Ontogenic pattern of dopamine, acetylcholine, and acetylcholinesterase in the brains of normal and hypothyroid rats.

The influence of neonatal thyroidectomy (Tx) on developmental changes in dopamine (DA), acetylcholine (ACh), and acetylcholinesterase (AChE) was studied in the whole brain of rats. In control animals, brain levels of ACh gradually increased and attained adult values at the 70th day. In contrast, AChE activity showed a rapid increase between the 7th and 30th days. Levels of DA were low during the early postnatal life but markedly increased to reach adult values of 1.47 mug/g at the 30th day, after which no further enhancement was noted. Neonatal Tx interfered with the normal growth of the animals, decreased brain weights, and markedly influenced the developmental pattern of both DA and ACh in the brain. The concentration of DA in 30-day-old hypothyroid rats was 46% of the control values. In contrast, brain ACh levels in Tx rats were consistently above those seen in controls, being significantly higher, by 49 and 64%, at 15 and 30 days, respectively. Activity of AChE in brains of hypothyroid animals was not significantly different from that in controls. Treatment of Tx rats with thyroid hormone virtually restored the levels of DA and ACh to values in control animals.     

A selective molecularly imprinted polymer for immobilization of acetylcholinesterase (AChE): an active enzyme targeted and efficient method

In the present study, we immobilized acetylcholinesterase (AChE) enzyme onto acetylcholine removed imprinted polymer and acetylcholine containing polymer. First, the polymers were produced with acetylcholine, substrate of AChE, by dispersion polymerization. Then, the enzyme was immobilized onto the polymers by using two different methods: In the first method (method A), acetylcholine was removed from the polymer, and then AChE was immobilized onto this polymer (acetylcholine removed imprinted polymer). In the second method (method B), AChE was immobilized onto acetylcholine containing polymer by affinity. In method A, enzyme‐specific species (binding sites) occurred by removing acetylcholine from the polymer. The immobilized AChE reached 240% relative specific activity comparison with free AChE because the active enzyme molecules bounded onto the polymer. Transmission electron microscopy results were taken before and after immobilization of AChE for the assessment of morphological structure of polymer. Also, the experiments, which include optimum temperature (25–65°C), optimum pH (3–10), thermal stability (4–70°C), kinetic parameters, operational stability and reusability, were performed to determine the characteristic of the immobilized AChE. Copyright © 2015 John Wiley & Sons, Ltd.     

Cholinesterases preceding major tracts in vertebrate neurogenesis

The role of acetylcholinesterase (AChE) in neurotransmission is well known. But long before synapses are formed in vertebrates, AChE is expressed in young postmitotic neuroblasts that are about to extend the first long tracts. AChE histochemistry can thus be used to map primary steps of brain differentiation. Preceding and possibly inducing AChE in avian brains, the closely related butyrylcholinesterase (BChE) spatially foreshadows AChE-positive cell areas and the course of their axons. In particular, before spinal motor axons grow, their corresponding rostral sclerotomes and myotomes express BChE, and both their neuronal source and myotomal target cells express AChE. Since axon growth has been found inhibited by acetylcholine, it is postulated that both cholinesterases can attract neurite growth cones by neutralizing the inhibitor. Thus, the early expression of both cholinesterases that is at least partially independent from classical cholinergic synaptogenesis, sheds new light on the developmental and medical significance of these enzymes.     

Acetylcholinesterase induces neuronal cell loss,astrocyte hypertrophy and behavioral deficits in mammalian hippocampus

Previous studies have demonstrated that acetylcholinesterase (AChE) promotes the assembly of amyloid-beta-peptides into neurotoxic amyloid fibrils and is toxic for chick retina neuronal cultures and neuroblastoma cells. Moreover, AChE is present in senile plaques in Alzheimer's disease (AD) brains. Here we have studied the effect of AChE on astrocytes and hippocampal neurons in vivo. Morphological as well as behavioral disturbances were analyzed after intrahippocampal injection of AChE. Rats were trained in the Morris water maze and assayed for behavioral parameters. Neuronal cell loss was found in the upper leaf of the dentate gyrus in rats injected with AChE in comparison with control animals. Glial fibrillary acidic protein immunoreactivity showed astrocytic hypertrophy and the magnitude of the response was associated with neuronal cell loss. Behavioral results show that injection of AChE produces cognitive impairment demonstrated by an altered water maze performance including (i) a higher escape latency score, (ii) a decreased spatial acuity and (iii) a shorter time of swimming in the platform quadrant. These findings indicate that a local increment in neuronal AChE concentration at the mammalian hippocampus, such as those present in amyloid deposits, may play a role in triggering neuropathological and behavioral changes such as those observed in AD brains.     

