Kaolin-induced hydrocephalus causes acetylcholinesterase activity dysfunction following hypothalamic damage in infant rats.

In hydrocephalus, the progressive accumulation of cerebrospinal fluid (CSF) causes dilatation of the lateral ventricles affecting the third ventricle and diencephalic structures such as the hypothalamus. These structures play a key role in the regulation of several neurovegetative functions by the production of the hormones. Since endocrine disturbances are commonly observed in hydrocephalic children, we investigated the impact of progressive ventricular dilation on the hypothalamus of infant rats submitted to kaolin-induced hydrocephalus. Seven-day-old infant rats were submitted to hydrocephalus induction by kaolin 20% injection method. After 14 days, the animals were decapitated and brain was collected to analyze mitochondrial function, neuronal activity by acetylcholinesterase (AChE) enzyme, oxidative damage, glial activation, and, neurotransmission-related proteins and anti-apoptotic processes in the hypothalamus. The hydrocephalic animals showed reduction in respiratory rates in the States of phosphorylation (P < 0.01) and non-phosphorylation (P < 0.05); increase in AChE activity in both the cytosol (P < 0.05) and the membrane (P < 0.01); decrease in synaptophysin (P < 0.05) and Bcl-2 (P < 0.05) contents and; increase in protein carbonyl (P < 0.01), GFAP (P < 0.01) and Iba-1 (P < 0.05) levels. The results demonstrate that ventricular dilation causes hypothalamic damage characterized by cholinergic dysfunction and suggests further investigation of the synthesis and secretion of hormones to generate new approaches and to assist in the treatment of hydrocephalic patients with hormonal alterations.

Early growth response-1 regulates acetylcholinesterase and its relation with the course of Alzheimer's disease.

Our previous studies showed that the transcription factor early growth response-1 (EGR1) may play a role in keeping the brain cholinergic function intact in the preclinical stages of Alzheimer's disease (AD). In order to elucidate the mechanisms involved, we first performed data mining on our previous microarray study on postmortem human prefrontal cortex (PFC) for the changes in the expression of EGR1 and acetylcholinesterase (AChE) and the relationship between them during the course of AD. The study contained 49 patients, ranging from non-demented controls (Braak stage 0) to late AD patients (Braak stage VI). We found EGR1-mRNA was high in early AD and decreased in late AD stages, while AChE-mRNA was stable in preclinical AD and slightly decreased in late AD stages. A significant positive correlation was found between the mRNA levels of these two molecules. In addition, we studied the relationship between EGR1 and AChE mRNA levels in the frontal cortex of 3-12-months old triple-transgenic AD (3xTg-AD) mice. EGR1- and AChE-mRNA were lower in 3xTg-AD mice compared with wild-type (WT) mice. A significant positive correlation between these two molecules was present in the entire group and in each age group of either WT or 3xTg-AD mice. Subsequently, AChE expression was determined following up- or down-regulating EGR1 in cell lines and the EGR1 levels were found to regulate AChE at both the mRNA and protein levels. Dual-luciferase assay and electrophoretic mobility shift assay in the EGR1-overexpressing cells were performed to determine the functionally effective binding sites of the EGR1 on the AChE gene promoter. We conclude that the EGR1 can upregulate AChE expression by a direct effect on its gene promoter, which may contribute significantly to the changes in cholinergic function in the course of AD. The 3xTg-AD mouse model only reflects later stage AD.

The preventive effects of ascorbic acid supplementation on locomotor and acetylcholinesterase activity in an animal model of schizophrenia induced by ketamine

ABSTRACT Studies have shown that schizophrenic patients seem to have nutritional deficiencies. Ascorbic acid (AA) has an important antioxidant effect and neuromodulatory properties. The aim of this study was to evaluate the effects of AA on locomotor activity and the acetylcholinesterase activity (AChE) in an animal model of schizophrenia (SZ). Rats were supplemented with AA (0.1, 1, or 10 mg/kg), or water for 14 days (gavage). Between the 9th and 15th days, the animals received Ketamine (Ket) (25 mg/kg) or saline (i.p). After the last administration (30 min) rats were subjected to the behavioral test. Brain structures were dissected for biochemical analysis. There was a significant increase in the locomotor activity in Ket treated. AA prevented the hyperlocomotion induced by ket. Ket also showed an increase of AChE activity within the prefrontal cortex and striatum prevented by AA. Our data indicates an effect for AA in preventing alterations induced by Ket in an animal model of SZ, suggesting that it may be an adjuvant approach for the development of new therapeutic strategies within this psychiatric disorder.

