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Alzheimer's disease (AD) is the most common neurodegenerative disorder in the elderly, and studies have suggested that β-asarone has pharmacological effects on beta-amyloid (Aβ) injected in the rat hippocampus. However, the effect of β-asarone on autophagy in the APP/PS1 transgenic mouse is unreported. APP/PS1 transgenic mice were randomly divided into six groups (n=10/group): an untreated group, an Aricept-treated group, a 3-MA-treated group, a rapamycin-treated group, an LY294002-treated group, a β-asarone-treated group. The control group consisted of wild-type C57BL/6 mice. All treatments were administered to the mice for 30 days. Spatial learning and memory were assessed by water maze, passive avoidance, and step-down tests. AChE and Aβ42 levels in the hippocampus were determined by ELISA. p-Akt, p-mTOR, and LC3B expression were detected by flow cytometry. The expression of p-Akt, p-mTOR, Beclin-1, and p62 proteins was assessed by western blot. Changes in autophagy were viewed using a transmission electron microscope. APP and Beclin-1 mRNA levels were measured by Real-Time PCR. The learning and memory of APP/PS1 transgenic mice were improved significantly after β-asarone treatment compared with the untreated group. In addition, β-asarone treatment reduced AChE and Aβ42 levels, increased p-mTOR and p62 expression, decreased p-Akt, Beclin-1, and LC3B expression, decreased the number of autophagosomes and reduced APP mRNA and Beclin-1 mRNA levels compared with the untreated group. That is, β-asarone treatment can improve the learning and memory abilities of APP/PS1 transgenic mouse by inhibiting Beclin-1-dependent autophagy via the PI3K/Akt/mTOR pathway.

β-glucan (polysaccharide) rich diet has been reported to enhance cognition in humans but the mechanism remained elusive. Keeping this in mind, the present study was designed to investigate the interaction of β-glucan with central cholinergic system. Briefly, in-silico analysis revealed promising interactions of β-glucan with the catalytic residues of acetylcholinesterase (AChE) enzyme. In line with this outcome, the in vitro assay (Ellman's method) also exhibited inhibition of AChE by β-glucan (IC50=0.68±0.08μg/µl). Furthermore, the in vivo study (Morris water maze) showed significant dose dependent reversal of the amnesic effect of scopolamine (2mg/kg i.p.) by β-glucan treatment (5, 25, 50 and 100mg/kg, i.p.). Finally, the hippocampi of aforementioned treated animals also revealed dose dependent inhibition of AChE enzyme. Hence, it can be deduced that β-glucan possesses potential to enhance central cholinergic tone via inhibiting AChE enzyme. In conclusion, the present study provides mechanistic insight to the cognition enhancing potential of β-glucan. Keeping in mind its dietary use and abundance in nature, it can be considered as economic therapeutic option against cognitive ailments associated with decline in cholinergic neurotransmission.

The objective of this study was to evaluate the effects of maternal caffeine intake on the neuromotor development of rat offspring and on acetylcholine degradation and acetylcholinesterase (AChE) expression in the hippocampus of 14-day-old infant rats. Rat dams were treated with caffeine (0.3 g/L) throughout gestation and lactation until the pups were 14 days old. The pups were divided into three groups: (1) control, (2) caffeine, and (3) washout caffeine. The washout group received a caffeine solution until the seventh postnatal day (P7). Righting reflex (RR) and negative geotaxis (NG) were assessed to evaluate postural parameters as an index of neuromotor reflexes. An open-field (OF) test was conducted to assess locomotor and exploratory activities as well as anxiety-like behaviors. Caffeine treatment increased both RR and NG latency times. In the OF test, the caffeine group had fewer outer crossings and reduced locomotion compared to control, while the washout group showed increased inner crossings in relation to the other groups and fewer rearings only in comparison to the control group. We found decreased AChE activity in the caffeine group compared to the other groups, with no alteration in AChE transcriptional regulation. Chronic maternal exposure to caffeine promotes important alterations in neuromotor development. These results highlight the ability of maternal caffeine intake to interfere with cholinergic neurotransmission during brain development.

Chronic exposure to low doses of organophosphates during brain development can induce persistent neurochemical and behavioral effects. This study sought to determine the long-lasting effects of developmental exposure to chlorpyrifos (CPF) and diazinon (DZN) on passive avoidance (PA) performance and neuronal nitric oxide synthase (nNOS)-containing neurons in the subnuclei within basolateral complex of amygdala (BLC). Developing rats were exposed to daily dose (1mg/kg) of CPF or DZN during gestational days 15-18 and postnatal days (PND) 1-4. PA performance was assessed in young adulthood (PND 60). Brain sections were also processed by NADPH-diaphorase (NADPH-d) and nNOS immunohistochemistry. Gestational exposure to CPF increased NADPH-d(+)/nNOS-immunoreactive (IR) neurons within the basolateral nucleus (BL) and medial paracapsular intercalated cluster, which was along with PA retention impairment in both male and female rats. Prenatal exposure to DZN did not significantly change the number of NADPH-d(+)/nNOS-IR neurons in the BLC while impaired PA retention in females. Postnatal exposure to CPF decreased NADPH-d(+)/NOS-IR neurons in the BL without affecting PA performance. Exposure to DZN during early postnatal period impaired PA retention in both sexes, albeit to a lesser extent in females, and was along with a considerable sex independent reduction of NADPH-d(+)/NOS-IR neurons in all BLC subnuclei. Our data suggest that developmental exposure to apparently subtoxic dose of CPF and DZN elicit long-lasting impairment in PA retention that are associated, but not necessarily correlated with effects on NADPH-d(+)/NOS-IR neurons in BLC of the amygdala.

