Vanadium improves brain acetylcholinesterase activity on early stage alloxan-diabetic rats

The present study is designed to screen the possible effects of sodium orthovanadate therapy on the kinetic parameters of brain membrane-bound and soluble acetylcholinesterase (AChE) forms in alloxan-induced diabetic rats. The diabetic rats were treated with 300 mg/kg sodium orthovanadate orally for 45 days. While diabetes significantly decreased the brain specific activity (V(max)) of AChE soluble form by 42%, it caused a fivefold increase of the K(m) of the membrane-bound form. Furthermore, the activity of brain glutathione-S-transferase (GST) was significantly decreased and this was associated with a remarkable increase in brain lipid peroxidative parameter, thiobarbituric acid reactive substances (TBARS), as compared to sham control. The alterations of both AChE forms observed in diabetic state could be attributed to hyperglycemia and lipid peroxidation that triggered brain dysfunction by disturbing the neurotransmitter acetylcholine level. Administration of sodium orthovanadate reversed the diabetic conditions by lowering the blood glucose level and normalized the blood Hb(A1C) level. It also normalized the levels of brain AChE, GST and TBARS as compared to diabetic state and control. Therefore, vanadate administration could protect against direct action of lipid peroxidation on brain AChE and in this way, it might be useful in the prevention of cholinergic neural dysfunction, which is one of the major complications in diabetes.     

Effect of acute noise stress on acetylcholinesterase activity in discrete areas of rat brain

Effect of various stressor agents on the adrenergic system in brain had been studied extensively. However, reports on the effect of stress on various parameters of central cholinergic system are scanty. And very little is known about the effect of noise stress on the cholinergic system in brain. Hence, it was decided to elucidate the effect of acute noise stress on the activity of the enzyme acetylcholinesterase in discrete areas of brain in albino rats. Male albino rats of Wistar strain were subjected to acute noise stress for 30 minutes. The noise of pure sine wave tone was produced by using a function generator and was amplified. The frequency of noise generated was 1 kHz and the intensity was set at 100 dB. The total acetylcholinesterase activity was determined in the tissues of cerebral cortex, corpus striatum, hypothalamus and hippocampus of brain in these rats. The enzyme activity was estimated by colorimetric method using acetylthiocholine iodide as the substrate. The values were compared with the enzyme activity in the control rats. The activity of the enzyme increased significantly in all the four regions of the brain in rats after exposure to noise stress for 30 minutes. The results of the study indicate that the exposure to acute noise stress could modulate the cholinergic system in these areas of brain in rat.     

Effects of prenatal alcohol on gamma-glutamyl transpeptidase in various brain regions

Prenatal exposure of rats to alcohol produces morphological, biochemical, behavioral and physiological abnormalities. The enzyme gamma-glutamyl transpeptidase activity is increased when an animal is exposed to alcohol chronically. We examined the effect of the in utero exposure to alcohol on the regional brain distribution of gamma-GTP. Pregnant rats were placed into the following treatment groups: LC (ad lib lab chow and water), PF (pair-fed), 10% EDC (ethanol derived calories), 20% EDC and 35% EDC. The LC group was fed lab chow and water ad lib, the PF, 10% EDC, 20% EDC and 35% EDC groups were fed a liquid diet containing either 0%, 2%, 4% or 6.7% v/v ethanol, respectively. All the treatment groups received the same volume of an isocaloric diet as was consumed by the 35% EDC group. At birth, litters were culled to six and cross fostered to untreated surrogate mothers. Pups were sacrificed at 30 days of age and brains dissected into 8 regions. Each brain region was homogenized and divided into soluble and membrane bound fractions by centrifugation and digestion with deoxycholic acid. gamma-GTP activity was then measured. It was determined that the in utero exposure to alcohol produces an increase in brain gamma-GTP activity. The increase in gamma-GTP activity in some areas of brain is maintained at least until the animals are 30 days old. Alcohol treatment had no effect on the activity associated with the soluble form of enzyme. However, the activity associated with the membrane bound form of the enzyme was increased in several brain regions.(ABSTRACT TRUNCATED AT 250 WORDS)     

