Effect of axotomy on cholinergic enzyme activities in the spinal cord and sciatic nerve of the dog

Choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activity measured in the ventral and dorsal part of the dog spinal cord (L6-S2) and in the stumps of the sciatic nerve 5, 10, 15 and 21 days after its transection were compared with the corresponding activities in the intact contralateral nerve and in sham-operated animals. AChE was also examined histochemically. Changes in the enzyme activities in the central nerve stump were correlated with activity changes in the spinal cord. In the central nerve stump, a marked (25%) increase in AChE activity was found on the fifth day after transection, but by the 21st day it fell below control value levels; up to the 15th day it showed good correlation with AChE activity in the ventral spinal cord. Histochemically, pronounced reduction of enzymatic activity was found in the ipsilateral part of the spinal cord. On the 15th day, ChAT activity in the ventral spinal cord was also significantly decreased and the accumulation of the enzyme in the central nerve stump was negligible. On the contrary, at the last 21-day interval examined, a significant increase in ChAT activity and a nonsignificant increase in AChE activity was found in the spinal cord, but their activities in the central nerve stump were decreased. In the degenerated peripheral nerve stump ChAT activity dropped by an average of 99% and AChE activity by 48% during the first 15 days after transection but, on the 21st day, AChE activity was 22% higher than at the preceding interval.     

Acetylcholinesterase activity in brain slices of rats subjected to cooling

The acetylcholinesterase (AChE) activity is studied in rat slices of the cerebral cortex, corpus striatum, hypothalamus and medulla oblongata of rats during hypothermia (20 degrees C) and also 1 and 7 days after the posthypothermal period. Cooling of animals down to 20 degrees C is accompanied by an increase in the AChE activity in the brain both under incubation temperature of 20 degrees and 37 degrees C. Under prolonged hypothermia the AChE activity in the investigated brain regions, except for corpus striatum, returns to the control level. By the 7th day of posthypothermal period the AChE activity in corpus striatum, hypothalamus and medulla oblongata does not restore completely. The most substantial changes in the AChE activity both under hypothermia and posthypothermal period occur in corpus striatum, which obviously reflects its complicated functional role.     

Na, K-ATP-ase and acetylcholinesterase activity of the membrane structures of the rat brain and spinal cord during the seizure process]

The activity of ATP-ase and acetylcholinesterase (AChE) in crude mitochondrial fraction (CMF) and microsomal fraction of rat brain cortex and the spinal cord was studied in clonic seizures evoked by electroshock and 5 min after them. Inhibition of the Na, K-ATP-ase activity of the CMF of the brain at the clonic phase of convulsions and an increase in the activity of this enzyme in all the fractions of the tissues under study at the postconvulsive period were revealed. The activity of Ca-ATP-ase in the CMF of the brain increased during the convulsions and decreased at the postconfulsive period. The activity of Mg-ATP-ase remained unchanged. The AChE activity, as a rule increased during the convulsions, and grew even more during the postconvulsive period; the spinal cord tissue displayed a reduction of the activation effect. A possibility of structural reconstructions in the excitable neuron membranes during the convulsive activity is discussed.     

Higher environmental temperature-induced change in synaptosomal acetylcholinesterase activity of brain regions

Expcsure of adult male albino rats to higher environmental temperature (HET) at 35° for 2–12 hr or at 45° for 1–2 hr increases hypothalamic synaptosomal acetylcholinesterase (AChE) activity. Synaptosomal AChE activity in cerebral cortex of rats exposed to 35° for 12 hr and in cerebral cortex and pons-medulla of rats exposed to 45° for 1–2 hr are also activated. AChE activity of synaptosomes prepared from normal rat brain regions incubated in-vitro at 39° or 41° for 0.5 hr increases significantly in cerebral cortex and hypothalamus. The activation of AChE in ponsmedulla is also observed when this brain region is incubated at 41° for 0.5 hr. Increase of (a) the duration of incubation at 41° and (b) the incubation temperature to 43° under in-vitro condition decreases the synaptosomal AChE activity. Lioneweaver-Burk plots indicate that (a) in-vivo and invitro HET-induced increases of brain regional synaptosomal AChE activity are coupled with an increase ofV _max without any change inK _m (b) very high temperature (43° under in-vitro condition) causes a decrease inV _max with an increase inK _m of AChE activity irrespective of brain regions. Arrhenius plots show that there is a decrease in transition temperature in hypothalamus of rats exposed to either 35° or 45°; whereas such a decrease in transition temperature of the pons-medulla and cerebral cortex regions are observed only after exposure to 45°. These results suggests that heat exposure increases the lipid fluidity of synaptosomal membrane depending on the brain region which may expose the catalytic site of the enzyme (AChE) and hence activate the synaptosomal membrane bound AChE activity in brain regions. Further the in-vitro higher temperature (43°C)-induced inhibition of synaptosomal AChE activity irrespective of brain regions may be the cause iof partial proteolysis/disaggregation of AChE oligomers and/or solubilization of this membrane-bound enzyme.To whom to address reprint requests:     