We investigated the effects of alcohol (EtOH) and acetaldehyde (ACe) on choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) in the frontal cortex of Aldh2-/- (KO) mice. KO mice were used as models of Aldh2-deficient humans to examine ACe effects. Brain samples were analyzed at 40 and 120 min after 2- and 4-g/kg intraperitoneal EtOH administration by RT-PCR and Western blot. Wild-type (WT) mice exhibited a remarkable decrease in ChAT and AChE mRNA expression at both time points only after 4-g/kg EtOH treatment compared with the naive control, whereas KO mice showed a considerable reduction in cholinergic markers after 2- and 4-g/kg EtOH treatment. The 4-g/kg EtOH-induced decrease in ChAT and AChE RNA expression at both time points was significantly greater than that in obtained with the administration of 2-g/kg at 40 min in WT mice. KO mice showed a significant difference in ChAT mRNA at 40 min between the EtOH groups. The findings regarding the ChAT mRNA levels are consistent with the results of Western blot in both types of mice, with some exceptions. EtOH-induced ChAT and AChE expression in KO mice was significantly lower than that in WT mice. This genotype effect occurred mostly at 40 min after EtOH dosing. Only ACe was quantified in the brains of KO mice, whereas EtOH was detected in both types of mice in vivo. These results suggest that EtOH and ACe combined or high EtOH alone alters cholinergic markers expression via changes in presynaptic and postsynaptic processes in the mice frontal cortex, thus indicating that central cholinergic neurons may be sensitive to EtOH and ACe.

Our previous studies have shown that a minor isoform of acetylcholinesterase (AChE) is increased in the cerebrospinal fluid (CSF) of Alzheimer's disease (AD) patients. In the present study, the glycosylation of butyrycholinesterase (BuChE) was found to be altered in AD CSF. By combining an analysis of CSF AChE and BuChE glycosylation, it is possible to identify cases of AD with more than 90% sensitivity and specificity.

To study the mechanisms underlying cholinotoxic brain damage, we examined ethylcholine aziridinium (AF64A) effects on cholinesterase genes. In vitro, AF64A hardly affected cholinesterase activities yet inhibited transcription of the G,C-rich AChE DNA encoding acetylcholinesterase (AChE) more than the A,T-rich butyrylcholinesterase (BChE) DNA. In vivo, intracerebroventricular injection of 2 nmol of AF64A decreased AChE mRNA in striatum and septum by 3- and 25-fold by day 7, with no change in BChE mRNA or AChE activity. In contrast, hippocampal AChE mRNA increased 10-fold by day 7 and BChE mRNA and AChE activity decreased 2-fold. By day 60 post-treatment, both AChE mRNA and AChE levels returned to normal in all regions except hippocampus, where AChE activity and BChE mRNA were decreased by 2-fold. Moreover, differential PCR displays revealed persistent induction, specific to the hippocampus of treated rats, of several unidentified G,C-rich transcripts, suggesting particular responsiveness of hippocampal G,C-rich genes to cholinotoxicity.

This study describes the cellular distribution of muscarinic acetylcholine receptors (mAChRs) in the medial septum (MS), employing the monoclonal antibody M35 raised against purified mAChR-protein. mAChR-positive neurons are found throughout the MS, but are predominantly located in the midline area and in the lateral compartments. The labeled cell bodies are variable in shape and size (largest diameter ranging from 10-30 microns), while both soma and the associated dendritic processes are densely stained for mAChRs. Astrocytes immunoreactive for mAChRs were frequently found associated with the large blood vessels in the midline area. To study the neurotransmitter nature of the mAChR-positive cells, immunofluorescence double-labeling experiments were performed for mAChRs and GABAergic and cholinergic markers. GABAergic cells were identified immunocytochemically using antisera against glutamic acid decarboxylase (GAD), parvalbumin (PARV) or calbindin protein (CaBP). The cholinergic transmitter nature of the mAChR-positive cells was studied using adjacent 8 microns thick serial sections stained immunocytochemically for choline acetyltransferase (ChAT), or histochemically for acetylcholinesterase (AChE). These experiments showed that approximately half (52.3%) of all mAChR-positive cells contain GAD, whereas the other half is cholinergic. Conversely, nearly all GABAergic (98.6%) and cholinergic (96.9%) cells are endowed with mAChRs. GAD-positive terminals were found surrounding mAChR-positive perikarya which were either GAD-positive or GAD-negative, indicating GABAergic innervation on both GABAergic and cholinergic MS neurons. In general, the staining intensity for mAChRs appeared to be considerably higher in GABAergic than in cholinergic neurons, suggesting a stronger cholinergic impact upon the GABAergic neurons. The current anatomical findings contribute to the concept that the MS neurons form a firmly interconnected cell group, in which cholinergic neurotransmission mediated through mAChRs seems to play a significant role.

We examined the effect of long-term exposure to radio frequency radiation 147 MHz and its sub-harmonics 73.5 and 36.75 MHz amplitude modulated at 16 and 76 Hz (30-35 days, 3 h per day) on cholinergic systems in developing rat brain. A significant decrease in acetylcholine esterase activity was found in exposed rats as compared to the control. Decrease in acetylcholine esterase (AChE) activity was independent of carrier wave frequencies. A short-term exposure did not have any significant effect on AChE activity.

