Relation of brain regional physostigmine concentration to cholinesterase activity and acetylcholine and choline levels in rat

The relationship between physostigmine (Phy) concentration, acetylcholine (ACh), choline (Ch) and cholinesterase (ChE) activity was examined in whole rat brain after the administration of [³H]Phy (650 g/kg i.m.). Cholinesterase inhibition was found to be inversely related to Phy levels. Maximal inhibition (80%) was seen at 5 min and by 2 hrs ChE activity had returned to control levels. Acetylcholine levels in whole brain peaked at 30 min at a concentration (80 nmol/g) 2.3 times higher than controls (33 nmol/g). Choline levels were not significantly altered. The regional distribution of Phy concentration and ChE activity was studied in six areas of the brain following i.m. administration of three different dosages of [³H]Phy. Physostigmine concentration and ChE activity showed a dose dependency in each area examined except in SP (medial septum). Striatum (ST) showed the greatest relative increase of ACh up to 30 min, when compared to other areas. Choline levels were not changed in any area with the exception of ST at 5 min where a decrease was seen. There was a relationship between ChE activity, Phy concentration and ACh levels in all areas examined with exception of the medulla oblongata (MO). Our results indicate that even though ChE was inhibited practically uniformly in all brain areas, the percent increase with respect to control animals and the relative increase of ACh varied widely from area to area. This finding has clinical implications in cases in which cholinomimetic therapy is used to elevate ACh levels in specific brain areas which show a cholinergic deficit.Special issue dedicated to Prof. Eduardo De Robertis.     

The relationships between brain,serum, and whole blood ChE activity in the wood mouse (Apodemus sylvaticus) and the common shrew (Sorex araneus) after acute sublethal exposure to dimethoate

Abstract

Inhibition of cholinesterase (ChE) activity produced by a single acute intraperitoneal administration of dimethoate was studied in the wood mouse, Apodemus sylvaticus, and the common shrew, Sorex araneus, under laboratory conditions. ChE values from serum and whole blood were compared with those obtained from brain in order to obtain a non-destructive tool for predicting the severity of brain acetylcholinesterase (AChE) inhibition. In addition, serum and brain inhibition following oral exposure to dimethoate was also measured in the wood mouse. Normal ChE activity was higher in the brain and whole blood of the shrews than in wood mice. There was no difference between species in serum ChE activity. Exposure to dimethoate caused a dose-dependent reduction in ChE activity and there was a significant recovery in activity with increasing time after administration. In both species, serum and whole blood were more sensitive than brain for revealing organophosphate-induced ChE inhibition and serum was more sensitive than whole blood. Statistically significant relationships were defined between whole blood and brain ChE activity and between serum and brain ChE activity. Compared with serum, whole blood ChE activity was the more accurate predictor of brain AChE levels. The relationships between brain and serum ChE activity did not appear to be affected by the route of administration of the pesticide.     

The relationships between brain, serum, and whole blood ChE activity in the wood mouse (Apodemus sylvaticus) and the common shrew (Sorex araneus) after acute sublethal exposure to dimethoate

Inhibition of cholinesterase (ChE) activity produced by a single acute intraperitoneal administration of dimethoate was studied in the wood mouse, Apodemus sylvaticus, and the common shrew, Sorex araneus, under laboratory conditions. ChE values from serum and whole blood were compared with those obtained from brain in order to obtain a non-destructive tool for predicting the severity of brain acetylcholinesterase (AChE) inhibition. In addition, serum and brain inhibition following oral exposure to dimethoate was also measured in the wood mouse. Normal ChE activity was higher in the brain and whole blood of the shrews than in wood mice. There was no difference between species in serum ChE activity. Exposure to dimethoate caused a dose-dependent reduction in ChE activity and there was a significant recovery in activity with increasing time after administration. In both species, serum and whole blood were more sensitive than brain for revealing organophosphate-induced ChE inhibition and serum was more sensitive than whole blood. Statistically significant relationships were defined between whole blood and brain ChE activity and between serum and brain ChE activity. Compared with serum, whole blood ChE activity was the more accurate predictor of brain AChE levels. The relationships between brain and serum ChE activity did not appear to be affected by the route of administration of the pesticide.     