The recovery of acetylcholinesterase activity in the superior cervical ganglion of the rat following its inhibition by diisopropylphosphorofluoridate: A biochemical and cytochemical study

Injection of diisopropylphosphorofluoridate into rats results in a marked inhibition of the acetylcholinesterase activity of the superior cervical ganglia and associated nerve trunks. We have followed the recovery of the activity of the enzyme in these two tissues. We have studied the recovery biochemically, analysing also the contribution of the various molecular forms of acetylcholinesterase, and cytochemically.Shortly after injection of the poison there is a rapid recovery of the acetylcholinesterase activity in the ganglia. This phase continues until about 16 h post-injection, and then it stops. The activity remains at this level for a further 80 h then there is another, but slower, accumulation of enzyme activity. Ganglia which had been decentralized prior to administration of diisopropylphosphorofluoridate show an identical pattern of recovery although the absolute amount of enzyme at each stage is lower than in normal tissues. In contrast, the acetylcholinesterase activity of the preganglionic nerve trunk does not begin to recover from the poison until at least 48 h after its injection. It reaches normal levels by 200h. Analysis of the multiple molecular forms of acetylcholinesterase show: a) that there are four major soluble forms and, chiefly, one membrane-bound form of the enzyme in the ganglion, b) there is one soluble and one membrane-bound form in the nerve trunk, c) that there is no preferential loss of any of the observed forms in response to decentralization of the ganglia and, d) that there is no preferential recovery of any form after diisopropylphosphorofluoridate poisoning. The cytochemistry reveals that the initial recovery phase is due to synthesis of the enzyme by the ganglion cells since under our incubation conditions these are the only structures which contain the enzyme for the first 48 h after injection. It is noteworthy that acetylcholinesterase activity could not be demonstrated in the synaptic region until at least 48 h after injection of the poison at which time it could also be detected in presynaptic axons; this indicates that the synaptic enzyme is derived from the preganglionic nerve.The results are discussed in terms of what proportion of normal ganglionic levels of acetylcholinesterase activity is derived from the presynaptic cholinergic nerves and how much from the postganglionic, but adrenergic, cells. It is suggested that appreciably less of the acetylcholinesterase is derived from the nerve trunk than is lost upon decentralization of the ganglia; it appears that the nerve might regulate the amount of enzyme that the postganglionic cells can synthesise and store.     

Large litters rearing changes brain expression of GLUT3 and acetylcholinesterase activity in adult rats

Effects of malnutrition in the brain are more pronounced during the period of growth spurt, corresponding to the suckling in rodents. Neuronal glucose transporter GLUT3 expression and acetylcholinesterase activity were studied in the brain of adult young rats (84 days old) suckled in litters formed by 6 (control group) or 12 pups (malnourished group). In the adult rats, brain weight, blood glucose levels and GLUT3 expression were decreased in malnourished group (5%, 18%, 58%, respectively, P < 0.001, Student's t test) compared to the control. Increased activity of acetylcholinesterase was found in cerebral cortex homogenates and a significant interaction (P = 0.019, ANOVA two-way, Tukey's test) was found between nutritional state and homogenate fraction. In summary, malnutrition during suckling period decreased GLUT3 expression and increased acetylcholinesterase activity in the rat brain that could contribute to possible cognitive deficits and changes of brain metabolic activity.     

Multiple forms of acetylcholinesterase in the ciliary ganglion and iris of the pigeon

Different methods of extraction and analysis have been used to investigate the cholinesterase activity of the ciliary ganglion and iris from the adult pigeon. The molecular forms of acetylcholinesterase in the extracts have also been separated either by gel electrophoresis or by sedimentation velocity in a density gradient. The activity and molecular forms in (i) a ‘soluble fraction’ (the high-speed supernatant of an isotonic sucrose homogenate) and (ii) a ‘Triton extract’ (as above, but with 0.2% Triton X-100 in the homogenate) were examined. The cholinesterase activity in whole homogenates and in the two extracts was characterized by enzyme kinetics and use of different substrates and inhibitors.In the iris, as distinct from the ganglion, the total cholinesterase activity involved a small but definite proportion attributable to a butyrylcholinesterase. In the soluble fraction from both ganglion and iris. which contained 13 and 29% of the total activity, respectively, either method of separation revealed three main molecular forms of acetylcholinesterase; the evidence suggests that these are size isomers. In addition, Triton X-100 extracts from ganglia were shown by electrophoresis to contain a fourth slow migrating component. This form contained most of the enzymic activity and appears to be a membrane-bound form of acetylcholinesterase; it was not detected in the iris.     