The acetylcholinesterase (AChE) activity in striatum rat was determined before and shortly after death using the in vivo microspectrophotometric method. This technique allowed us to monitor the Ellman colorimetric reaction directly inside the brain using an optical probe implanted in a live animal and to determine locally the AChE activity. Whatever the cause of the animals death, we observed a drastic postmortem decrease of the AChE activity of about 35-50%, 10 min after death. We have verified that the postmortem decrease of brain temperature or pH and postmortem optical properties changes could only explain a fraction of the AChE activity fall (16%). This phenomenon seems to be related to events strictly localized at the cellular level, since local injection of cyanide at the measuring site promotes a decrease of the enzymatic activity (40%) close to the levels observed after death. The origin of this rapid postmortem fall of the AChE activity is discussed. The technical properties of the microspectrophotometric method exclude a decrease of the ectocellular pool of enzyme after death. Our results allow us to envisage the existence of an in vivo endogenous regulation of the AChE activity which disappears shortly after death.

Male Wistar rats subjected to unilateral fimbria-fornix transection by mechanical knife cut or to sham operations were tested in a water maze and in an open field. Half the animals in each group were treated with either 0.06 mg/kg nimodipine or vehicle, administered i.p. for 7 days, beginning the day of surgery. Animals were sacrificed and brains were processed for acetylcholine esterase (AChE) histochemistry. In the water maze, lesioned rats showed a significant impairment relative to the sham-operated animals. Nimodipine treatment did not improve performance. There were no differences among the groups in the observed frequencies of the open field behaviors of locomotion, hole-poke, rearing and grooming. A significant reduction of AChE-positive cell bodies was found in the medial septal region on the side of the lesion. There were no differences in water maze performance among groups of rats treated with 0.0, 0.5, 1.0, or 5.0 mg/kg nimodipine for 7 days, beginning the day of fimbria-fornix transection, in an attempt to determine any dose-dependent effect of the drug.

Acetylcholinesterase (AchE) is an important peripheral membrane-bound enzyme, crucial for cholinergic neurotransmission. Changes in AchE activity, kinetic and thermotropic properties were studied in hepatic encephalopathy (HE) associated with acute liver failure induced experimentally by the administration of the hepatotoxin thioacetamide (TAA). Activity of AchE decreased significantly following TAA administration. AchE from cerebral cortex membrane isolates of TAA-treated rats also showed a decrease in Vmax and an increase in Km. Arrhenius plots revealed considerable changes in the thermotropic behavior of AchE from membrane isolates obtained from TAA-treated rats as evident from the decreased transition temperature. A positive correlation was observed between changes in membrane cholesterol (r2 = 0.987), sphingomyelin (r2 = 0.99) levels and AchE activity, thus indicating that membrane lipid changes could underlie the observed changes in kinetic and thermotropic properties of membrane-bound AchE during TAA-induced HE.

Chronic ethanol intake causes various types of neural damage and behavioral impairments, probably acting through oxidative stress and excitotoxicity, while dietary restriction is considered by some authors to protect the central nervous system from these kinds of damage. In the present study, a factorial experimental design was used to investigate the effects of chronic ethanol and dietary restriction treatments, associated or not, on Wistar rats' exploratory behavior, spatial memory aspects and cortical and hippocampal acetylcholinesterase (AChE) activity. Dietary restriction lasted for the whole experiment, while ethanol treatment lasted for only 3 weeks. Despite the short ethanol treatment duration, for two behavior categories assessed, moving and rearing, an interaction was observed between the effects of chronic ethanol and dietary restriction. There were no significant differences in AChE activities among the groups. Cerebellar neural nitric oxide synthase (nNOs) activity was measured as a first step to assess oxidative stress. Dietary restriction significantly reduced NO formation. The present results indicate that dietary restriction might exert a protective effect against chronic-ethanol-induced changes in exploratory behavior. It is hypothesized that the mechanisms underlying this protection can involve prevention of oxidative stress.

To examine whether the selected antisense oligodeoxynucleotides (AS-ODN) targeting against human brain acetylcholinesterase (AChE) mRNA could improve the cognitive deficit in the Alzheimer's disease (AD) model mice induced by amyloid-β peptide (Aβ), we determined the time–effect relationship of AChE activity and the learning and memory after AS-ODN delivery. The results showed that the AChE activity decreased gradually along with time, initiating at 8 h and lasting 42 h. The time–effect curves of acetylcholine (ACh) behaved consistency with that of AChE activity. The animal cognition studies showed that in step-through test, the error number of the AS-ODN-treated AD model mice was significantly decreased, and the memory retention was increased. In the water maze performance, the swimming time obviously shortened. Our results indicated that antisense therapy is of potential use in the treatment of cognitive deficit in the Aβ model mice.

Galantamine is a rather weak acetylcholinesterase (AChE) inhibitor, currently approved for the symptomatic treatment of Alzheimer's disease, with possible additional allosteric potentiating effects at the nicotinic ACh receptor (nAChR). Earlier data from in vitro biochemical tests suggest that donepezil is 40- to 500-fold more potent than galantamine in inhibiting AChE. In this study, both brain levels and Ki values for AChE inhibition for donepezil and galantamine in rat, mouse, and rabbit after subcutaneous application were determined. Clearance of galantamine from the brain is in general faster that donepezil and is faster in rabbits compared to rats and mice. The brain-to-plasma ratio for galantamine and donepezil, respectively, ranges from 1.2 to 1.5 in the rabbit, 3.3 to 5.2 in the mouse, and 6.6 to 13 in the rat. Galantamine doses between 1.5 and 5 mg/kg are appropriate to reach brain levels within the documented optimal allosteric potentiating ligand dose-response. Ki values of brain AChE inhibition for galantamine and donepezil, respectively, are 7.1 and 2.3 microg/g in rats, 8.3 and 0.65 microg/g for mice, and 19.1 and 1.3 microg/g in rabbits. The data also suggest that for a similar degree of brain AChE inhibition, 3-15 times higher galantamine than donepezil doses are needed.