Non-classical actions of cholinesterases: Role in cellular differentiation, tumorigenesis and Alzheimer''s disease

The cholinesterases are members of the serine hydrolase family, which utilizes a serine residue at the active site. Acetylcholinesterase (AChE) is distinguished from butyrylcholinesterase (BChE) by its greater specificity for hydrolysing acetylcholine. The function of AChE at cholinergic synapses is to terminate cholinergic neurotransmission. However, AChE is expressed in tissues that are not directly innervated by cholinergic nerves. AChE and BChE are found in several types of haematopoietic cells. Transient expression of AChE in the brain during embryogenesis suggests that AChE may function in the regulation of neurite outgrowth. Overexpression of cholinesterases has also been correlated with tumorigenesis and abnormal megakaryocytopoiesis. Acetylcholine has been shown to influence cell proliferation and neurite outgrowth through nicotinic and muscarinic receptor-mediated mechanisms and thus, that the expression of AChE and BChE at non-synaptic sites may be associated with a cholinergic function. However, structural homologies between cholinesterases and adhesion proteins indicate that cholinesterases could also function as cell-cell or cell-substrate adhesion molecules. Abnormal expression of AChE and BChE has been detected around the amyloid plaques and neurofibrillary tangles in the brains of patients with Alzheimer's disease. The function of the cholinesterases in these regions of the Alzheimer brain is unknown, but this function is probably unrelated to cholinergic neurotransmission. The presence of abnormal cholinesterase expression in the Alzheimer brain has implications for the pathogenesis of Alzheimer's disease and for therapeutic strategies using cholinesterase inhibitors.     

Prevention of scopolamine-induced memory deficits by schisandrin B, an antioxidant lignan from Schisandra chinensis in mice

The preventive effect of schisandrin B (Sch B), an antioxidant ingredient of Schisandra chinensis, was studied on scopolamine-induced dementia in mouse. Scopolamine developed oxidative stress in the brain with the decreased levels of antioxidant enzymes and increased nitrite level. At the same time, a significant impairment of learning and memory occurred when evaluated by passive avoidance task (PAT) and Morris water maze (MWM) with concomitant increase of acetylcholinesterase (AChE) activity and decreased acetylcholine levels. Pre-treatment by Sch B (10, 25, 50 mg/kg) effectively prevented scopolamine-induced oxidative stress and improved behavioural tasks. Further, the scopolamine-induced increase in AChE activity was significantly suppressed and the level of acetylcholine was maintained as normal by Sch B treatment. These results suggest that Sch B have protective function against cerebral functional defects such as dementia not only by antioxidant prevention but also exerting its potent cognitive-enhancing activity through modulation of acetylcholine level.     

Parallel measurement of brain acetylcholinesterase and the muscarinic cholinergic receptor in the diagnosis of acute, lethal poisoning by anti-cholinesterase pesticides

The activity of acetylcholinesterase (AChE) and the density of muscarinic cholinergic binding receptors (mCBR) were measured in brains from normal Japanese quail (Coturnix coturnix japonica) and from quail after lethal intoxication with diazinon. These were measured in brains from whole heads held at 25 C for 0 to 8 days after death. The maximum relative loss of activity due to post mortem decomposition alone during 8 days was 13% and 10% for AChE and mCBR, respectively. During post mortem decomposition, the ratio of AChE: mCBR activities remained constant at approximately 1.3:1 in normal brains while it was always less than or equal to 0.5:1 after intoxication with diazinon. Normal AChE activity could be estimated from mCBR density. Parallel measurement of AChE and mCBR may assist in the post mortem diagnosis of death due to acute poisoning with anti-cholinesterase pesticides when control specimens are not available.     

Developmental changes in the brain levels of neurotransmitters as influenced by maternal ethanol consumption in the rat.