Acetylcholinesterase and butyrylcholinesterase activity in the atria of the heart of adult albino rats

In experiments on adult albino rats the authors used the substances BW 284 C51 (1.5-bis(allyldimethylammoniumphenyl)-pentane-3-one-dibromide) as a specific inhibitor of acetylcholinesterase (AChE) and ethopropazine (10-(2-diethylaminopropyl) phenothiazine hydrochloride) as a specific inhibitor of butyrylcholinesterase (BuChE) to determine the two enzyme activities in atrial homogenates and to investigate changes after AChE or BuChE inhibition of the negative chronotropic effect of acetylcholine (ACh) on atria incubated in vitro. AChE accounted for only 12% and BuChE for 88% of the total ability of atrial homogenates to hydrolyse acetylcholine. The concentration of exogenous ACh needed to reduce the spontaneous frequency of contractions of the isolated right atrium by 30, 60, or 90/min fell by 78%, 79% and 84% respectively after BW 284 C51 inhibition of AChE and by 95%, 94% and 94% after simultaneous inhibition of AChE and BuChE. The significance of AChE in control of the negative chronotropic effect of ACh is thus evidently significantly greater than would correspond to the percentual proportion of AChE in cholinesterase activities in the atria of the rat heart. In can be assumed that AChE is functionally associated with parasympathetic innervation of the heart and that it is probably present in a high concentration in the primary pacemaker region.     

Cholinesterases: New Roles in Brain Function and in Alzheimer's Disease

The most important therapeutic effect of cholinesterase inhibitors (ChEI) on approximately 50% of Alzheimer's disease (AD) patients is to stabilize cognitive function at a steady level during a 1-year period of treatment as compared to placebo. Recent studies show that in a certain percentage (approximately 20%) of patients this cognitive stabilizing effect can be prolonged up to 24 months. This long-lasting effect suggests a mechanism of action other than symptomatic and cholinergic. In vitro and in vivo studies have consistently demonstrated a link between cholinergic activation and APP metabolism. Lesions of cholinergic nuclei cause a rapid increase in cortical APP and CSF. The effect of such lesions can be reversed by ChEI treatment. Reduction in cholinergic neurotransmission–experimental or pathological, such as in AD–leads to amyloidogenic metabolism and contributes to the neuropathology and cognitive dysfunction. To explain the long-term effect of ChEI, mechanisms based on -amyloid metabolism are postulated. Recent data show that this mechanism may not necessarily be related to cholinesterase inhibition. A second important aspect of brain cholinesterase function is related to enzymatic differences. The brain of mammals contains two major forms of cholinesterases: acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). The two forms differ genetically, structurally, and for their kinetics. Butyrylcholine is not a physiological substrate in mammalian brain, which makes the function of BuChE of difficult interpretation. In human brain, BuChE is found in neurons and glial cells, as well as in neuritic plaques and tangles in AD patients. Whereas, AChE activity decreases progressively in the brain of AD patients, BuChE activity shows some increase. To study the function of BuChE, we perfused intracortically the rat brain with a selective BuChE inhibitor and found that extracellular acetylcholine increased 15-fold from 5 nM to 75 nM concentrations with little cholinergic side effect in the animal. Based on these data and on clinical data showing a relation between cerebrospinal fluid (CSF) BuChE inhibition and cognitive function in AD patients, we postulated that two pools of cholinesterases may be present in brain, the first mainly neuronal and AChE dependent and the second mainly glial and BuChE dependent. The two pools show different kinetic properties with regard to regulation of ACh concentration in brain and can be separated with selective inhibitors. Within particular conditions, such as in mice nullizygote for AChE or in AD patients at advanced stages of the disease, BuChE may replace AChE in hydrolizing brain acetylcholine.     