In order to develop the specific oligobodies against human brain acetylcholinesterase (AChE) and distinguishes between human erythrocyte and brain AChEs, we applied the strategy of 'target switching' to obtain the specific polyclonal and monoclonal oligobodies. The specificity between human brain AChE and other ChEs was identified by Western blotting, dot blotting and enzyme protein binding assay (EPBA). The results showed that the oligobodies against the human brain AChE specifically immunoreacted with the human brain AChE and Torpedo AChE, not showing significant binding to AChE from human erythrocyte and butyrylcholinesterase (BChE) from human serum.

The cellular origin of the acetylcholinesterase (AChE) associated with amyloid plaques in the Alzheimer’s disease (AD) brain is unknown. In this study we report that amyloid β-peptides (Aβ) increased AChE levels in both neuronal and astrocytic primary cultures, supporting the possibility that both neurons and glia may make a direct contribution to the pool of AChE seen around amyloid deposits in the AD brain., JS-V was supported by a grant from the MCyT of Spain (Ramón y Cajal Program). DHS was supported by grants from the NH&MRC of Australia, Axonyx Inc. (New York), and the Rebecca L. Cooper Medical Research Foundation Ltd. (Australia).

The mechanism by which (-) deprenyl enhances cognitive function in Alzheimer's disease (AD) is not yet understood. (-) Deprenyl (0.2 mg/kg/day) was administered intramuscularly to adult male monkeys (n = 6) for 25 days. Control monkeys (n = 6) received physiological saline by the same route. The activity of acetylcholinesterase (AChE) in different brain regions and the dendritic arborization in CA3 pyramidal neurons of hippocampus were analysed, (-) Deprenyl-treated monkeys showed a significant increase in the AChE activity by 43% (p < 0.001) in the frontal cortex, by 39% (p < 0.025) in the motor cortex, by 66% (p < 0.001) in the hippocampus and by 26% (p < 0.05) in the striatum compared to controls. The branching points and the intersections of both apical and basal dendrites of CA3 hippocampal pyramidal neurons were also significantly increased in (-) deprenyl-treated monkeys. Enhanced AChE activity may increase dendritic arborization in the hippocampus and it may also play a role in improving cognitive functions observed in AD, following(-) deprenyl treatment.

A novel method for visualization of brain acetylcholinesterase (AChE) in vivo has been developed. Following intravenous administration of a radiolabelled acetylcholine analog, N-methyl-3-piperidyl acetate, there was very good agreement between the distribution of radioactivity and AChE activity in the brain of rat and monkey. The method would be applicable for in vivo studies of human brain AChE activity in disorders of central cholinergic systems such as Alzheimer's disease.

Patches of high nicotinamide-adenine dinucleotide phosphate diaphorase (NADPH-d) activity were found in the mediodorsal and midline thalamic nuclei of cats. These patches matched acetylcholinesterase (AChE)-rich patches within the medial thalamus, whereas other AChE-rich patches were NADPH-d negative. There were also patches of NADPH-d activity in the lateral habenula, but these did not match the AChE staining. These results suggest that the functional role of discrete thalamic regions may require the joint presence of AChE and NADPH-d enzymatic activities.

We have studied the behavior of a mammalian brain tetrameric acetylcholinesterase (AChE) form released by proteinase K from a crude membrane fraction of bovine caudate nucleus. The solubilization of active AChE indicated the presence of a protease-sensitive site in the anchored protein. Unexpectedly, the solubilized AChE maintained its capacity to form aggregates in detergent-free gradients. We demonstrate here that this property was due neither to the presence of the hydrophobic membrane-anchoring domain still linked to the enzyme, nor to the presence of AChE activity trapped in small plasma membrane vesicles. Moreover, we found that the proteinase K-treated extract, devoid of AChE activity, induced the aggregation of purified hydrophilic AChE which usually does not form aggregates. Our results suggest the presence of an AChE aggregating factor in bovine brain extracts prepared in the presence of proteinase K. It is possible that this aggregation may reflect a process of AChE clustering on neurons.

Caliber and microtubular density of myelinated fibers, acetylcholinesterase (AChE) content and its accumulation at a ligature were studied in the phrenic nerve of mature (3–4 months) and aging (2-year-old) rats. The number of axons remained constant. The cross-sectional area of the nerve was 67% greater in the older group; the axoplasm, though, constituted about 20% of the nerve tissue irrespective of age. The mean cross-sectional area of myelinated axons was twice as big in aging compared to mature rats. All axons grew in the same proportion irrespective of their original caliber. The microtubular density of 3-μm axons was about 22 microtubules/μm2 in mature and aging rats. The AChE activity of aging rats was half as much as that of mature rats if it was expressed per wet weight of nerve tissue but did not change if it was expressed per nerve fiber. Twenty-four hours after ligation of the nerve, total AChE activity rose in mature and aging rats by ca. 168%; the molecular forms - asymmetric and globular — accumulated in the same proportion in both age groups. We conclude that myelinated axons grow in the adult stage of life but the structure of axoplasm, content of AChE per axon, and rate of fast transport remain lifelong features of nerve fibers.

