Poster Sessions AP05: Neuronal and Glial Cell Biology

Acetylcholinesterase (AChE) is classically known for terminating cholinergic transmission, however, increasing evidence shows that the biological function of AChE is not limited to this role. Interestingly, the AChE gene is either amplified, mutated and/or aberrantly expressed by cells in a variety of human tumors despite the fact that AChE is not present in their normal counterparts. Studies from our laboratory and others have shown that AChE is transiently expressed during normal neural development when cells are invariably engaged in intense growth and movement. Together, these observations support the idea that AChE may play a role in tumorigenesis, however, this hypothesis has not been tested before. In support of this idea, we have found that neuroblastoma cells genetically engineered to over-express AChE developed tumors in vivo at a notably greater rate compared with transfection controls. Tumor cells were implanted into the brains of irradiated Fischer rats and following a 10-day survival period, macroscopic examination revealed that the tumor mass generated from the AChE over-expressing cells was six-fold greater than transfection controls. Moreover, histochemical and Western blot analyses of two human glioma cell lines revealed that the different levels of AChE expression in these cells directly correlated with their proliferation rates. These studies showed that the high AChE-expressing cells divided at a 50% greater rate than the low AChE-expressing cells. Interestingly, the high AChE-expressing cells also exhibited increased MAPK phosphorylation, a key step in the regulation of cell growth. These findings together with the consistent observation that AChE is misexpressed in human tumors, and the fact that AChE levels are up-regulated during normal development, support the idea that AChE plays a role in tumor cell growth.     

Poster Sessions AP05: Neuronal and Glial Cell Biology. Novel roles for acetylcholinesterase in brain tumors

Acetylcholinesterase (AChE) is classically known for terminating cholinergic transmission, however, increasing evidence shows that the biological function of AChE is not limited to this role. Interestingly, the AChE gene is either amplified, mutated and/or aberrantly expressed by cells in a variety of human tumors despite the fact that AChE is not present in their normal counterparts. Studies from our laboratory and others have shown that AChE is transiently expressed during normal neural development when cells are invariably engaged in intense growth and movement. Together, these observations support the idea that AChE may play a role in tumorigenesis, however, this hypothesis has not been tested before. In support of this idea, we have found that neuroblastoma cells genetically engineered to over‐express AChE developed tumors in vivo at a notably greater rate compared with transfection controls. Tumor cells were implanted into the brains of irradiated Fischer rats and following a 10‐day survival period, macroscopic examination revealed that the tumor mass generated from the AChE over‐expressing cells was six‐fold greater than transfection controls. Moreover, histochemical and Western blot analyses of two human glioma cell lines revealed that the different levels of AChE expression in these cells directly correlated with their proliferation rates. These studies showed that the high AChE‐expressing cells divided at a 50% greater rate than the low AChE‐expressing cells. Interestingly, the high AChE‐expressing cells also exhibited increased MAPK phosphorylation, a key step in the regulation of cell growth. These findings together with the consistent observation that AChE is misexpressed in human tumors, and the fact that AChE levels are up‐regulated during normal development, support the idea that AChE plays a role in tumor cell growth.     

Asymmetric distribution of acetylcholinesterase in gravistimulated maize seedlings

Acetylcholinesterase (AChE) activity has previously been studied by this laboratory and shown to occur at the interface between the stele and cortex of the mesocotyl of maize (Zea mays L.) seedlings. In this work we studied the distribution of AChE activity in 5-d-old maize seedlings following a gravity stimulus. After the stimulus, we found an asymmetric distribution of the enzyme in the coleoptile, the coleoptile node, and the mesocotyl of the stimulated seedlings using both histochemical and colorimetric methods for measuring the hydrolysis of acetylthiocholine. The hydrolytic capability of the esterase was greater on the lower side of the horizontally placed seedlings. Using the histochemical method, we localized the hydrolytic capability in the cortical cells around the vascular stele of the tissues. The hydrolytic activity was inhibited 80 to 90% by neostigmine, an inhibitor of AChE. When neostigmine was applied to the corn kernel, the gravity response of the seedling was inhibited and no enzyme-positive spots appeared in the gravity-stimulated seedlings. We believe these results indicate a role for AChE in the gravity response of maize seedlings.     