Abstract— Effects of prolonged maternal ethanol consumption by pregnant and lactating rats were investigated on the cerebral contents of certain neurotransmitters and the activities of their enzymes in fetal and neonatal brains. Ethanol consumption by the pregnant rats resulted in a significant decrease in the fetal brain contents of acetylcholine and an increase in γ-aminobutyric acid and glutamate. The cerebral levels of 5-hydroxytryptamine (5-HT), norepinephrine of choline in the fetal brains from the ethanol group were not significantly different from those observed in the control group. With the increase in gestational age, an increase in the levels of 5-HT, norepinephrine, acetylcholine, γ-amino-butyric acid (GABA) and glutamate was observed in the fetal brains from both ethanol and control groups. The brains of neonates suckling on ethanol-fed mothers showed a significant decrease in acetylcholine levels and an increase in GABA and glutamate levels as compared to the corresponding controls. The activities of L-glutamate decarboxylase and GABA-aminotransferase in the fetal as well as neonatal brains were decreased in the ethanol-fed group as compared to the controls. A single injection of acetaldehyde resulted in a decrease in acetylcholine, GABA and glutamate levels in both neonatal and adult brains.     

Neurite Differentiation Is Modulated in Neuroblastoma Cells Engineered for Altered Acetylcholinesterase Expression

Abstract: Previous observations from several groups suggest that acetylcholinesterase (AChE) may have a role in neural morphogenesis, but not solely by virtue of its ability to hydrolyze acetylcholine. We tested the possibility that AChE influences neurite outgrowth in nonenzymatic ways. With this aim, antisense oligonucleotides were used to decrease AChE levels transiently, and N1E.115 cell lines were engineered for permanently altered AChE protein expression. Cells stably transfected with a sense AChE cDNA construct increased their AChE expression 2.5-fold over the wild type and displayed significantly increased neurite outgrowth. Levels of the differentiation marker, tau, also rose. In contrast, AChE expression in cell lines containing an antisense construct was half of that observed in the wild type. Significant reductions in neurite outgrowth and tau protein accompanied this effect. Overall, these measures correlated statistically with the AChE level ( p < 0.01). Furthermore, treatment of AChE-overexpressing cells with a polyclonal antibody against AChE decreased neurite outgrowth by 43%. We conclude that AChE may have a novel, noncholinergic role in neuronal differentiation.     

The effect of galactose metabolic disorders on rat brain acetylcholinesterase activity

To evaluate whether in classical galactosemia galactose (Gal), galactose-1-phosphate (Gal-1-P) and galactitol (Galtol) affect brain acetylcholinesterase (AChE) activity, various concentrations (1-16 mM) of these compounds were preincubated with brain homogenates of suckling rats as well as with pure eel Electroforus electricus AChE at 37 degrees C for 1 h. Initially, Galtol (up to 2.0 mM) increased (25%) AChE activity which decreased. thereafter, reaching the control value in high Galtol concentrations. Gal-1-P decreased gradually the enzyme activity reaching a plateau (38%), when incubated with 8-16 mM. However, when the usually found 2 mM of Galtol and 2 mM of Gal-1-P, concentrations in galactosemia were added in the incubation mixture simultaneously, brain AChE was stimulated (16%). Galtol or Gal-1-P modulated brain AChE as well as enzyme activity of E.electricus in the same way. Gal, Glucose (Glu) and glucose-1-phosphate (Glu-1-P) had no effect on AChE activity. It is suggested that Galtol as well as Gal-1-P can affect acetylcholine degradation acting directly on AChE molecule. Consequently the direct action of these substances on the enzyme might explain the brain cholinergic dysfunction in untreated galactosemia patients.     

Acetylcholinesterase in Huntington''s and Alzheimer''s Diseases: Simultaneous Enzyme Assay and Immunoassay of Multiple Brain Regions

A newly developed enzyme-linked immunosorbent assay for acetylcholinesterase (AChE) protein was combined with conventional measures of enzyme activity in a study of 15 brain regions from six control cases (non-neurological deaths), six cases of Alzheimer's disease, and six cases of Huntington's disease. In the control brains, the mean AChE activity varied 100-fold from region to region (cortex lowest, striatum highest). The variation in enzyme activity was exactly paralleled by a variation in protein immunoreactivity. Overall, the homospecific activity of AChE averaged 0.26 +/- 0.007 mU/pg, close to the value for electrophoretically homogeneous enzyme isolated from red blood cells. Similar homospecific activities were observed in samples from Huntington's and Alzheimer's brains. Evidently, AChE that is immunoreactive but enzymatically inactive does not accumulate in any of the three conditions examined. Huntington's brain samples showed normal total contents of AChE, but Alzheimer's brains showed significant decreases of both enzyme activity and immunoreactivity in all seven cortical regions and in two out of the eight subcortical structures examined, hippocampus and nucleus accumbens.     