Rat muscle nerves were examined histochemically for their activity of acetylcholinesterase (AChE). The corresponding muscles were stained for myofibrillar ATPase and for NADH diaphorase. The nerves to the extensor digitorum longus (EDL) muscle and to the medial head of the gastrocnemius (MG) muscle consist of a motor axons of high AChE activity. Both muscles are characterized by the prevalence of type II muscle fibres. On the other hand, the soleus muscle and the quandratus femoris muscle, both mainly composed of type I muscle fibres, are innervated by a motor axons of low AChE activity. Since it is well established that EDL and MG are typical fast-twitch muscles and that the soleus, and probably also the auadratus femoris, is a typical slow-twitch muscle, it is suggested that, in rat, fast muscles are innervated by motor nerve fibres of high AChE activity and slow muscles are innervated by motor axons of low AChE activity.

The axonal transport of slowly migrating [3H]leucine labelled proteins was studied in regenerating hypoglossal and vagus nerves of the rabbit 1–4 weeks after a nerve crush. In normal nerves, as well as in the nerves contralateral to nerve crush, a transport rate of 4–5 mm/day for the hypoglossal nerve and 20–25 mm/day for the vagus nerve was calculated. The axonal transport of slowly migrating labelled proteins was increased in the regenerating hypoglossal nerve as compared to the contralateral 1 week after a nerve crush; this was mainly due to an increased transport rate. In contrast, the axonal transport of slowly migrating labelled proteins was decreased in the regenerating vagus nerve as compared to the contralateral nerve 1 week after a nerve crush. The results indicate a decreased transport rate and a decreased amount of material transported on the regenerating side. Changes in axonal transport in both hypoglossal and vagus nerves were less pronounced after 4 weeks of regeneration. Rapidly migrating [3H]leucine labelled proteins were transported into regenerating hypoglossal and vagus nerves distal to the crush zone, indicating axonal transport in the growing axons. The regeneration rate was estimated to be about 3 mm/day in the vagus nerve after 2–4 weeks of regeneration and about 4 mm/day in the hypoglossal nerve after 1 week of regeneration. The levels of acetyl-CoA: choline O-acetyltransferase (ChAc, E.C. 2.3.1.6) and acetylcholinesterase (AChE, E.C. 3.1.1.7) activity were decreased in regenerating vagus nerve the first week after a nerve crush.

A quantitative pharmacohistochemical technique has been used in the present study to assay acetylcholinesterase (AChE) activity in the neostriatum of C57BL/6 and DBA/2 mice. This technique permits the measurement of enzyme activity into microscopically defined compartments and is suitable for the study of striatal AChE-containing, putatively cholinergic, neurons. Microphotometric measurements have been performed in the cytoplasm of AChE-containing perikarya and in the striatal matrix: in both compartments, AChE activity was significantly higher in DBA/2 than in C57BL/6 mice. The present data show that AChE quantitative pharmacohistochemistry is suitable for studying the enzyme activity in nervous tissue and, particularly, in the cytoplasm of individual AChE-containing neurons. In addition, interstrain comparison indicates the presence of a genetically determined higher AChE content in striatal neurons of the DBA/2 strain.

A period of 1 or 24 h of cold stress (5 degrees C) resulted in a significant decrease of acetylcholine (ACh) concentration in the hypothalamus and hippocampus in rats. In the hippocampus the activity of the choline acetyltransferase (ChAT) was significantly increased after 24 h and that of acetylcholinesterase (AChE) after 1 and 24 h exposure to cold, whereas in the hypothalamus, AChE activity was found to be decreased, albeit only after 24 h exposure. Separate investigation of the dorsal and ventral hippocampus under 24 h of cold revealed that the ACh decreased in the dorsal hippocampus only, where no change in ChAT activity was observed. On the other hand, ACh showed no change in the ventral hippocampus where an increase of ChAT activity was found. Forced swimming for 20 min also induced a significant decrease of ACh in the hippocampus and cerebral cortex, along with a significant increase of choline concentration in the given regions. We conclude that under certain stress situations the cholinergic system in rat brain areas, mainly in the hippocampus and hypothalamus, is activated.

Long-Evans rats received septal lesions or sham operations and were tested for performance in a radial arm maze, level of activity and water intake in order to test whether recovery of function was mediated by sprouting of peripheral sympathetic fibers. Animals receiving septal lesions displayed an initial deficit in radial arm maze performance followed by recovery. No critical changes occurred in activity level and no recovery was seen in water intake. Subsequent superior cervical ganglionectomies had no effect on recovery of radial arm maze performance. There was a significant relationship between behavioral recovery and the degree of hippocampal AChE depletion. It is concluded that recovery of radial arm maze performance is not mediated by sympathetic sprouting following septal lesions but might be mediated by residual septohippocampal fibers.

The effectiveness of testosterone (T) and metabolites to increase activity of cholinergic enzymes in the androgen-sensitive syrinx of male zebra finches was assessed. Administration of aromatizable (T and androstenedione) or non-aromatizable (androsterone) androgens to castrates increased total activity of acetylcholinesterase (AChE) in the syrinx. Estradiol was ineffective in altering AChE activity. Results, when compared with behavioral data from the birds 5 , support the hypothesis that expression of male reproductive behaviors requires peripheral target organ activation by androgens and central target area activation by both androgens and estrogens.