A comparison of the localization of acetylcholinesterase in the rat brain as demonstrated by enzyme histochemistry and immunohistochemistry

The localization of acetylcholinesterase (AChE) as revealed either by enzyme-histochemical or by immunohistochemical methods was compared in distinct regions of the rat brain. In general, the localization of AChE observed was nearly the same, whether revealed by histochemical demonstration of its catalytic activity or by immunohistochemical detection of the enzyme molecule itself, in all regions investigated. Penetration problems of the antibodies, however, arose on strong myelin sheaths of the facial nerve, for instance, where no immunohistochemical staining was found though there was a relatively strong histochemical reaction. These problems could be partly solved by increasing the normal concentration of Triton X-100 added to the immunohistochemical solutions (0.1%) to 2.5%. Furthermore, it seems that sites containing low amounts of AChE could be better detected by the enzyme-histochemical method, whereas the depiction of structures (particularly of nerve fibres) was somewhat sharper with the immunohistochemical method.     

Developmental changes and acetylcholinesterase activity in the metamorphosing brain of Tenebrio molitor: Correlation to ecdysteroid titers

The brain of Tenebrio molitor exhibited marked fluctuations in acetylcholinesterase (AChE) activity throughout metamorphosis. This was true AChE activity, since it was inhibited by high substrate concentrations and by 10 μM of the specific AChE inhibitor BW284C51 [(1,5-bis'4-allyldimethylammoniumphenyl)-pentan-3-one dibromide] but not by iso-OMPA (tetraisopropylpyrophosphoramide), a cholinesterase (but not AChE) inhibitor. The histochemical AChE activity was localized in the neuropile and the nuclear envelope of neurons and glial cells. The enzyme extracted from brains with 1% Triton X-100 and 1 M NaCl sedimented as a single peak in a sucrose density gradient, with a sedimentation coefficient of 5.4S. This single AChE sedimentation peak was mainly due to an amphiphilic dimeric form. AChE activity per brain increased in newly ecdysed pupa. AChE activity per milligram of protein exhibited a peak in the mid-pupa which could be correlated to the increase in ecdysteroid titers. © 1994 Wiley-Liss, Inc.     

Acetylcholinesterase activity in visual cortex structures during early visual deprivation

By means of quantitative histochemical methods it has been shown that an early photic deprivation (animals kept in a dark chamber for two months after their birth) leads to a decrease in the activity level of acetylcholinesterase (AChE) in the visual area of the cerebral cortex. With the recovery of the visual function (animals kept in normal photic conditions for two weeks) the AChE activity becomes markedly normalized. The obtained data allow to suggest that the decrease in AChE activity due to deprivation is functionally determined.     

A comparison of the localization of acetylcholinesterase in the rat brain as demonstrated by enzyme histochemistry and immunohistochemistry

Summary The localization of acetylcholinesterase (AChE) as revealed either by enzyme-histochemical or by immunohistochemical methods was compared in distinct regions of the rat brain. In general, the localization of AChE observed was nearly the same, whether revealed by histochemical demonstration of its catalytic activity or by immunohistochemical detection of the enzyme molecule itsclf, in all regions investigated. Penetration problems of the antibodies, however, arose on strong myelin sheaths of the facial nerve, for instance, where no immunohistochemical staining was found though there was a relatively strong histochemical reaction. These problems could be partly solved by increasing the normal concentration of Triton X-100 added to the immunohistochemical solutions (0.1%) to 2.5%. Furthermore, it seems that sites containing low amounts of AChE could be better detected by the enzymehistochemical method, whereas the depiction of structures (particularly of nerve fibres) was somewhat sharper with the immunohistochemical method.Dedicated to Professor Dr.T.H. Schiebler on the occasion of his 65th birthday     