Effects on Cholinergic Markers in Rat Brain and Blood after Short and Prolonged Administration of Donepezil

Donepezil is a selective inhibitor of acetylcholinesterase (AChE) clinically used for treating Alzheimer’s disease. Cholinergic effects after short-term exposure of donepezil (up to 12 h) have been extensively studied in rats, but few have addressed the potential long-term effects. After 14 days administration (1×3 mg/kg, decapitation 4 h after the last injection) the cerebral acetylcholine level was increased by 35% and the AChE activity was decreased by 66% and 32% in brain and blood, respectively. No change was detected in choline acetyltransferase activity, or the levels of vesicular acetylcholine transporter, choline transporter, or muscarinic receptors. Expression of various cholinergic genes was unaffected. Preliminary results of AChE activity in human blood showed 60–97% and 43–89% of pre-exposed level after one and three days of donepezil administration (5 mg daily), respectively. In conclusion, donepezil exposure in rats at doses that do not inhibit brain AChE continuously during the day, will not lead to tolerance development. Special issue dedicated to Professor Simo Oja     

Recovery of the acetylcholinesterase activity in a monolayer culture of chick myoblasts after irreversible inhibition by an organophosphorus inhibitor

The recovery of the acetylcholine esterase (AChE) activity after the irreversible inhibition with an organophosphorus inhibitor B-156 was studied in a developing monolayer culture of chick myoblasts. The culture was obtained from muscles of posterior limbs of the 11 day old chick embryos. The AChE activity was estimated by the modified Ellman method from the moment of inoculation to the stage of spontaneous contractions of muscle fibres. After the B-156 treatment the AChE activity of muscle cells decreased, then started to increase and the maximum recovery of activity, below the initial level, was attained within roughly 2 days after the treatment. The AChE activity in the treated culture somewhat decreased thereafter. The lower the inhibitor concentration, i.e. the lower the value of the initial AChE inhibition, the higher the starting rate and degree of recovery of the AChE activity. The results obtained suggest that, unlike the multilayer culture of muscle tissue at later stages of differentiation no compensatory enhancement of AChE biosynthesis after irreversible inhibition of this enzyme by an organophosphorus inhibitor is observed in the monolayer culture of chick myoblasts at the early stages of myogenesis.     

A protease activity associated with acetylcholinesterase releases the membrane-bound form of the amyloid protein precursor of Alzheimer's disease.

Amyloid deposits in the brains of patients with Alzheimer's disease (AD) contain a protein (beta A4) which is abnormally cleaved from a larger transmembrane precursor protein (APP). APP is believed to be normally released from membranes by the action of a protease referred to as APP secretase. Amyloid deposits have also been shown to contain the enzyme acetylcholinesterase (AChE). In this study, a protease activity associated with AChE was found to possess APP secretase activity, stimulating the release of a soluble 100K form of APP from HeLa cells transfected with an APP cDNA. The AChE-associated protease was strongly and specifically inhibited by soluble APP (10 nM) isolated from human brain. The AChE-associated protease cleaved a synthetic beta A4 peptide at the predicted cleavage site. As AChE is decreased in AD, a deficiency of its associated protease might explain why APP is abnormally processed in AD.     

On Some Structural Analogies between Acetylcholinesterase and the Macromolecular Receptor of Acetylcholine

Several properties of the enzyme acetylcholinesterase (AChE) isolated in vitro are compared with those of the membrane receptor(s) of acetylcholine expressed by the in vivo electrical response of the electroplax membrane. AChE strongly binds in vitro effectors of the electroplax: agonists e.g., decamethonium or antagonists, e.g., d-tubocurarine and flaxedil. It also reacts covalently with an affinity labeling reagent of the acetylcholine receptor site(s) in vivo (TDF). Two classes of sites on AChE molecule account for the binding of these quaternary nitrogen containing compounds: (1) the anionic site of the active center and (2) noncatalytic "peripheral anionic centers" located outside the active center. A disulfide bond breaking agent, dithiothreitol (DTT) alters in a parallel manner the reaction of AChE and the excitable membrane of the electroplax to TDF. The irreversibility of TDF action is lost in both cases, after exposure to DTT. Both AChE and the acetylcholine receptor thus contain disulfide bonds—they are closely related but not necessarily identical proteins.     