The preventive effects of ascorbic acid supplementation on locomotor and acetylcholinesterase activity in an animal model of schizophrenia induced by ketamine.

Studies have shown that schizophrenic patients seem to have nutritional deficiencies. Ascorbic acid (AA) has an important antioxidant effect and neuromodulatory properties. The aim of this study was to evaluate the effects of AA on locomotor activity and the acetylcholinesterase activity (AChE) in an animal model of schizophrenia (SZ). Rats were supplemented with AA (0.1, 1, or 10 mg/kg), or water for 14 days (gavage). Between the 9th and 15th days, the animals received Ketamine (Ket) (25 mg/kg) or saline (i.p). After the last administration (30 min) rats were subjected to the behavioral test. Brain structures were dissected for biochemical analysis. There was a significant increase in the locomotor activity in Ket treated. AA prevented the hyperlocomotion induced by ket. Ket also showed an increase of AChE activity within the prefrontal cortex and striatum prevented by AA. Our data indicates an effect for AA in preventing alterations induced by Ket in an animal model of SZ, suggesting that it may be an adjuvant approach for the development of new therapeutic strategies within this psychiatric disorder.

An acetylcholinesterase-independent mechanism for neurobehavioral impairments after chronic low level exposure to dichlorvos in rats.

The present study was designed to explore an alternate mechanism of action other than acetylcholinesterase inhibition for the chronic, low-level exposure to dichlorvos, an organophosphate, in vivo. Dichlorvos, at dose of 1.0 as well as 6.0 mg/kg b. wt. for 12 weeks to rats showed impairment in neurobehavioral indices viz. rota rod, passive avoidance and water maze tests. Though higher dose of dichlorvos had a detrimental effect on acetylcholinesterase activity, no significant inhibition was seen with lower dose of dichlorvos for the same period of exposure i.e. 12 weeks. Muscarinic acetylcholine receptor binding studies revealed a decrease in the number of binding sites (B(max)) in low as well as high dose groups but the dissociation constant (K(d)) value was unaffected with both doses of dichlorvos. Use of selective ligands against M(1), M(2) and M(3) receptor subtypes indicated that M(2) is the major receptor subtype being affected by chronic low-level exposure to dichlorvos. Western blot analysis and immunofluorescence studies also confirmed these biochemical findings. Thus, the present study suggests that M(2) receptors may play a major role in the development of neurobehavioral impairments after chronic exposure to dichlorvos.

The effect of the readthrough acetylcholinesterase variant (AChE-R) on uterine muscle and leiomyomas.

Acetylcholine signaling and acetylcholinesterase (AChE) function(s) are pivotal elements in muscle development. The effects of the stimulus-dependent readthrough AChE variant, AChE-R, on leiomyomas and normal myometrium proliferation were assessed in vivo and in vitro. Histological preparations and cell cultures therefrom were obtained during hysterectomies or myomectomies and included both the leiomyoma sample and the adjacent normal uterine muscle as control. In situ hybridization procedures were performed using AChE cRNA probes complementary to the human AChE-R transcript. Antibodies against the AChE-R variant served for immunohistochemical staining. To determine the biological function of AChE-R on the uterine muscle cell cultures, we used a synthetic peptide representing the potentially cleavable morphogenically active C-terminus of AChE-R (ARP). Cell proliferation was assessed using the incorporation of 5'-bromo-2-deoxyuridine (BrDU). Leiomyomas expressed an excess of AChE-R mRNA and the AChE-R protein compared with the normal myometrium. Cell cultures originating from leiomyomas proliferated significantly faster than cultures from the adjacent myometrium (P = 0.027 at BrDU incorporation). Addition of ARP (2-200 nM) caused a dose-dependent decrease in the proliferation of cell cultures from both leiomyomas and the myometrium. The effect on the myometrium reached statistical significance (at 20 and 200 nM, P = 0.02), whereas the variability of the rapidly proliferating primary cultures was high and precluded statistical significance in the leiomyoma cultures. AChE-R is involved in the proliferation of the myometrium. The inhibitory effect of ARP on the myometrium may suggest a future therapeutic role of ARP.