Comparison of changes in AChE activity in the brain of the laboratory rat after soman and tabun intoxication

The aim of this study was to compare changes in activity of acetylcholinesterase (AChE) in the brain and motor endplates of rat after administration of soman and tabun. We took brain and diaphragm from laboratory rats administered a median lethal dose (LD(50)) of soman or tabun. Enzyme activity of AChE was studied in selected structures of brain and in motor endplates in the diaphragm. Histochemical detection of AChE by Karnovski and Roots with simultaneous histochemical detection of alkaline phosphatase in case of brain sections was used. The highest activity of AChE in the control group was found in the striatum, amygdaloid nuclei, substantia nigra, superior colliculi, and motor nuclei of cranial nerves V, X a XII. LD(50) of both nerve agents dramatically decreased the activity of AChE in the structures studied--both brain and diaphragm. After intoxication by either agent, activity in above mentioned nuclei was characterized as low or focally moderate. Very low activity was seen in some structures (CA3 field of hippocampus, some nuclei of the tegmentum and cerebellar cortex). We found minimal differences in the histochemical picture of soman or tabun intoxication, apart from the striatum and the superior colliculi which showed stronger inhibition by tabun.     

TRANSPORT OF CHOLINE ACETYLTRANSFERASE AND ACETYLCHOLINESTERASE IN THE RAT SCIATIC NERVE: A BIOCHEMICAL AND ELECTRON HISTOCHEMICAL STUDY

The transport of acetylcholinesterase (AChE) and choline acetyltransferase (ChAc) were investigated by biochemical and histochemical methods. After ligature of one of the sciatic nerves of the rat for varying times—4, 14, 20 and 44 h—the normal levels and the accumulation of AChE and ChAc activities were investigated. It can be inferred from the results that there is a rapid accumulation of AChE activity just proximal to the ligature, while the increase in ChAc activity is less pronounced. Distal to the ligature the level of AChE is above the control value whereas, in contrast to this, the ChAc activity is significantly decreased. Histochemical demonstration of the two enzymes indicates that they are present in the cholinergic axons. The reaction end-product produced by AChE occurs within vesicles and neurotubules, while the endproduct due to ChAc appears to be free in the axoplasm, bound to neurofilaments and on the outer surface of vesicles and tubules.     

A histochemical study of the distribution of choline acetyltransferase and acetylcholinesterase activity in sensory ganglia and nerve roots of the bullfrog

Synopsis Histochemical techniques were employed for the localization of choline acetyltransferase (ChAc; EC 2.3.1.6.), acetylcholinesterase (AChE; EC 3.1.1.7) and cholinesterase (ChE; EC 3.1.1.8) activities in dorsal and ventral roots and dorsal root ganglia of the bullfrog. AChE activity was present in most of the neuronal elements of dorsal root ganglia, in some nerve fibres in the dorsal roots, and in all nerve fibres in ventral roots. ChE activity in dorsal root ganglia and in the dorsal roots was confined to non-neuronal elements. No ChE activity was demonstrable in the ventral roots. ChAc activity was localized in many neurons of the dorsal root ganglia and in some nerve fibres of the dorsal roots; however, none of the ventral root fibres were visibly reactive. Some supportive cells of the dorsal roots and ganglia contained small amounts of ChAc activity. Except for the ventral roots, the histochemical distribution of AChE and ChAc activity was similar. The results of solubility studies indicated that under the histochemical conditions, approximately 50% of the ChAc remained bound to the dorsal roots and ganglia, whereas more than 90% of the ChAc in the ventral roots was soluble. This would account for the lack of reactivity in ventral root fibres. Differences in ChAc solubility are discussed in relation to the interpretation of histochemical data and in relation to the concept of multiple forms of ChAc. The results of this study indicate that at least one-third of the neurons of the dorsal root ganglia contain significant levels of the enzymes involved in both the synthesis and hydrolysis of acetylcholine.     

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.     