Oral Administration of Gintonin Attenuates Cholinergic Impairments by Scopolamine,Amyloid-β Protein,and Mouse Model of Alzheimer’s Disease

Gintonin is a novel ginseng-derived lysophosphatidic acid (LPA) receptor ligand. Oral administration of gintonin ameliorates learning and memory dysfunctions in Alzheimer’s disease (AD) animal models. The brain cholinergic system plays a key role in cognitive functions. The brains of AD patients show a reduction in acetylcholine concentration caused by cholinergic system impairments. However, little is known about the role of LPA in the cholinergic system. In this study, we used gintonin to investigate the effect of LPA receptor activation on the cholinergic system in vitro and in vivo using wild-type and AD animal models. Gintonin induced [Ca²⁺]_i transient in cultured mouse hippocampal neural progenitor cells (NPCs). Gintonin-mediated [Ca²⁺]_i transients were linked to stimulation of acetylcholine release through LPA receptor activation. Oral administration of gintonin-enriched fraction (25, 50, or 100 mg/kg, 3 weeks) significantly attenuated scopolamine-induced memory impairment. Oral administration of gintonin (25 or 50 mg/kg, 2 weeks) also significantly attenuated amyloid-β protein (Aβ)-induced cholinergic dysfunctions, such as decreased acetylcholine concentration, decreased choline acetyltransferase (ChAT) activity and immunoreactivity, and increased acetylcholine esterase (AChE) activity. In a transgenic AD mouse model, long-term oral administration of gintonin (25 or 50 mg/kg, 3 months) also attenuated AD-related cholinergic impairments. In this study, we showed that activation of G protein-coupled LPA receptors by gintonin is coupled to the regulation of cholinergic functions. Furthermore, this study showed that gintonin could be a novel agent for the restoration of cholinergic system damages due to Aβ and could be utilized for AD prevention or therapy.     

The time dependent UV resonance Raman spectra, conformation, and biological activity of acetylcholine analogues upon binding to acetylcholine binding proteins.

In order to obtain quantitative data on the relation between the conformation of acetylcholine and its interaction with biologically significant proteins, a series of acetylcholine analogues with absorption bands in the region 200-300 nm have been synthesized or obtained commercially. Each of these compounds were assayed to measure its activity as an ion channel activator of the nicotinic acetylcholine receptor protein (AChR). In addition, the suitability of some of these compounds as substrates for hydrolysis by acetylcholine esterase (AChE) was determined. One of these analogues, dimethylthionocarbamylcholine (DMTC-Ch), has the ester carbonyl oxygen replaced by a thionyl sulfur. DMTC-Ch has been found to be quite active as an ion channel activator when bound to AChR and was found to react with the enzyme AChE as a suicide substrate. It forms a thionoester of the serine at the AChE active site by an ester exchange reaction that releases the choline as the first product. However, the second or acid product is not released even at pH 7.5 over a period of days. This acetylcholine analog has an absorption band at about 240 nm and exhibits very strong ultraviolet resonance Raman (UVRR) spectra using 239 nm excitation from a frequency modified Nd:YAG laser. This technique allows observation of both conformational changes of the ligand molecule that result in frequency changes as well as changes in the excited state electronic structure that results in changes in the relative intensity of the Raman bands. The time dependence of the UVRR spectrum of the ligand upon binding to both AChE and AChR has been studied from 0.1 msec to minutes. Some time dependence in the conformation of DMTC-Ch upon binding to AChE has been found for very short (0.1-0.5 msec) times. However, no change in the conformation of this neurotransmitter analog is found in the available time range upon binding to AChR. From these data it is concluded that a previous suggestion that acetylcholine has a conformational change upon binding to AChR may be incorrect since the solution behavior of the carbamyl cholines and acetylcholine are similar. Even if acetylcholine does change conformation upont binding to AChR, it is unlikely that such a conformational change plays a significant role in channel activation. We present strong evidence that acetylcholine and its analogues can be active in a variety of conformations.(ABSTRACT TRUNCATED AT 400 WORDS)     

Diet-Induced Changes in AChE Activity after Long-Term Exposure

In the present study we investigated a potential mechanism by which high sugar (HS) and high fat (HF) diets could affect acetylcholinesterase (AChE) activity. The treatment with HS and HF diet was done for six months on male and female rats. The results showed decreased hippocampal AChE activity in male and females receiving HS and HF diets (HS 24% and 36%; HF 38% and 32%, males and females, respectively; P < 0.05). The activity in the cerebral cortex was reduced in males (49 and 40%) and females (19 and 17%) (P < 0.05) on HS and HF diets, respectively. In the hypothalamus AChE activity was decreased on HS diet in males (46%) and female (25%) (P < 0.05) and also on HF diet in males (34%) and females (21%) (P < 0.05). However, in the cerebellum no changes in AChE activity were observed. These results indicate that HS and HF diets produced mainly inhibition in acetylcholine degradation. It probably indicates a chronic alteration induced by these diets on the cholinergic system.     