Specification of the structure of oximes able to reactivate tabun-inhibited acetylcholinesterase.

The efficacy of various oximes to reactivate acetylcholinesterase phosphorylated by tabun (O-ethyl-N,N-dimethyl phosphoramidocyanidate) was tested by in vitro and in vivo methods. The oximes commonly used for the treatment of acute poisonings with highly toxic organophosphates appeared to be almost ineffective (HI-6, pralidoxime, methoxime) or just slightly effective (obidoxime) against tabun. On the other hand, trimedoxime seemed to be a significantly more efficacious reactivator than the others in the case of tabun poisonings. In vitro, the concentration of trimedoxime corresponding to 1.0 mmol/l was able to reach 50% reactivation of tabun-inhibited brain acetylcholinesterase. Higher reactivating potency of trimedoxime in comparison with the other commonly used oximes was demonstrated by in vivo method, too. In addition, other structural analogues of trimedoxime were found to be efficacious in counteracting tabun-induced acetylcholinesterase inhibition although not as efficacious as trimedoxime itself. Some effective acetylcholinesterase reactivators were characterised by dissociation constant of enzyme-reactivator complex as well as enzyme-inhibitor-reactivator complex and by rate constant of reactivation.

Estradiol decreases the acetylcholine-elicited airway reactivity in ovariectomized rats through an increase in epithelial acetylcholinesterase activity.

Estrogen replacement therapy (ERT) is frequently prescribed for postmenopausal women. Epidemiological data suggest that sex hormones may play a role in the expression of asthma, but the mechanism(s) whereby this influence is mediated remain(s) unclear. To better understand the role of physiologic doses of estrogens in airway function, we tested the hypothesis that 17beta-estradiol (E(2), 10 microg/kg per d for 21 d) given to oophorectomized female rats modifies airway responsiveness to cholinergic agonists, compared with oophorectomized rats given placebo. In vivo, the concentration of inhaled acetylcholine (ACh) required to double pulmonary resistance (EC(200)RL) in anesthetized spontaneously breathing tracheotomized rats was calculated as an index of airway responsiveness. E(2)-treated rats were less responsive to ACh than placebo-treated rats (EC(200)RL, 9.40 +/- 1.48 vs. 1.52 +/- 0.85 mg. ml(-1), respectively). Ex vivo airway responsiveness was evaluated with the cumulative concentration-response curve (CCRC) of isolated tracheal segments. Compared with placebo, E(2) treatment significantly increased the EC(50) of ACh (p = 0.01) but did not alter the CCRC to carbachol. Removing the epithelium or treatment with physostigmine abolished the difference in EC(50) of ACh between the groups. Acetylcholinesterase (AChE) activity of homogenized whole trachea was 1.4-fold greater in the E(2)-treated group compared with placebo (p = 0.02), whereas no difference was found in homogenized epithelium-free trachea. We conclude that E(2) treatment decreases airway responsiveness to ACh in ovariectomized rats at least in part by increasing AChE activity dependent on the presence of the epithelium.

Treatment options for Alzheimer's disease.