Activity of Acetylcholinesterase in Olfactory Bulb of the Pike <Emphasis Type="Italic">Esox lucius</Emphasis> and Its Role in Development of Long-Term Posttetanic Potentiation

A complex electrophysiological, biochemical, and histochemical study is carried out for determination of activity and distribution of acetylcholinesterase (AChE) in olfactory bulb (OB) of the pike during long-term posttetanic potentiation (PTP). Between the 30th and 60th min after tetanus, a stable increase of enzymatic activity in parallel with a rise of potentiation is observed. Sixty min after tetanus, at the point of maximal development of long-term PTP (the potentiation value is 170%), the specific activity of AChE rises by 89%. This increase was found to be due to synthesis of the enzyme de novo, with involvement of the majority of mitral cells and a significant part of granular cells.     

Histopathology, histochemistry and acetylcholinesterase activity after repetitive administration of fluostigmine to albino rat

Histopathological, histochemical and biochemical investigations were performed on the brain, sciatic nerve, skeletal muscle, heart, liver and kidney of rats which were given 5% of LD50 dose of DFP for 10 days. A decrease in AChE activity, degeneration of neurons and necrotic changes in the nuclei of hypothalamus, degeneration of myelin sheaths in sciatic nerve, a decrease in succinic dehydrogenase activity in the myocardium, and a minimal decrease of acid phosphatase activity (AcPh) in the liver were found. The biochemical determination of AChE level indicated about 30% AChE activity in erythrocytes and tibialis muscle, and 40% in the brain 1 hr after the last dose of the inhibitor and 80% and 50% respectively on the 7th day after poisoning in relation to normal values.     

Histo- and immunocytochemical characterization of the neurons of the mucosal plexus in the rat colon.

The mucosal plexus of the rat colon descendens is constituted of a network of nerves that, in contrast to most other segments of the digestive tract, contains also ganglia. The ganglia, consisting of neurons and glial cells, are located in the basal part of the lamina propria at distances between 100 and 1,200 microns. They are not vascularized. The neurons in these ganglia were characterized by means of: (1) the histochemical demonstration of acetylcholinesterase (AChE) activity, (2) the immunocytochemical identification of neurofilament proteins (NFP; 200 kD) and (3) their ultrastructure. The glial cells, which were AChE negative, could be distinguished from the neurons by differences in size and chromatin pattern. All neurons of the mucosal plexus reveal AChE activity in the perikaryon, but only parts of the axons are AChE positive. NFP-like immunoreactivity was detected in the perikarya but only in a minor part of the axons. These findings confirm previous light-microscopical observations and add new evidence for the existence of neurons (ganglia) in the mucosal plexus of the rat colon.     

A comparison of tabun-inhibited rat brain acetylcholinesterase reactivation by three oximes (HI-6, obidoxime, and K048) in vivo detected by biochemical and histochemical techniques

Tabun belongs to the most toxic nerve agents. Its mechanism of action is based on acetylcholinesterase (AChE) inhibition at the peripheral and central nervous systems. Therapeutic countermeasures comprise administration of atropine with cholinesterase reactivators able to reactivate the inhibited enzyme. Reactivation of AChE is determined mostly biochemically without specification of different brain structures. Histochemical determination allows a fine search for different structures but is performed mostly without quantitative evaluation. In rats intoxicated with tabun and treated with a combination of atropine and HI-6, obidoxime, or new oxime K048, AChE activities in different brain structures were determined using biochemical and quantitative histochemical methods. Inhibition of AChE following untreated tabun intoxication was different in the various brain structures, having the highest degree in the frontal cortex and reticular formation and lowest in the basal ganglia and substantia nigra. Treatment resulted in an increase of AChE activity detected by both methods. The highest increase was observed in the frontal cortex. This reactivation was increased in the order HI-6 < K048 < obidoxime; however, this order was not uniform for all brain parts studied. A correlation between AChE activity detected by histochemical and biochemical methods was demonstrated. The results suggest that for the mechanism of action of the nerve agent tabun, reactivation in various parts of the brain is not of the same physiological importance. AChE activity in the pontomedullar area and frontal cortex seems to be the most important for the therapeutic effect of the reactivators. HI-6 was not a good reactivator for the treatment of tabun intoxication.     