In vivo dose response relationship between physostigmine and cholinesterase activity in RBC and tissues of rats

Dose response of physostigmine (Phy) was studied in rat using various doses (25-500 micrograms/kg i.m.). Rats were sacrificed 15 min after Phy administration. Blood and tissues were analyzed for ChE activity by radiometric method and Phy concentration by HPLC method. A comparison of ChE values in different tissues of rats indicated that ChE activity was highest in brain (7.11 mumol/min/g) and least in diaphragm (0.67 mumol/min/g). The enzyme activity was eleven times more in brain as compared to diaphragm. Phy produced a dose-dependent inhibition of ChE in RBC (18-42%), brain (23-35%) and diaphragm (25-35%) from 50 to 200 micrograms/kg, then ChE inhibition was plateaued from 200 to 500 micrograms/kg in these tissues. A dose related ChE inhibition was seen in heart (16-50%) and thigh muscle (8-53%) from 50 to 500 micrograms/kg. Phy concentration increased linearly from 50 to 400 micrograms/kg in plasma, brain, heart and thigh muscle. These results indicate that ChE inhibition is linear up to 200 micrograms/kg in RBC, 150 micrograms/kg in brain and 300 micrograms/kg in heart. This linearity is not consistent in other tissues.     

Characterization of cholinesterase of muscularis muscle of Bufo marinus

We have characterized the cholinesterase (ChE) of muscularis muscle of Bufo marinus by selectively using specific inhibitors of acetylcholinesterase and pseudocholinesterase and observing susceptibility to inhibition when substrate is present in excess. The ChE activity in this preparation due to acetylcholinesterase (AChE) and pseudocholinesterase (BuChE) was 90 and 10%, respectively. The optimum temperature and pH for the ChE were 38 degrees C and 7.4, respectively and the excess substrate inhibition was noted above a pS of 2.6. The Km for acetylthiocholine (ASCh) was 0.76 X 10(-4) M.     

Metrifonate and dichlorvos: Effects of a single oral administration on cholinesterase activity in rat brain and blood

Cholinesterase activities in rat forebrain, erythrocytes, and plasma were assessed after a single oral administration of metrifonate or dichlorvos. In 3-month-old rats, the dichlorvos (10 mg/kg p.o.)-induced inhibition of cholinesterase reached its peak in brain after 15–45 min and after 10–30 min in erythrocytes and plasma. Cholinesterase activity recovered rapidly after the peak of inhibition, but did not reach control values in brain and erythrocytes within 24 h after drug administration. The recovery of plasma cholinesterase activity, in contrast, was already complete 12 h after dichlorvos treatment. Metrifonate (100 mg/kg p.o.) had qualitatively similar inhibition kinetics as dichlorvos, albeit with a slightly delayed onset. Peak values were attained 45–60 min (brain) and 20–45 min (blood), after drug administration. Apparently complete recovery of cholinesterase activity was noted in both tissues 24 h after treatment. The dose-dependence of drug-induced inhibition of cholinesterase in rat blood and brain was determined at the time of maximal inhibition, i.e., 30 min after dichlorvos treatment and 45 min after metrifonate treatment. The oral ED₅₀ values obtained for dichlorvos were 8 mg/kg for brain and 6 mg/kg for both erythrocyte and plasma cholinesterase. The corresponding oral ED₅₀ values for metrifonate were 10 to 15 times higher, i.e., 90 mg/kg in brain and 80 mg/kg in erythrocytes and plasma. In rats deprived of food for 18 h before drug treatment, the corresponding ED₅₀ values for metrifonate were 60 and 45 mg/kg, respectively, indicating an about two-fold higher sensitivity of fasted rats to metrifonate-induced cholinesterase inhibition compared to non-fasted rats. Compared to 3-month-old rats, 19-month-old rats showed a higher sensitivity towards metrifonate and dichlorvos. At the time of maximal inhibition, there was a strong correlation between the degree of cholinesterase inhibition in brain and blood. These results demonstrate that single oral administration of metrifonate and dichlorvos induces an inhibition of blood and brain cholinesterase in the conscious rat in a dose-dependent and apparently fully reversible manner. While the efficiency of a given dose of inhibitor may vary with the satiety status or age of the animal, the extent of brain ChE inhibition can be estimated from the level of blood ChE activity.     