An understanding of the basic pathophysiology and molecular mechanisms of Alzheimer's disease is essential to effective treatment of the disease. Despite multiple hypotheses related to the development and progression of Alzheimer's disease, no unifying theory is currently available. Inflammation, oxidation stress, estrogen hormone status, pathways for production of beta-amyloid42, apolipoprotein E state, cholinergic neuron depletion, and head injury are all possible contributors and therefore provide points of intervention or potential intervention in the development and progression of Alzheimer's disease. Thus, this article reviews current therapeutic modalities, including estrogen replacement therapy, Ginkgo biloba, and the two cholinesterase inhibitors approved in the United States, tacrine and donepezil.

Acetylcholinesterase promotes regeneration of neurites in cultured adult neurons of Aplysia.

Aplysia, a marine mollusc, has significant amounts of acetylcholinesterase in its hemolymph, reaching maximum levels in the adults with reproductive maturity [Srivatsan M., et al. (1992) J. comp. Physiol. 162, 29-37]. Since hemolymph of mature Aplysia is neurotrophic to Aplysia neurons in culture [Schacher S. and Proshanski E. (1983) J. Neurosci. 3, 2403-2413], we examined whether acetylcholinesterase is a hemolymph neurotrophic factor. Dopaminergic neurons from the pedal ganglia of young adult Aplysia were maintained in culture in defined medium or defined medium supplemented with hemolymph. After 24 h, neurons in defined medium supplemented with hemolymph were well attached to the substratum and exhibited multiple, long neurites. In contrast, neurons in defined medium alone attached poorly and exhibited one or two short neurites. When acetylcholinesterase was inhibited with a specific, membrane-impermeable inhibitor (1,5-bis(4-allyldimethylammoniumphenyl)-pentan-3-one dibromide) which binds to its catalytic and peripheral anionic sites, the neurotrophic effect of hemolymph was significantly reduced. However, inhibition of the catalytic site alone with membrane impermeable echothiophate still resulted in enhanced neurite growth. An analogue of acetylcholine, carbachol, which is not hydrolysed by acetylcholinesterase, did not interfere with neurite growth when added to the supplemented medium. Acetylcholinesterase isolated from the hemolymph and highly purified human acetylcholinesterase also promoted neurite growth in Aplysia neurons. These results show that i) acetylcholinesterase circulating in the hemolymph promotes neurite growth of adult neurons in culture; ii) the growth promoting action of acetylcholinesterase is independent of its function of hydrolysing acetylcholine and iii) the peripheral anionic site of acetylcholinesterase appears to be involved in neurite regeneration.

Reduction of transiently expressed acetylcholinesterase activity in developing thalamocortical projections does not affect the mature pattern of basal forebrain projections to visual cortex.

Experiments tested the hypothesis that acetylcholinesterase (AChE) activity, expressed transiently in developing thalamocortical projections, serves to limit the growth of basal forebrain cholinergic projections into thalamocortical recipient zones. Newborn rats were subjected to enucleation, a procedure that eliminates transient AChE activity in developing visual cortex. After 3-8 weeks survival, AChE histochemical techniques revealed no alteration in the pattern of AChE positive basal forebrain axons in visual cortex. These data indicate that transient AChE activity in developing sensory cortex does not limit ingrowth of basal forebrain cholinergic axons.

Neonatal infraorbital nerve transection and blockade of axoplasmic transport reduce expression of acetylcholinesterase by thalamocortical axons.

Acetylcholinesterase (AChE) is transiently expressed by ventrobasal thalamic neurons and their axons in the primary somatosensory cortex and this enzyme has been used as a marker for these axons in studies concerned with cortical development and plasticity. The present experiment evaluated the effects of both peripheral nerve transection and blockade of axoplasmic transport upon the expression of AChE in thalamic axon terminals in the primary somatosensory cortex. Both manipulations resulted in marked reductions in the density of AChE in thalamocortical axons and less dramatic decreases in the density of this enzyme in thalamic neurons. These results indicate that AChE may not invariably provide unequivocal information about the distribution of somatosensory thalamocortical axon terminals in developing rodents.

Cholinesterases regulate neurite growth of chick nerve cells in vitro by means of a non-enzymatic mechanism.