The modular organization of the monkey cerebellum is revealed in normal material by histochemical methods for demonstrating the presence of the enzymes acetylcholinesterase (AChE) and cytochrome oxidase (CO). In the cerebellar white matter, AChE- and CO-rich fibers are distributed in longitudinal compartments, which are aligned with AChE- and CO-rich bands in the granule cell layer, and with narrower AChE-rich strips in the molecular layer. The number and disposition of zones demonstrated histochemically are consistent with, and extend, the basic scheme of cerebellar zonation established by Voogd and Voogd and Bigare´.

Summary The activities of three enzymes of neurotransmitter metabolism (choline acetyltransferase, CAT; acetylcholinesterase, AChE; and glutamic acid decar☐ylase, GAD) were studied in normal, transected, and organ cultured crayfish nerves. CAT (to a lesser extent AChE) was dramatically decreased in activity when the nerve was cut proximal to the nerve cell bodies. GAD activity was unaffected by such procedures. In organ cultured nerve, where both motor and sensory axons degenerated, the CAT and AChE activities were virtually absent, whereas GAD activity remained close to normal levels. Inhibition of protein synthesis in cultured nerve caused the GAD activity to decrease rapidly. In view of these data, and well documented fact that motor axons survive axotomy whereas sensory axons do not, a hypothesis that GAD is synthesized in the peripheral nerve is presented.

The neonatal and early postnatal development of choline acetyltransferase (ChAc) and acetylcholinesterase (AChE) activities, possibly the neurochemical correlates for the formation of cholinergic neurons or synapses, was studied using the ciliary and the superior cervical ganglion of the chicks from the 7th day (stage 31 of Hamburger and Hamilton 10 ) and the 13th day (stage 39) of incubation, respectively, to the 29th day after hatching. In the ciliary ganglion, where both the pre- and the postganglionic nerves are cholinergic, a sharp increase in the ChAc activity was observed from the 14th day of incubation and reached a maximum on the 29th day after hatching where the value amounted to 450-fold (per pair of ganglia) or 47-fold (per unit weight of protein content) the initial value. On the other hand, the total activity of AChE increased only 7-fold and the specific activity remained practically unchanged. In the superior cervical ganglion, where only the preganglionic nerves are cholinergic, the ChAc and AChE activities began to increase at the 15th day in the embryo, exhibiting an almost similar time course and reached a maximum on the 29th day after hatching. The alteration of both enzyme activities in the ciliary ganglion during development is discussed in connection with the morphological and electrophysiological findings reported to date.

Choline acetyltransferase (CAT) was assayed in the optic tectum of 4 teleost species with different visual powers. The results showed a close relationship between the enzyme levels in the optic tectum and the development of the visual system. In the more visual species, the trout, CAT activity in the optic tectum was about 30-fold higher than in the catfish, whose visual system is much less developed. Two species with intermediate development of the visual system, the goldfish and the tench, showed intermediate levels of CAT activity. Kainic acid treatment caused a significant decrease of both CAT and acetylcholinesterase (AChE) in the goldfish optic tectum. Concomitant histological examination showed, among other effects, the disappearance of most neurons belonging to the pyramidal and fusiform type in the stratum fibrosum and griseum superficiale of the tectum. The comparative and experimental data therefore suggest that the relationship between cholinergic mechanisms and the visual function is, to a significant extent, connected with the presence of intrinsic cholinergic circuits in the optic tectum. The relevance of these findings, also in relation to the problem of the identification of the retino-tectal transmitter, is discussed.

The Koelle copper thiocholine method was used to evaluate the effects of early postnatal septal lesions on histochemically demonstrable acetylcholinesterase (AChE) activity in the hippocampal region of the rat. Septal lesions made on the 5th or 8th day of life resulted in an almost total and permanent prevention of development of AChE-containing neuropil in the hippocampus and the dentate area. This effect was more pronounced in these areas than in the posterior parts of the hippocampal region (the entorhinal area, para-and presubiculum). The effect of early lesions was already discernible 2 days post-operatively, reducing the rapid onset of the neuropil stain in all areas of the hippocampal region observed in the normal animals during the second and third postnatal week. Adult animals which had received septal lesions in early postnatal life, showed stronger AChE activity in the presubiculum than did adults with fresh lesions (e.g. 6 days survival). This may indicate that in the former animals AChE-positive afferents had sprouted in the presubiculum.

The unilateral injection of kainic acid into the nucleus basalis magnocellularis (NBM) resulted in an alteration of the distribution of acetylcholinesterase (AChE) molecular forms in frontoparietal cortex ipsilaterally to the lesion. The G 4 G 1 ratio fell from 5.4 ± 0.8 in contralateral to 3.0 ± 0.5 in ipsilateral cortex. The NBM lesion effect thus, mimicks, the loss of tetrameric G4 form reported for various brain cortical areas of Alzheimer's disease (AD) patients. The data support the suggestion that G4 form is enriched in presynaptic nerve terminals.

Choline acetyltransferase (ChAT), acetylcholinesterase (AChE) and glutamic acid decar☐ylase (GAD) activities were measured in 20 brain regions from autopsied control and senile dementia of the Alzheimer type (SDAT) cases. Large, widespread reductions in the activities of ChAT and AChE were found in tissues from SDAT cases, while GAD activities were reduced in 3 of the 20 regions investigated. AChE activity in cerebrospinal fluid from SDAT cases was similar to that found in samples from non-demented patients.