A comparison of tabun-inhibited rat brain acetylcholinesterase reactivation by three oximes (HI-6, obidoxime,and K048) in vivo detected by biochemical and histochemical techniques

Tabun belongs to the most toxic nerve agents. Its mechanism of action is based on acetylcholinesterase (AChE) inhibition at the peripheral and central nervous systems. Therapeutic countermeasures comprise administration of atropine with cholinesterase reactivators able to reactivate the inhibited enzyme. Reactivation of AChE is determined mostly biochemically without specification of different brain structures. Histochemical determination allows a fine search for different structures but is performed mostly without quantitative evaluation. In rats intoxicated with tabun and treated with a combination of atropine and HI-6, obidoxime, or new oxime K048, AChE activities in different brain structures were determined using biochemical and quantitative histochemical methods. Inhibition of AChE following untreated tabun intoxication was different in the various brain structures, having the highest degree in the frontal cortex and reticular formation and lowest in the basal ganglia and substantia nigra. Treatment resulted in an increase of AChE activity detected by both methods. The highest increase was observed in the frontal cortex. This reactivation was increased in the order HI-6 < K048 < obidoxime; however, this order was not uniform for all brain parts studied. A correlation between AChE activity detected by histochemical and biochemical methods was demonstrated. The results suggest that for the mechanism of action of the nerve agent tabun, reactivation in various parts of the brain is not of the same physiological importance. AChE activity in the pontomedullar area and frontal cortex seems to be the most important for the therapeutic effect of the reactivators. HI-6 was not a good reactivator for the treatment of tabun intoxication.     

Histochemical localization of acetylcholinesterase in blood cells

Cholinesterase activity was detected by histochemical methods in normal human blood smears. In erythrocytes, acetylcholinesterase was found to be localized in the cortical region of the cells and, to a lesser degree, in the inner cytoplasm. In leucocytes, cholinesterase activity was found around the nuclear membrane and in the cytoplasm. The specificity of the enzymic activity was ascertained by using selective inhibitors.     

大鼠脊髓不完全性损伤后前角运动神经元的酶细胞化学改变

以改良Ａｌｅｎ氏法造成Ｗｉｓｔａｒ大鼠不完全性脊髓损伤,采用神经学功能评分法评定大鼠运动功能,应用定量酶细胞化学方法观察脊髓前角运动神经元内乙酰胆碱酯酶（ＡＣｈＥ）和酸性磷酸酶（ＡｃＰ）活性变化。结果显示：１．脊髓损伤后大鼠运动功能障碍,随后逐渐恢复。２．前角运动神经元内ＡＣｈＥ活性减弱、ＡｃＰ活性增强;随后酶活性呈逐渐恢复,四周时ＡＣｈＥ活性基本恢复正常。结果说明：大鼠脊髓不完全性损伤后运动功能变化与前角运动神经元的功能状态具有较强的相关性;前角运动神经元在不完全性脊髓损伤运动功能恢复中起重要作用。     

Dolichos biflorus agglutinin receptors in mouse muscle. II. Biochemical properties in relation to molecular forms of acetylcholinesterase

A biochemical analysis has been performed on the relationship between the receptors for Dolichos biflorus agglutinin (DBA) and collagen tailed acetylcholinesterase (16S AChE) in mouse skeletal muscle. The molecular forms of AChE were separated by differential salt extraction and by gradient centrifugation. DBA binding activity was measured using a microtiter plate binding assay and affinity chromatography. The 16S form of AChE was bound to DBA, whereas globular forms of AChE were not. However, only a small proportion of 16S AChE was capable of binding to DBA, and most of the DBA binding capacity in muscle extracts was not associated with the 16S AChE. The possible association with the neuromuscular synapse of DBA binding molecules other than 16S AChE is discussed with respect to our previous histochemical study on DBA binding sites in mouse muscle.