Cerebrospinal fluid gradients of acetylcholinesterase and butyrylcholinesterase activity in healthy aging

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities were measured in 13 sequential 2 ml aliquots of cerebrospinal fluid (CSF) obtained by lumbar puncture from 7 young and 7 elderly healthy normal subjects. The slopes of the rostrocaudal gradients of AChE and BChE were calculated and compared to those of total protein concentration and the major dopaminergic metabolite homovanillic acid (HVA), for which a pronounced rostrocaudal gradient (with highest concentrations of HVA in more rostral CSF) is consistent with HVA originating primarily from the brain. AChE activity was higher in more caudal fractions of young, but not elderly subjects and there was a significant difference between the mean AChE gradient slopes in the young and old groups. These results suggest that the spinal cord makes an important contribution to AChE activity in lumbar CSF. Furthermore, the absence of a negative AChE gradient in elderly subjects may be the result of a greater rate of entry of cerebral AChE into CSF, possibly as a consequence of an increased ventricular surface area and shorter diffusion distances in atrophic elderly brains. In contrast to AChE, BChE activity and total protein concentrations were higher in more caudal CSF fractions of not only young but also old subjects. In addition, there was a significant correlation between the gradient slopes of BChE activity and total protein concentrations, suggesting that the majority of BChE activity in lumbar CSF derives from the same source as the majority of total protein, namely plasma. The diffuse (i.e. brain and spinal cord) origin of AChE in lumbar CSF would explain the relatively modest changes in lumbar CSF AChE activity in diseases involving certain central cholinergic systems, most notably Alzheimer's disease.     

Glutamic Acid Decarboxylase in Sea Lamprey (Petromyzon marinus): Characterization, Localization, and Developmental Changes

Abstract: We have carried out assays for glutamic acid decarboxylase (GAD) in homogenates of brain and spinal cord from larval and adult sea lamprey ( Petromyzon marinus ). The enzyme had similar characteristics in both stages. Optimal pH was 6.8; optimal temperature was 27–30° C; K _m at 27°C was 5 mM. GAD activity was distributed uniformly along the length of the spinal cord. Specific activities for the larval cord and brain were 26 and 63 nm CO₂/mg protein/h. respectively. The specific activities for the adult cord and brain were 29 and 236 nm CO₂/mg protein/h, respectively. Thus, the activity of cord homogenates did not change significantly between larval and adult stages, but that of the brain increased about fourfold.     

Promoting glutathione synthesis after spinal cord trauma decreases secondary damage and promotes retention of function.

The study aimed to 1) quantify oxidative stress in spinal cord after crush injury at T6, 2) determine whether the administration of the procysteine compound L-2-oxothiazolidine-4-carboxylate (OTC) would up-regulate glutathione (GSH) synthesis and decrease oxidative stress, and 3) determine whether decreased oxidative stress results in better tissue and function retention. We demonstrate that spinal cord compression (5 s with a 50 g aneurysm clip) at T6 in rats results in oxidative stress that is extensive (significant increases in oxidative stress seen at C3 and L4) and rapid in onset. Indices of oxidative stress used were GSH content, protein carbonyl content, and inactivation of glutathione reductase. Administration of OTC resulted in a marked decrease in oxidative stress associated with a sparing of white matter at T6 (16+/-1.9% retained in OTC-treated animals vs. less than 1% in saline-treated). Behavioral indices in control, saline-treated, and OTC-treated animals after 6 wk were respectively: angle board scores (59 degrees, 32 degrees, and 42 degrees ), modified Tarlov score (7, 2.4, and 4.1), and Basso-Beattie-Bresnahan score (21, 5.3, and 12.9). We conclude that administration of OTC after spinal cord trauma greatly decreases oxidative stress and allows tissue preservation, thereby enabling otherwise paraplegic animals to locomote.     