The effects of some porphyrinogenic drugs on the brain cholinergic system.

In central nervous system, acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) hydrolyse acetylcholine. Diminished cholinesterase activity is known to alter several mental and psychomotor functions. The symptoms of cholinergic crisis and those observed during acute attacks of acute intermittent porphyria are very similar. The aim of this study was to investigate if there could be a link between the action of some porphyrinogenic drugs on brain and the alteration of the cholinergic system. To this end, AChE and BuChE activities were assayed in whole and different brain areas. Muscarinic acetylcholine receptor (mAChR) levels were also measured. Results obtained indicate that the porphyrinogenic drugs tested affect central cholinergic transmission. Quantification of mAChR gave quite different levels depending on the xenobiotic. Veronal administration inhibited 50% BuChE activity in whole brain, cortex and hippocampus; concomitantly cortex mAChR was 30% reduced. Acute and chronic isoflurane anaesthesia diminished BuChE activity by 70-90% in whole brain instead cerebellum and hippocampus mAChR levels were only altered by chronic enflurane anaesthesia. Differential inhibition of cholinesterases in the brain regions and their consequent effects may be of importance to the knowledge of the mechanisms of neurotoxicity of porphyrinogenic drugs.     

The effect in vitro of volatile anesthetics on the activity of cholinesterases.

Because the mechanism of anesthesia is unknown, the relationship between anesthetics and enzymes essential to brain function may be an important one. Therefore, the effect of 8 volatile anesthetics on the enzymatic activity of solubilized, purified dog brain and human erythrocyte acetylcholinesterase (AChE) and human serum cholinesterase (ChE) was studied in vitro. Serum ChE was found to be insensitive to saturated solutions of all the anesthetics studied. However, brain and erythrocyte AChE were reversibly inhibited in a dose-dependent manner by all 8 anesthetics in concentrations exceeding those used in clinical practice. Kinetic analysis revealed a mixed (competitive, non-competitive) type of inhibition with the exception of the ether-crythrocyte AChE interaction which was characterized by competitive inhibition. Ether and methoxyflurane were found to depress the AChE activity the most and isoflurane and enflurane the least. The concentrations of anesthetic in the gas phase necessary for 50% inhibition of erythrocyte AChE activity (I₅₀) were calculated for 5 anesthetics and found to correlate with their water-gas partition coefficients. These data suggest that the effect in vitro of volatile anesthetics on the catalytic activity of cholinesterases is a variable one and may be unrelated to anesthetic potency in vivo. The implications of these data concerning anesthetic-active site interactions are discussed.     

A comparative study of the interaction of the cholinesterase from the brain of the cabbage fly, the acetylcholinesterase from bovine erythrocytes and the butyrylcholinesterase from horse blood serum with organophosphorus inhibitors

Studies have been made of the effect of several organophosphorus inhibitors, R1(R2)P(O) . SCH2CH2SR and R1(R2)P(O)SCH2CH2SRR . -O4SCH3 (or -I), which differ by the structure of split (R, P) and phosphoryl (R1, R2) parts of the molecule, on cholinesterase (ChE) from the brain of the fly Delia brassicae, acetylcholinesterase (AChE) of the bovine erythrocytes and butyrylcholinesterase (BuChE) from the blood serum of the horse. For fly ChE, higher values of a constant (kII) of the inhibition rate (at pH 7.5 and temperature 25 degrees C) were obtained both with thiophosphates and with thiophosphonates. This finding reveals higher reactivity of the active centre of this enzyme, as well as significantly lower selectivity of the latter to the structure of organophosphorus inhibitors. The data obtained suggest the existence of differences in the size of hydrophobic regions of anionic and esterase parts of the active centre in ChE of the fly and AChE of mammals, as well as the existence of some similarity between ChE of the fly and BuChE.     

