionic cell bodies and to the arches of its fenestrations and the precipitations appear to be in intimate contact with the deep side of the satellite cell sheath. The authors believe that the different results obtained in the first case may be explained by admitting that at pH 6,4 a massive staining of the satellite cells' capsules and sheathes may occur as the result of diffusion of the enzyme or of intermediate products from the preganglionic nerve fibers, which are the site of high acetylcholinesterase activity.