Effects of ambient temperature on brain acetylcholinesterase activity and protein content in three Egyptian vertebrates

Exposure to cold caused an increase in AChE activity of the different brain regions of both Arvicanthis and Columbia and a decrease in the enzyme activity of Scincus midbrain. Heat exposure provoked variable changes in AChE activity of the various brain regions of the three experimental species. The changes in AChE activities may be one of the mechanisms by which birds and mammals tend to acclimatize themselves to various forms of stress. In reptiles, AChE activity varied with changes in ambient temperature and this is probably due to the adaptive significance of thermally directed changes in enzyme substrate affinity.     

神经生长因子对兔坐骨神经损伤后脊髓前角运动神经元乙酰胆碱酯酶的影响

钳夹损伤兔右坐骨神经,于损伤处注射蛇毒ＮＧＦ４００Ｂｕ／ｋｇ／日,损伤术后１,３,７天和２,３,４,６,８周动态观察脊髓腰段伤侧第Ⅸ板层外侧群的大型运动神经元的ＡＣｈＥ活性改变。结果表明术后１,３天实验组（指损伤给药组）和对照组（指损伤对照组）ＡＣｈＥ活性均下降（Ｐ＞００５）;术后１,２,３周对照组ＡＣｈＥ活性明显下降,而实验组ＡＣｈＥ活性逐渐趋于恢复（Ｐ＜００１）;术后６周实验组ＡＣｈＥ活性恢复至正常水平（Ｐ＜００１）。本研究显示蛇毒ＮＧＦ对坐骨神经损伤后脊髓前角运动神经元ＡＣｈＥ活性恢复有促进作用,从而对运动神经元可起一定的保护作用和促进恢复的作用     

Effects of dorsal root section and occlusion of dorsal spinal artery on the neurotransmitter candidates in rat spinal cord

In order to obtain further evidence of putative neurotransmitters in primary sensory neurons and interneurons in the dorsal spinal cord, we have studied the effects of unilateral section of dorsal roots and unilateral occlusion of the dorsal spinal artery on cholinergic enzyme activity and on selected amino acid levels in the spinal cord. One week after sectioning dorsal roots from caudal cervical (C₇) to cranial thoracic (T₂) levels, the specific activity of choline acetyltransferase (ChAT) was significantly decreased and acetylcholinesterase (AChE) showed a tendency to decrease in the dorsal quadrant on the operated side of the spinal cord. Dorsal root sectioning had little effect on the levels of free glutamic acid or other amino acids in the dorsal spinal cord. These results suggest that primary sensory neurons may include some cholinergic axons, and that levels of putative amino acid transmitters are not regulated by materials supplied by axonal transport from the dorsal root ganglia. By contrast, one week following unilateral occlusion of the dorsal spinal artery, the activities of ChAT and AChE were unchanged in the operated quadrant of the spinal cord, while decreases of Asp, Glu, and GABA, and an increase in Tau were detected. These findings are consistent with the proposals that such amino acids, but not ACh, may function as neurotransmitter candidates in interneurons of the dorsal spinal cord.Abbreviation used ACh acetylcholine - AChE acetylcholinesterase - Asp aspartic acid - ChAT choline acetyltransferase - GABA -aminobutyric acid - Glu glutamic acid - Gly glycine - SP substance P - Tau taurine     

Lipid content in brain and spinal cord during experimental allergic encephalomyelitis in rats

Abstract— The content of cerebrosides, sulphatides, gangliosides, cholesterol and phospholipids was evaluated in the brain and spinal cord of rats during the acute and recovery stages of experimental allergic encephalomyelitis (EAE). During the acute stage there was a significant decrease of sulphatides and gangliosides in spinal cord; in brain, only sulphatides were diminished. In the recovery stage, cerebrosides and gangliosides were decreased in the brain, whereas the lipid content of the spinal cord was similar to that in control animals. Cholesterol esters were detected in the brain and spinal cord during both periods. The results show that the changes are not the same for brain and spinal cord during the acute and recovery stages and that glycosphingolipids from either white or grey matter seem to be preferentially altered.     