Organophosphorus compounds as tools for the pharmaco-histochemical study of cholinesterase in the rat striatum

A series of organophosphorous compounds (OP) was tested using a pharmacohistochemical method applied in vitro on the rat striatum, the central structure which contains the highest levels of acetylcholine and its metabolic enzymes; the OP showed a great variety of action towards the specific cholinesterase (AChE) and non-specific cholinesterase (BuChE). Except for iso-OMPA which is specific for BuChE localized in the microvessels endothelium, all the OP doses used in the present study were more or less potent inhibitors of cholinesterases (ChE). 15 mn after LD 50 doses of OP administered by subcutaneous route, a partial inhibition of the neurophile AChE occurred, revealing some striatal neurons which displayed high residual activity, i.e. the cholinergic interneurons. During the recovery phase following the inhibition of AChE by 1.5 LD 50 doses (the animals being treated with atropine) the AChE reaction product was detected almost simultaneously in some axo-spinous synapses probably non-cholinergic. The partial inhibition and the de novo synthesis of AChE also revealed the presence of small and less reactive non-cholinergic neurons. Among all the OP tested, soman was remarkable for its patchy inhibition of AChE in the striatum. The significance of the alternation of reactive and non-reactive areas is discussed.     

Synthesis and molecular docking studies of some 4-phthalimidobenzenesulfonamide derivatives as acetylcholinesterase and butyrylcholinesterase inhibitors

A series of 4-phthalimidobenzenesulfonamide derivatives were designed, synthesized and evaluated for the inhibitory activities against acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). Structures of the title compounds were confirmed by spectral and elemental analyses. The cholinesterase (ChE) inhibitory activity studies were carried out using Ellman’s colorimetric method. The biological activity results revealed that all of the title compounds (except for compound 8) displayed high selectivity against AChE. Among the tested compounds, compound 7 was found to be the most potent against AChE (IC₅₀=?1.35?±?0.08?μM), while compound 3 exhibited the highest inhibition against BuChE (IC₅₀=?13.41?±?0.62?μM). Molecular docking studies of the most active compound 7 in AChE showed that this compound can interact with both the catalytic active site (CAS) and the peripheral anionic site (PAS) of AChE.     

人脑和人血清胆碱酯酶三维结构的计算机模拟研究

本文以同源的电鳐胆碱酯酶（Ｔ．ＡＣｈＥ）的三维结构为模板,模拟预测了人脑和血清胆碱酯酶（Ｈ。ＡＣｈＥ和Ｈ．ＢｕＣｈＥ）的三维结构和活力中心的组成。指Ｔ．ＡＣｈＥ,Ｈ．ＡＣｈＥ和Ｈ．ＢｕＣｈＥ宁间结构差异,并讨论了ＡＣｈ和Ｈ。ＡＣｈＥ的对接（ｄｏｃｋｉｎｇ）。Ｈ。ＡＣｈＥ和Ｈ．ＢｕＣｈＥ三维结构的确定将为进一步深入研究它的中毒机理和合理药物设计提供靶子。     

Gender-related differences in circadian rhythm of rat plasma acetyl- and butyrylcholinesterase: Effects of sex hormone withdrawal