Postnatal development of nitrergic and cholinergic structures in rat spinal cord

Nitric oxide (NO) is known to be a freely diffusible gaseous neurotransmitter that is not requiring synaptic connection to exert its effects. Nitric oxide synthase (NOS), the enzyme responsible for NO synthesis can be visualised by nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry. Other neurotransmitter is a classical neurotransmitter acetylcholine (ACh), regulated by enzyme acetylcholinesterase (AChE) that hydrolyses the acetylcholine after its releasing. This work is presenting results of histochemical study of the NADPH-d and AChE expression (nitrergic and cholinergic neurons) in the spinal cord (SC) during various periods in its development. Specimens from Wistar rat pups in the age ranging from 1st to 21st postnatal days (P1-P21) have been compared with those of adult rats (P90). Transverse sections of the SC were evaluated by light microscope. In adults, the NADPH-d positivity was detectable in the neurons of superficial and deep layers of the dorsal horn, pericentral area and in the area of preganglionic autonomic nuclei. AChE positive structures were seen in the same locations as previous ones with the exception of two locations: in superficial layers of the dorsal horn AChE staining was absent, while in the ventral horn the groups of AChE positive motoneurons were found. At the perinatal period both NADPH-d and AChE positive neurons were stained from slight to moderate intensity only. During later developmental periods the staining gradually increased and achieved adult level of intensity on the day P21. Our results confirmed the presence of nitrergic and cholinergic neurons in investigated areas of the SC and indicated their fully functioning of NADPH-d and AChE positive structures in SC from the third postnatal week.     

Evolution and ultrastructure of the bovine spermatogonia precursor cell line

We have used a cytochemical technique to investigate the distribution of acetylcholinesterase (AChE) activity in the deutocerebrum of the brain of the sphinx moth Manduca sexta. To distinguish between extra-and intracellular pools of the enzyme, some brains were treated prior to histochemical staining with echothiophate, an irreversible AChE inhibitor which penetrates cell membranes very slowly and, therefore, inhibits only extracellular AChE. In the antennal nerve, fascicles of presumably mechanosensory fibers show echothiophateinsensitive AChE activity. They bypass the antennal lobe and project to the antennal mechanosensory and motor center of the deutocerebrum. In the antennal lobe, fibers in the coarse neuropil, cell bodies in the lateral cell group, and all glomeruli exhibit AChE activity. In most ordinary glomeruli, echothiophate-sensitive AChE activity is concentrated in the outer cap regions, corresponding to the terminal arborizations of olfactory afferents. A previously unrecognized glomerulus in the ventro-median antennal lobe shows uniform and more intense AChE-specific staining that the other glomeruli. No AChE activity appeared to be associated with malespecific pheromone-sensitive afferents in the macro-glomerular complex. About 67 interneurons with somata in the lateral cell group of the antennal lobe show echo-thiophate-insensitive AChE activity. These neurous seem to be members of two types of antennal-lobe projection neurons with fibers passing through the outer-antenno-cerebral tract to the protocerebrum. AChE-stained arborizations of these neurons appear to invade all glomeruli, including three distinguishable subunits of the male-specific macroglomerular complex. In echothiophate-treated animals, the projections of one of these types of fiber form large terminals in the lateral horn of protocerebrum, which partly protrude into the adjacent glial cell layer. The results suggest that extracellularly accessible AChE is associated with ordinary olfactory receptor terminals but apparently not with pheromone-sensitive afferents. Intracellular AChE appears to be present in antennal mechanosensory fibers and in two types of olfactory projection neurons of the antennal lobe. The study provides further evidence for cholinergic neurotransmission of most antennal afferents. The AChE-containing interneurons might be cholinergic as well or use the enzyme for functions unrelated to hydrolysis of acetylcholine.Abbreviations ACh acetylcholine - AChE acetylcholinesterase - AL antennal lobe - AMMC antennal mechanosensory and motor center - ChAT choline acetyltransferase - IACT inner antenno-cerebral tract - MGC macroglomerular complex