MYELIN PROTEINS FROM DIFFERENT REGIONS OF THE CENTRAL NERVOUS SYSTEM

—The protein composition of myelin prepared from specific anatomical regions of the bovine brain and spinal cord was studied by a modification of the method of Gonzalez -Sastre (1970). Spinal cord myelin contained lesser amounts of chloroform-methanol soluble protein and proteolipid protein and had a lower activity of the enzyme 2′,3′-cyclic nucleotide 3′-phosphohydrolase than did myelin from subcortical white matter. There was no difference, however, in the protein composition of myelin from the various levels of the spinal cord. The amino acid composition of both proteolipid and basic protein showed no significant regional differences. Myelin preparations from both brain and spinal cord contained DM-20 protein.     

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。     

Isolation and characterization of axolemma-enriched fractions from discrete areas of bovine CNS

Myelinated axons were isolated by flotation from bovine pons, middle cerebellar peduncle, cervical spinal cord and three regions of the subcortical white matter. The myelinated axons were osmotically and mechanically shocked, followed by fractionation on a linear 15% sucrose to 45% sucrose density gradient. Axolemma-enriched fractions (AEF) found in the 28% to 32% sucrose region of the gradient from brainstem and cord white matter had high acetylcholinesterase (AChE) while little or nil AChE activity was found in corresponding AEF derived from the subcortical white matter. Morphologically, the subcortical white matter from all regions contained a heterogeneous population of well-myelinated to thinly myelinated axons, while brainstem and cord regions contained a more homogeneous population of well-myelinated axons. Histochemical analysis of AChE localized this enzyme to axonal elements. The AEF derived from any white matter source had similar polypeptide compositions. AEF derived from subcortical white matter contained two-fold more myelin basic protein and a three-fold greater content of 2 3 cyclic nucleotide 3 phosphodiesterase (CNP) compared with AEF derived from well myelinated white matter. We conclude that the purity of the AEF is related to the degree of myelination of the white matter from which the AEF is derived. Homogeneously well myelinated white matter (pons, cerebellar peduncle, cervical spinal cord) yields the highest purity AEF, as judged by the low CNP and myelin basic protein content and highest enrichment in AChE specific activity.     

Activity of Na, K-ATPase and the enzymes of intermediate metabolism in the brains of rats exposed to electroshock]

Activity of Na, K -ATPase, acetylcholinesterase (AChE) and glutamic acid decarboxylase (GAD) in the fractions of the rat brain and spinal cord tissue were studied in rats during a single electroshock (ES) and 5 and 30 minutes after it. GAD activity of the synaptosome fraction was shown to decrease insignificantly, but activity of AChE, Na, K -ATPase and possibly of proteolytic enzymes increased 5 minutes after electroshock and became normal in 30 minutes. It is supposed that the revealed inhibition of Na, K -ATPase activity in the "synaptosomes" of the rat brain cortex could be of pathogenetic significance in the origination of the convulsive process.     

An attempt to assess functionally minimal acetylcholinesterase activity necessary for survival of rats intoxicated with nerve agents

Acetylcholinesterase (AChE, EC 3.1.1.7) is an important enzyme for cholinergic nerve transmission. The action of toxic organophosphates such as nerve agents is based on AChE inhibition. The death following acute nerve agent poisoning is due to central or peripheral respiratory/cardiac failure. Therefore, the changes in AChE activity following nerve agents acting predominantly on the central (sarin, soman) or peripheral (VX) level were studied. It is known that AChE activity in different structures exists in relative excess. Female Wistar rats intoxicated with sarin, soman, and VX in different doses (0.5-2.0xLD(50)) were divided into groups of survived and died animals. AChE activities in diaphragm, brain parts (pontomedullar area, frontal cortex, basal ganglia, in some cases other parts of the brain) were determined and the rest of activity (in %) was correlated with survival/death of animals. More precise elucidation of action of nerve agents and the assessment of minimal AChE activity in different organs compatible with the survival of organism poisoned with nerve agents were the aims of this study.     