Fear induced neuronal alterations in a genetic model of depression: An fMRI study on awake animals

Previous human imaging studies used facial stimuli to explore the potential association between depression and fear. This study aimed at investigating brain alterations in a rodent model of depression when innate fear was induced in the form of the predator odor trimethylthiazoline (TMT). Flinders sensitive line (FSL) rats, a genetic animal model of depression, and their control counterpart Flinders resistant line (FRL), were used in this functional magnetic resonance imaging (fMRI) assessment. Compared to FRL, FSL rats exhibited greater BOLD activation in the cortical amygdala and hypoactivation in the prefrontal cortex in response to TMT, suggesting cortico-amygdalar dysfunction in the depressed strain. In addition, the hyperactivation in the insular cortex in FSL rats may be the basis for enhanced neuronal responses to fear and aversion in depression. These results are evidence for the value of translational models of depression in expanding understanding of the neural circuitries sub-serving common human co-morbidities like depression and fear.     

Effect of aflatoxicosis on acetylcholinesterase activity in the brain and adenohypophysis of the male rat

The effects of 7 or 42 48-hourly i.p. injections of 20 micrograms aflatoxin B1 (AFB) on the acetylcholinesterase activity (AChE) in 8 brain areas and the adenohypophysis of the adult male rat were studied. Aflatoxicosis increased adenohypophysial AChE in direct proportion to the duration of intoxication, which also altered the distribution of AChE in the rat brain. With acute treatment, AChE was depressed in the cerebellum and hippocampus while in the chronically dosed rats AChE was drastically elevated in the mesencephalon and amygdala. Results suggest that AFB changes the ACh turnover and hence the cholinergic transmission in the brain and adenohypophysis. This may result in behavioural deficits and/or performance decrements via a disturbance of the hypothalamo-hypophysial axis.     

Release of acetylcholinesterase from the guinea-pig cerebellum in vivo

In the cerebellum there is scant evidence for cholinergic transmission but a large amount of acetylcholinesterase. Although it exists most commonly in a membrane-bound form, the release of a soluble form of this enzyme, within the cerebrum, has indicated that it may have a novel non-cholinergic role. In order to understand why the cerebellum is rich in acetylcholinesterase, the first step has been to investigate the possibility of its release in this structure. Following unilateral application of a depolarizing concentration of potassium ions, there was a large, sustained, calcium-dependent increase in release of acetylcholinesterase, specifically in the local cerebellar cortex; a marked enhancement of acetylcholinesterase release also occurred in the contralateral cerebellum, suggesting that the phenomenon reflected polysynaptic neuronal events. Indeed, systemic administration of harmaline, which modifies activity in certain cerebellar afferent pathways, induced a significant increase in acetylcholinesterase release in the cerebellar cortex. Local administration of the cholinomimetic, carbachol, had no effect. It is concluded that acetylcholinesterase is released from cerebellar neurons in association with physiological events, yet unrelated to cholinergic transmission.     

Characterization of tissue enzyme activities in rats with dorsomedial hypothalamic lesions and their sham-operated controls

The dorsomedial hypothalamic lesions (DMNL) resulted in a depression in food intake and linear and ponderal growth. The present study was designed to examine basic enzymatic adaptations to the lesions produced in the dorsomedial hypothalamus. Liver glucose-6-PO₄ dehydrogenase, citrate cleavage enzyme and malic enzyme were depressed in DMNL rats. These observations suggest a depression in fatty acid synthesis in liver tissue; however, no change in the enzyme fatty acid synthetase was observed. Adipose tissue enzymes involved in the conversion of dietary carbohydrate to fatty acid were not significantly altered in the DMN syndrome. Subtle changes in enzyme activity may be responsible for the development of the DMN syndrome. The mechanisms of the induced changes in enzyme activity remain to be elucidated.     