The role of acetylcholinesterase (AChE) in the termination of the cholinergic response through acetylcholine (ACh) hydrolysis and the involvement of plasma butyrylcholinesterase (BuChE), mainly of hepatic origin, in the metabolism of xenobiotics with ester bonds is well known. Besides, BuChE has a crucial role in ACh hydrolysis, especially when selective anticholinesterases inhibit AChE. Herein, we analyzed the gender-related differences and the circadian changes of rat plasma cholinesterases. Plasma and liver cholinesterase activities were evaluated in control or 2–30-day castrated adult male and female rats. Plasma and liver AChE activities did not differ between genders and were not influenced by sex hormone deprivation. BuChE plasma activity was 7 times greater in female, reflecting gender differences in liver enzyme expression. Castration increased liver and plasma BuChE activity in male, while reduced it in female, abolishing gender differences in enzyme activity. Interestingly, female AChE and BuChE plasma activities varied throughout the day, reaching values 27% and 42% lower, respectively, between 2 p.m. and 6 p.m. when compared to the morning peaks at 8 a.m. Castration attenuated daily female BuChE oscillation. On the other hand, male plasma enzymes remained constant throughout the day. In summary, our results show that liver and plasma BuChE, but not AChE, expression is influenced by sex hormones, leading to high levels of blood BuChE in females. The fluctuation of female plasma BuChE during the day should be taken into account to adjust the bioavailability and the therapeutic effects of cholinesterase inhibitors used in cholinergic-based conditions such Alzheimer's disease.     

Design,synthesis and evaluation of 2,4-disubstituted pyrimidines as cholinesterase inhibitors

A group of 2,4-disubstituted pyrimidine derivatives (7a–e, 8a–e and 9a–d) that possess a variety of C-2 aliphatic five- and six-membered heterocycloalkyl ring in conjunction with a C-4 arylalkylamino substituent were designed, synthesized and evaluated as cholinesterase (ChE) inhibitors. The steric and electronic properties at C-2 and C-4 positions of the pyrimidine ring were varied to investigate their effect on ChE inhibitory potency and selectivity. The structure–activity relationship (SAR) studies identified N-benzyl-2-thiomorpholinopyrimidin-4-amine (7c) as the most potent cholinesterase inhibitor (ChEI) with an IC₅₀ = 0.33 μM (acetylcholinesterase, AChE) and 2.30 μM (butyrylcholinesterase, BuChE). The molecular modeling studies indicate that within the AChE active site, the C-2 thiomorpholine substituent was oriented toward the cationic active site region (Trp84 and Phe330) whereas within the BuChE active site, it was oriented toward a hydrophobic region closer to the active site gorge entrance (Ala277). Accordingly, steric and electronic properties at the C-2 position of the pyrimidine ring play a critical role in ChE inhibition.     

Butyrylcholinesterase in Human Brain and Acetylcholinesterase in Human Plasma: Trace Enzymes Measured by Two-Site Immunoassay

Enzyme-linked immunosorbent assays for acetylcholinesterase (AChE) and for butyrylcholinesterase (BuChE) were markedly more specific than conventional assays using selective enzyme inhibitors. The new assays were used with blood and brain samples containing traces of one enzyme dominated by large amounts of the other. The results showed that human plasma does contain AChE (8 ng/ml), even though its major cholinesterase is BuChE (3,300 ng/ml). BuChE immunoreactivity was not detected in human red blood cells but occurred in all brain regions. The cerebellum was the richest region tested (540 ng of BuChE/g of tissue), whereas the cerebral cortex was the poorest (240 ng of BuChE/g). However, because of the small local AChE content (99 ng/g), BuChE was the major cortical cholinesterase. The picture was reversed in the putamen, where BuChE immunoreactivity (340 ng/g) was far outweighed by that of AChE (6,100 ng/g).     