Alterations in acetylcholinesterase (AChE) activity were investigated in middle endplate (MEP), distal endplate (DEP) and non-endplate (NEP) regions of rat diaphragm muscle after phrenic nerve transection either intrathoracically (low, 3–5 mm nerve stump) or in the neck (high, 50–55 mm nerve stump). When the phrenic nerve was severed low, the time of initial loss of AChE activity in MEP and DEP regions was 20 h. In contrast, when the nerve was transected in the neck, the onset time of AChE activity loss was delayed in both MEP and DEP regions to 24 h. Therefore, the onset time of loss of AChE activity was dependent on the nerve stump length. The rate of loss of AChE activity was not significantly different for MEP or DEP regions after either high or low nerve transection and averaged a rate of 1.54%/h. Loss of AChE activity in the NEP region was independent of nerve stump length and enzyme activity decreased at a rate of 0.62–0.63%/h, beginning immediately after nerve transection. AChE activity in phrenic nerve was also measured and was found to contribute significantly to measurements of endplate enzyme activity in diaphragm. Data indicate a nerve length-dependent loss of AChE in MEP and DEP regions of muscle with an apparent velocity of 300 mm/day, suggesting a relationship to fast axoplasmic transport of either AChE itself or a trophic factor.

Batrachotoxin (BTX), which causes increased Na+ permeability and blocks axoplasmic transport, or 6-aminonicotinamide (6-AN), which causes neuronal damage, was injected into the subarachnoid space of rat lumbar spinal cord. The activity of acetylcholinesterase (AChE) was measured in homogenates of the fast-twitch extensor digitorum longus (EDL) muscle and the slow-twitch soleus (SOL) muscle 10 days after injection. Both drug treatments significantly decreased AChE in EDL and SOL. Correlative electrophysiological measurements were made in intact EDL and SOL after injection of BTX or 6-AN. The results support the hypothesis that AChE in muscle is neurotrophically controlled.

Summary The reaction of septal and commissural-associational afferents in the dentate gyrus was examined at various times following a unilateral entorhinal lesion in 2- and 3-month-old, 12- to 18-month-old and 25–30-month-old rats. The response of septohippocampal fibers was examined histochemically by staining for acetylcholinesterase (AChE) activity; and that of commissural-associational fibers by the Holmes' fiber stain. In 2- and 3-month-old rats, AChE staining fibers, which project to the outer three-fourths of the molecular layer of the dentate gyrus, increased their staining intensity within 5–6 days following lesion of the entorhinal cortex. The rate of the response and the eventual magnitude declined progressively with the age of the subject. In 2- and 3-month-old rats, the commissural-associational fiber plexus appeared to expand partially into the entorhinal zone within 6 days following the lesion. This response also decreased progressively in rate and magnitude with age. Animals in the oldest age group showed at 12 days after the lesion a greater variability in the expansion of the commissural-associational fiber plexus than all younger groups. Astrocytes in the dentate molecular layer appeared to become more abundant and more hypertrophied in unoperated animals with age. The appearance of astrocytes in 25- to 30-month-old rats was similar to that seen in 2- and 3-month-old animals following an entorhinal lesion. An entorhinal lesion in the aged animals did not appear to cause a marked change in the appearance of astrocytes.

An effective state of malnutrition was imposed on rat pups throughout their postnatal period of development by feeding the lactating dams a 12% casein diet. Controls were fed a 24% casein diet. The malnourished pups exhibited such signs of retarded physical development as reduced body growth and delayed eye opening. At weaning brain weights were reduced and regional brain analysis revealed distortions of chemical composition. These changes included a general reduction of cholesterol concentration, decreased cerebellar DNA content (indicative of cell number), and reduced telencephalic and brain stem protein: DNA ratios (reflecting cell size). The cerebellum also displayed reduced AChE activity suggesting an appreciable effect on its synaptic elements, particularly involving the cholinergic system. Brain stem concentrations of 5-HT and 5-HIAA were increased, implying activation of the central serotonergic neurohumoral system. Possibly related to this, a concomitant increase of adrenal corticosterone was revealed, implicating the additional involvement of the pituitary-adrenal axis in the consequences of postnatal malnutrition. Behaviorally, the previously malnourished weanlings displayed a state of heightened ‘emotionality’. Their performance in a 2-way shuttle task was also markedly abnormal. It is proposed that the enhanced activation of the brain stem serotonergic system may, at least partially, underlie the state of heightened ‘emotionality’, which is the most characteristic behavioral manifestation of perinatal malnutrition.

Summary Axonal transport of choline acetyltransferase (ChAc, E.C.:2.3.1.6) and acetylcholinesterase (AChE, E.C.:3.1.1.7) was studied in the peroneal fascicles of rabbit sciatic nerves. The accumulation of ChAc in the central nerve stump proceeded 5 times more slowly than that of ChE and occurred at a distance of 2–4 mn proximally from the end, whereas AChE accumulated in the last 2 mm of the stump. In double-ligated segments of the nerve in situ the activity of ChAc decreased at the proximal and increased at the distal end; the activity of AChE rose at both ends. The increase of ChAc activity did not cease until 22 h, whereas that of AChE stopped before 10 h. The intensity of ChAc transport is considerably diminished in the part of axon separated from the nerve cell body. Differences between the behavior of ChAc and AChE are interpreted by the assumption that the axonal transport of ChAc is slow, unidirectional, concerns all of the enzyme in the nerve, and that most of the transported enzyme is not associated with intraaxonal organelles. In contrast to ChAc, the transport of AChE is fast, bidirectional, and concerns a minor proportion of enzyme in the nerve; the transported enzyme is associated with organelles. The rat of proximodistal transport of ChAc is estimated at 4 mm/day (based on the assumption that 100% of the enzyme moves proximo-distally) and that of AChE at 480 mm/day (based on the estimate that 5% of enzyme moved proximo-distally in the present experiments).