THE EFFECTS OF PENTOBARBITONE-SODIUM ANAESTHESIA, SHOCK AVOIDANCE CONDITIONING AND ELECTRICAL SHOCK ON ACETYLCHOLINESTERASE ACTIVITY AND PROTEIN CONTENT IN REGIONS OF THE RAT BRAIN

Abstract— Pentobarbitone sodium anaesthesia was found to produce an increase in protein content in some regions of the rat brain, i.e. posterior cortex, caudate nucleus, and a decrease in protein content in the ventral cortex.
Acetylcholinesterase expressed in terms of wet weight was found to increase in the cerebellum, medulla, and to decrease in the medial cortex, hippocampus, thalamus and caudate nucleus. The changes in activity were not explicable in terms of a direct effect of the anaesthetic on the enzyme. A decrease in protein content of rat brain was observed in the frontal cortex, ventral cortex, hippocampus and caudate nucleus after electrical shocks. Following shock avoidance conditioning procedure (shuttle-box), decreases in protein content were observed in the medial cortex, posterior cortex, cerebellum and ventral cortex; in the thalamus an increase in protein content was observed.
Changes in AChE activity were observed following footshock in the frontal cortex and medulla where there was an increase in activity and in the caudate nucleus, hypothalamus, thalamus, and olfactory tubercle where there was a decrease in activity.
Following shock avoidance conditioning the activity of the AChE increased in posterior cortex, hippocampus, thalamus and hypothalamus and the activity of the enzyme decreased in the ventral cortex.     

Proteolytic activity in brains of rabbits treated with aluminum

Aluminum injection in rabbits leads to neurofibrillary changes which are at light microscopic level similar to those found in Alzheimer's disease. We used this animal model to see whether changes in proteolytic activity occur that may affect protein degradation in the altered neurofibrillary structure. Rabbits were injected via the cisterna magna with aluminum chloride, and after ten days tissue was excised from the spinal cord, hippocampus, occipital lobe, and cerebellum. Sections from the hippocampus and spinal cord were examined for neurofibrillary changes; enzyme activity was measured in all four areas. The enzymes studied were cathepsins A, B, and D, and the angiotensin-converting enzyme. No significant differences could be established in enzymatic activity in aluminum-injected animals compared to controls. However, a significant decrease in Triton-soluble proteins was observed in the treated animals, which correlated with changes in neurofibrillary structure. This decrease was most noticeable in the spinal cord (from 16.6 to 12.5 mg/g).     

Two-Site Immunoassay for Acetylcholinesterase in Brain, Nerve, and Muscle

Two-site methods were developed for immunoassay of acetylcholinesterase (AChE; EC 3.1.1.7) in crude extracts of rat and human tissues. A radiometric assay for human AChE utilized a specific monoclonal AChE antibody adsorbed to polystyrene microtiter wells at alkaline pH. AChE bound strongly to this antibody after 24 h at 4 degrees C. Bound enzyme was detected with an 125I-labeled antibody against a different AChE epitope. The assay signal was quasi-linearly related to AChE concentration in purified and crude samples, with a detection threshold near 100 pg. Tetrameric and dimeric AChE behaved equivalently in the assay. Two-site methods with a different pair of species-selective antibodies worked equally well for immunoassay of rat AChE. Assays of the rat enzyme showed that immunoreactivity was lost as rapidly as enzyme activity during heating to 54 degrees C. On the other hand, immunoreactivity was preserved despite loss of enzyme activity after exposure to anticholinesterases or trypsin. A biotinylated second antibody detected by alkaline-phosphatase-conjugated avidin was used to develop an AChE enzyme-linked immunosorbent assay (ELISA) with a sensitivity similar to that of the radiometric assay. Either the ELISA or the radiometric immunoassay may be useful whenever proteolysis or other mechanisms are suspected of dissociating enzyme activity and immunoreactivity. In denervated muscle and ligated peripheral nerve, application of the two-site method showed closely parallel variations in immunoreactivity and enzyme activity.