Age-dependent changes in plasma and brain cholinesterase activities of house wrens and European starlings

We determined age-dependent changes in plasma and brain cholinesterase (ChE) activity for two species of passerines: house wren (Troglodytes aedon) and European starling (Sturnus vulgaris, starling). In plasma from nestlings of both species, total ChE activity increased with age, acetycholinesterase (AChE, EC 3.1.1.7) activity declined rapidly immediately after hatching, and butyrylcholinesterase (BChE, EC 3.1.1.8) activity increased steadily. For both species, total ChE and BChE activities and the BChE:AChE ratio in plasma were significantly greater in adults than nestlings suggesting trends observed in nestlings continue post fledging. In older nestlings and adults, AChE activity in plasma was significantly greater and BChE:AChE ratio less in house wrens than starlings. For house wrens as compared with starlings, ChE activity in brain increased at a significantly greater rate with age in nestlings and was significantly greater in adults. However, ChE activity in brain was similar at fledging for both species suggesting that the increase in ChE in brain is more directly related to ontogeny than chronologic age in nestlings of passerines. For both species, ChE activity increased significantly with brain weight of nestlings but not adults. House wrens hold similar patterns of age-dependent change in ChE activity in common with starlings but also exhibit differences in AChE activity in plasma that should be considered as a factor potentially affecting their relative toxicologic response to ChE inhibitors.     

Kinetics of Brain Cholinesterase Inhibition Following Metrifonate Administration

In previous metrifonate (MTF) studies, there has been evidence for a preferential functional effect of the drug in cortical but not in striatal regions. In the present study we investigated the kinetics of brain cholinesterase (ChE) inhibition following an acute administration of MTF (100 mg/kg) in various brain regions of young and old Fischer 344 rats. The main objective was to test the hypothesis that the functional regional selectivity, observed in previous studies, was correlated with the extent of ChE inhibition. Using Karnovsky's method for histochemical staining, the highest staining intensity in control rats was found in the striatum and hippocampus, compared to a low basal activity in the frontal and frontoparietal cortices. In the striatum of drug treated old rats, enzyme inhibition was somewhat greater than that found in young rats. However, in the hippocampus, four to eight hours following MTF administration, the inhibition was greater in young compared to old rats. The differences in the sensitivity of various brain regions towards MTF induced ChE inhibition could not be correlated with the regional variation of MTF functional effects.     

Age-related changes in acetylcholinesterase and its molecular forms in various brain areas of rats

A previous study conducted in this laboratory revealed a decrease in total cholinesterase (total ChE) in the cerebral cortex, hippocampus and striatum in aged rats (24 months) of various strains, as compared with young animals (3 months). The purpose of the present experiments was to extend the study to other brain areas (hypothalamus, medulla-pons and cerebellum) and to assess whether this decrease was dependent on the reduction of either specific acetylcholinesterase (AChE) or butyrylcholinesterase (BuChE) or both. By using ultracentrifugation on a sucrose gradient, the molecular forms of AChE were evaluated in all the brain areas of young and aged Sprague-Dawley rats. In young rats the regional distribution of total ChE and AChE varied considerably with respect to BuChE. The age-related loss of total ChE was seen in all areas. Although there was a reduction of AChE and, to somewhat lesser extent, of BuChE in the cerebral cortex, hippocampus, striatum, and hypothalamus (but not in the medulla-pons or the cerebellum), the ratio AChE/BuChE was not substantially modified by age. Two molecular forms of AChE, namely G4 (globular tetrameric) and G1 (monomeric), were detected in all the brain areas. Their distribution, expressed as G4/G1 ratio, varied in young rats from about 7.5 for the striatum to about 2.0 for the medulla-pons and cerebellum. The age-related changes consisted in a significant and selective loss of the enzymatic activity of G4 forms in the cerebral cortex, hippocampus, striatum, and hypothalamus, which resulted in a significant decrease of the G4/G1 ratio. No such changes were found in the medullapons or the cerebellum. Since G4 forms have been proposed to be present presynaptically, their age-related loss in those brain areas where acetylcholine plays an important role in neurotransmission may indicate an impairment of presynaptic mechanisms.