Acetylcholinesterase (AChE) ultracytochemistry with thiocholine method was studied in the rat neostriatum. AChE synthesizing neurons were identified by observing reappearance of the enzyme after application of an AChE inhibitor, DFP. Neurons in the neostriatum were classified into 6 types. all such types were demonstrated to synthesize AChE. Two types of efferent neuron (types III and V) have shown a relatively large amount of AChE and resynthesized the enzyme in the earlier stage after DFP. A tentative model of the neuronal network in the neostriatum was schematically shown.

The effects of chronic bombesin (BBS) on [3H]spiperone (SPD) binding activity, choline acetyltransferase (ChAT), acetylcholinesterase (AChE) and glutamate decarboxylase (GAD) were investigated in the rat brain corpus striatum (CS). The chronic i.p. administration of BBS to rats increased: (1) the specific [3H]SPD binding to the striatal Pm (plasma membrane) (16%, P less than 0.03 and 34%, P less than 0.008 at 5 micrograms/kg respectively), (2) the specific GAD activity in the CS by 52% (5 micrograms/kg, n.s.) and 46% (10 micrograms/kg, P less than 0.05) respectively, (3) the specific ChAT activity in the CS by 54% (10 micrograms/kg, P less than 0.002), and (4) the specific AChE activity by 23% (10 micrograms/kg, P less than 0.02) after 14 days. It increased only: (1) the specific [3H]SPD binding by 29% (P less than 0.001, at 10 micrograms/kg) and (2) the specific GAD activity by 23% (P less than 0.015, 10 micrograms/kg), after 7 days. Neither ChAT nor AChE activity was affected after 7 days treatment of BBS at 10 micrograms/kg. In vitro study showed that BBS at 0.2 microM did not affect any of the neurochemical parameters examined in the CS. Thus, the changes in brain chemistry caused by chronic BBS were not due to direct effects of BBS but may be mediated through its metabolites or CCK release. Data indicate that the central effects of peripherally administered BBS are dependent on both the duration and the dosage of the drug treatment and that the dopaminergic and GABAergic systems seem to be more vulnerable to chronic BBS than the cholinergic system in the rat brain CS.(ABSTRACT TRUNCATED AT 250 WORDS)

The projection of the septum to the dentate gyrus has been demonstrated autoradiographically and the pattern of acetylcholinesterase (AChE) staining in the dentate gyrus has been mapped histochemically, in a series of normal young adult rats and in a group of animals in which the entorhinal cortex had been ablated or its efferents to the dentate gyrus interrupted, some weeks earlier. It is clear from this material that the normal disposition of the septal projection to the dentate gyrus differs significantly from the pattern of AChE staining; however, in the denervated region of the molecular layer in the experimental animals there is a marked increase in the density of the septal projection which precisely coincides with the zone of intensification of AChE staining. It follows from this that although the distribution AChE does not accurately reflect the organization of the septo-dentate projection in normal animals, the intensification of AChE staining provides a good indication of the reorganization which occurs in this pathway following entorhinal deafferentation.

Headless cockroaches were trained to hold a metathoracic leg in a raised position in order to avoid electric shock, and acetylcholinesterase (AChE) activity in the metathoracic ganglion was measured. The following factors were varied: age of the animals (nymph or adult), shock size and duration (0.8 mA, 2.0 msec or 0.4 mA, 0.2 msec), method of immobilisation (CO 2 anaesthesia or cooling), position of the shock electrodes on the preparation (both in the leg, or one in the leg and one in the neck), and the treatment following training (none, CO 2 anaesthesia or cooling). All AChE assays were performed both immediately after the ganglion had been homogenised, and again after 24 h. No evidence could be found that AChE is altered by training.

Summary (1) The distributions of true cholinesterase (AChE) and pseudocholinesterase (ChE) in the spinal cord of the rat have been studied histochemically, by means of a thiocholine technique, at the light and electron microscope levels. (2) The somatic motoneurones in the ventral gray column contain high concentrations of AChE and are well demonstrated by the technique; the enzyme occupies the clefts of the rough endoplasmic reticulum and, in places, the nuclear envelope. The motoneurones are grouped into sub-columns which lie at the periphery of the ventral horn, the central portion of which is devoid of AChE-containing cells. Loss of enzyme following sciatic nerve section at mid-thigh level is restricted to the motoneurones in the retrodorsolateral sub-column in the last lumbar and first sacral segments. (3) The thoraco-lumbar part of the intermediolateral gray column contains AChE only, while the sacral part of the column contains both AChE and ChE. The enzyme content of the sacral intermediolateral column is unaffected by sciatic nerve section but is reduced following interruption of pelvic splanchnic nerves. (4) Apart from the neurones in the ventral and intermediolateral gray columns, cholinesterases are also found in certain other varieties of cells. AChE is present in neurones of the intermediomedial column and in cells lying in the lateral white funiculus adjacent to the substantia gelatinosa, while ChE is found in several small neurones scattered in the central portion of the ventral gray horn. These 3 varieties of cells are found throughout the length of the cord and they are all probably propriospinal as they are unaffected by peripheral nerve section.