Inhibition of acetylcholinesterase and butyrylcholinesterase by the organophosphorus insecticide methylparathion in the central nervous system of the golden hamster (Mesocricetus auratus).

The toxic effects of the organophosphorus pesticide methylparathion are primarily caused by the inhibition of acetylcholinesterase activity in the central nervous system, whereas the relationship between butyrylcholinesterase and poisoning symptoms is unclear. The presumed different effects of methylparathion on acetylcholinesterase in various regions of brain and spinal cord suggest differences in the distribution of molecular enzyme forms. In the present work, the in vitro and in vivo effects of methylparathion on acetylcholinesterase and butyrylcholinesterase were studied in whole brain homogenates of golden hamsters with biochemical methods. Furthermore, acetylcholinesterase activity was determined in regions of the nervous system by quantitative histochemistry (microdensitometry). Biochemically, very low IC50 values of the hydrophilic and lipophilic fractions of both enzymes were measured. Analysis of the time course of enzyme inhibition revealed maximum inhibition 45 min after methylparathion application. Using microdensitometry different degrees of acetylcholinesterase inhibition were found in various areas of the brain. The highest inactivation was observed in the Substantia nigra and in thalamic nuclei; in several regions of the cerebellum, the inhibition rate was comparatively lower. In conclusion, methylparathion acts as an potent inhibitor of acetylcholinesterase and butyrylcholinesterase in the hamster nervous system. The region-specific different inactivation of acetylcholinesterase might be caused by the existence of multiple forms of the enzyme in various brain regions.     

Amphiphilic and hydrophilic forms of choline-O-acetyltransferase in cholinergic nerve endings of the Torpedo

In the purely cholinergic nerve endings isolated (i.e. synaptosomes) from the electric organ of the fish Torpedo, the enzyme choline acetyltransferase was found to exist not solely in its well-known soluble form but also in a form which is non-ionically bound to the plasma membrane; this activity could not be solubilized in solutions of high ionic strength (0.5 M NaCl). The non-ionic detergent Triton X-114 was used to solubilize synaptosomes isolated from either the electric organ of Torpedo or rat brain. This detergent allows to separate hydrophilic from amphiphilic proteins of cells or subcellular fractions. Twelve per cent of the synaptosomal choline acetyltransferase partitioned as amphiphilic and 80-97% as hydrophilic activity. The percentage of amphiphilic activity present in synaptosomes was significantly higher than that of the form of activity (4.4%) extracted from samples containing only the soluble form of choline acetyltransferase but was significantly lower than the percentage of amphiphilic enzyme present in preparations of synaptosomal plasma membrane (20-22%) which were enriched in the non-ionically membrane-bound form of choline acetyltransferase. These results indicate that the soluble and the non-ionically membrane-bound enzymes differ in their capacity to interact with non-ionic detergents. The preparations of synaptosomal plasma membranes contained significantly higher proportions of detergent-insoluble choline acetyltransferase activity than did the whole synaptosomes; the difference was more striking for the Torpedo than for the rat enzyme. This detergent-insoluble activity was not due to aggregates of the enzyme. Some properties of the hydrophilic and amphiphilic choline acetyltransferase of Torpedo were analyzed. The two forms of the enzyme did not exhibit different affinities for their substrates; they were found to differ with respect to their sensitivity to inhibition by increasing concentrations of the two products of the reaction, acetylcholine and coenzyme A and heat inactivation at 45 degrees C. Most probably the hydrophilic and amphiphilic activities correspond to what was referred to as soluble and non-ionically membrane-bound choline acetyltransferase, respectively. The amphiphilic form may be an integral enzyme of the plasma membrane of cholinergic nerve endings or may be tightly bound to a specific protein in this membrane which may act as a "receptor" for choline acetyltransferase.     

Acetylcholinesterase activities in hippocampus, frontal cortex and striatum of Wistar rats after pilocarpine-induced status epilepticus

Experimental manipulations suggest that in vivo administration of cholinergic agonists or inhibitors of acetylcholinesterase (AChE) increases the concentration of acetylcholine. Biochemical studies have proposed a role for AChE in brain mechanisms responsible by development to status epilepticus (SE) induced by pilocarpine. The present study was aimed at investigating the changes in AChE activities in hippocampus, striatum and frontal cortex of adult rats after pilocarpine-induced SE. The control group was treated with 0.9% saline (s.c., control group) and another group received pilocarpine (400 mg/kg, s.c.). Both groups were sacrificed 1 h after treatment. The results have shown that pilocarpine administration and resulting SE produced a significant decrease in the AChE activity in the hippocampus (63%), striatum (35%) and frontal cortex (27%) of adult rats. Our results demonstrated a direct evidence of a decrease in the activity of the AChE in rat brain regions during seizure activity that could be responsible by regulation of acetylcholine levels during the establishment of SE induced by pilocarpine.     

Influence of emotional-algic stress on the activity of carboxypeptidase H — An enzyme involved in processing rat brain neuropeptides

It was established that the activity of carboxypeptidase H increases in divisions of the brain of rats during a one-time emotional-algic stress. The most substantial increase in the activity of the enzyme is observed in the hypophysis and the hypothalamus. The changes in activity were less marked with chronic emotional-algic stress. The involvement of carboxypeptidase H in the development of stress reactions, as well as the possibility of the participation of the soluble form of the enzyme in the processing of secretory peptides, and of the membrane-bound form in the processing, of neuropeptides with a central action, is hypothesized. Department of Chemistry, V. G. Belinskii State Pedagogical Institute, Penza. Translated from Fiziologicheskii Zhurnal imeni I. M. Sechenova, Vol. 80, No. 3, pp. 23–27, March, 1994     

Cardiac sarcoplasmic reticulum function in insulin- or carnitine-treated diabetic rats

Cardiac sarcoplasmic reticulum (SR) function and SR levels of long-chain (LC) acylcarnitines were determined in streptozotocin-induced diabetic rats treated with insulin or D,L-carnitine. ATP-dependent calcium transport was significantly depressed in cardiac SR isolated from untreated diabetic rats compared with control rats. Diabetic rat cardiac SR levels of LC acylcarnitines were also significantly elevated. Various parameters of heart function (left ventricular developed pressure, +dP/dT, and -dP/dT), as determined on an isolated working heart apparatus, were found to be depressed in untreated diabetic rats. Cardiac SR isolated from diabetic rats treated throughout the study period with insulin or D,L-carnitine did not have elevated levels of LC acylcarnitines associated with SR membrane nor was SR calcium transport activity depressed. Heart function in the diabetic rats treated with insulin was similar to control rat hearts but heart function remained depressed in diabetic rats treated with D,L-carnitine. The data suggest that the LC acylcarnitines are involved in the observed impairment of cardiac SR function in diabetic rats. Other factors, however, must be contributing to the depression in heart function noted in these animals.     

Acute starvation decreases acetylcholinesterase activity in different regions of rat brain.

The activity of acetylcholinesterase (AChE) was assayed spectrophotometrically in four brain regions of rats that had been deprived of food for 96 h. A significant decrease in the total AChE activity (by 4-45%) as well as in its specific activity (by 14-28%) was observed in the supernatant and total particulate fractions from cerebral hemispheres, cerebellum, brainstem and diencephalon + basal ganglia. Similarly, blood glucose, body weight and protein content of subcellular fractions from most brain regions showed decreases after starvation.     

Solubilization of 20S acetylcholinesterase from the chick central nervous system

The ionic detergent sodium cholate, in the presence of 1 M NaCl, solubilizes a 20S acetylcholinesterase from chick retina and other brain tissues previously extracted with a buffered solution containing 1% Triton X-100 and 1 M NaCl. This 20S acetylcholinesterase appears to be a tailed form of the enzyme which, upon collagenase digestion, is converted to a 22S (mainly) form. This finding suggests that the vertebrate central nervous system does contain asymmetric, collagen-tailed forms of acetylcholinesterase, as is the case in skeletal muscle and cholinergic ganglia.     

Membrane-bound choline acetyltransferase in Torpedo electric organ: A marker for synaptosomal plasma membranes?

The enzyme choline-O-acetyltransferase catalyses the biosynthesis of acetylcholine from acetyl coenzyme A and choline and is considered as one of the best markers for cholinergic nerve endings. The distribution of this enzymatic activity was analysed during the purification of plasma membranes of purely cholinergic nerve endings isolated from the electric organ of the fish Torpedo marmorata. This tissue, which receives a profuse and purely cholinergic innervation, can be considered as being a "giant" neuromuscular synapse. The isolated nerve endings (synaptosomes) were first osmotically disrupted and their plasma membranes isolated by equilibrium density centrifugation (discontinuous followed by continuous sucrose gradients). Choline acetyltransferase activity was found to exist in three forms: (1) a soluble form (the major one) present in the cytoplasm of the nerve endings, (2) a form which is ionically associated with membranes and which can be solubilized by washing exhaustively the membrane fraction with solutions of high ionic strength (0.5 M NaCl) and (iii) a form which is non-ionically bound to membranes and cannot be solubilized with high salt solution. The soluble and the non-ionically bound activities exhibited very similar affinities for choline (1.34 and 1.64 mM, respectively). The non-ionically membrane-associated form of choline acetyltransferase was found to "copurify" with the cholinergic synaptosomal plasma membranes of Torpedo, its specific activity being increased from 122 (crude fraction) to 475 (purified membrane fraction) nmol/h/mg protein. An enrichment was also observed for another cholinergic marker, the enzyme acetylcholinesterase, but not for the nicotinic receptor to acetylcholine, a marker for postsynaptic membranes. No choline acetyltransferase activity could be detected in preparations of synaptic vesicles that were highly purified from the electric organ. Also, the non-ionically associated form of choline acetyltransferase activity was hardly detectable (2.4 nmol/h/mg protein) in fractions enriched in axonal membranes prepared from the cholinergic electric nerves innervating the electric organ. The partition into soluble and membrane-bound activity was also analysed for choline acetyltransferase present in human placenta, a rich source for the enzyme but a non-innervated tissue. In this case the great majority of the enzyme appeared as soluble activity. Very low levels of non-ionically membrane-bound activity were found to be present in a crude membrane fraction from human placenta (2.8 nmol/h/mg protein).(ABSTRACT TRUNCATED AT 400 WORDS)     

Distribution of the glucocorticoid receptor in the human amygdala; changes in mood disorder patients

Exposure to stress activates the hypothalamic–pituitary–adrenal (HPA) axis that stimulates glucocorticoid (GC) release from the adrenal. These hormones exert numerous effects in the body and brain and bind to a.o. glucocorticoid receptors (GR) expressed in the limbic system, including the hippocampus and amygdala. Hyperactivity of the HPA axis and disturbed stress feedback are common features in major depression. GR protein is present in the human hypothalamus and hippocampus, but little is known—neither in healthy subjects nor in depressed patients—about GR expression in the amygdala, a brain structure involved in fear and anxiety. Since chronic stress in rodents affects GR expression in the amygdala, altered GR protein level in depressed versus healthy controls can be expected. To test this, we investigated GR-α protein expression in the post-mortem human amygdala and assessed changes in ten major or bipolar depressed patients and eight non-depressed controls. Abundant GR immunoreactivity was observed in the human amygdala, both in neurons and astrocytes, with a similar pattern in its different anatomical subnuclei. In major depression, GR protein level as well as the percentage of GR-containing astrocytes was significantly higher than in bipolar depressed patients or in control subjects. Taken together, the prominent expression of GR protein in the human amygdala indicates that this region can form an important target for corticosteroids and stress, while the increased GR expression in major, but not bipolar, depression suggests possible involvement in the etiology of major depression.     

Short-term REM sleep deprivation increases acetylcholinesterase activity in the medulla of rats

Involvement of cholinergic ponto-medullary brainstem mechanism regulating rapid eye movement (REM) sleep is known. Recently it was found that though short term REM deprivation influenced brainstem neuronal excitability, the activity of the brainstem acetylcholinesterase was not affected until after 96 h deprivation. Therefore, it was hypothesized that short-term REM deprivation might influence acetylcholinesterase in a restricted brainstem region. Results of this study show that the enzyme activity increased only in the medulla after 24 and 48 h REM deprivation. The flower pot technique was used for depriving the experimental rats of REM sleep. Suitable control experiments were conducted to rule out the possibility of non-specific effects. Thus, the medullary cholinergic mechanism probably is more important for REM.