Cholinesterases present homologies with some cell adhesion molecules; however, it is unclear whether and how they perform adhesive functions. Here, we provide the first direct evidence showing that neurite growth in vitro from various neuronal tissues of the chick embryo can be modified by some, but not all, anticholinesterase agents. By quantifying the neuritic G4 antigen in tectal cell cultures, the effect of anticholinesterases on neurite growth is directly compared with their cholinesterase inhibitory action. BW 284C51 and ethopropazine, inhibiting acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), respectively, strongly decrease neurite growth in a dose-dependent manner. However, echothiophate which inhibits both cholinesterases, does not change neuritic growth. These quantitative data are supplemented by morphological observations in retinal explant cultures grown on striped laminin carpets, viz., defasciculation of neurite bundles by BW 284C51 and Bambuterol occurs, indicating that these drugs disturb adhesive mechanisms. These data strongly suggest that a) cholinesterases can participate in regulating axonal growth, b) both AChE and BChE can perform such a nonsynaptic function, and c) this function is not the result of the enzyme activity per se, since at least one drug was found that inhibits all cholinesterase activities but not neurite growth. Thus, a secondary site on cholinesterase molecules must be responsible for adhesive functions.

[Acetylcholinesterase activity during the cortical development of the cat brain]. / Actividad de acetilcolinesterasa durante el desarrollo cortical del cerebro de gato.

Acetylcholinesterase (AChe) hydrolyses acetylcholine to choline and acetate, thereby inactivating the neurotransmitter. However, the possible non-cholinergic function of this enzyme has been recently suggested. In the present work, changes in AChe activity during the postnatal development of the cat brain are described, in order to show if the enzyme could play a part in the maturing processes of the CNS. The study was performed in the frontal and parietal cortices, area 17 and areas 18 and 19 as a whole in the 15 and 30 days postnatal stages. Significant increases with age were observed in all the brain areas under study. It is suggested that this enzyme activity play a part in the maturation of the cat brain cortices, possibly in the cholinergic development and/or as a cell growth regulatory factor.

Effects of BIM-22015, an analog of ACTH4-10, on functional recovery after frontal cortex injury.

Male rats, 90-100 days old, with frontal cortex lesions were given either subcutaneous sterile water (SW) as a vehicle control or 1, 10, or 100 micrograms of BIM-22015 every other day for 20 days. Brain-injured subjects tested in the Morris water maze with either 10 micrograms BIM-22015 or SW took significantly more trials than sham-operated rats to locate a submerged platform eight consecutive times within 60 s. The animals given 1 or 100 micrograms BIM-22015 took significantly fewer trials to reach criterion than brain-injured animals in the other drug treatment groups. On a percentage of savings, measured 8 days after reaching criterion, the brain-injured subjects given 1, 10, or 100 micrograms BIM-22015 did not differ from sham-operated rats. In contrast, the brain-injured animals given SW took longer to find the submerged platform than they did during the initial training. To assess long-term effects of the ACTH analog treatment, rats were trained on a delayed spatial alternation task 30 days after receiving the last injection. On this task, brain-injured rats treated with the 10-micrograms dose performed significantly better than those given sterile water. Acetylcholinesterase (AChE)-labeled neurons counted in the nucleus basalis magnocellularis indicated that rats with frontal cortex damage given the 10-micrograms treatment did not differ from the sham controls and had significantly more AChE-positive neurons than injured counterparts treated with SW or 100 micrograms.

Phylogeny of putative cholinergic visual pathways through the pretectum to the hypothalamus in teleost fish.

Three patterns of pretectal organization can be discerned morphologically in teleosts. The taxonomic distribution of these pretectal patterns suggests that the intermediately complex pattern (seen in most teleost groups) has given rise to both the elaborate pattern (seen in percomorphs) and the simple pattern (seen in cyprinids). Two pretectal patterns (intermediately complex and elaborate) form part of similar, homologous visual pathways to the hypothalamus; the third pattern is involved in a nonhomologous pathway to the hypothalamus. Acetylcholinesterase (AChE) histochemistry was used in the present study in order to characterize these pretectal patterns further. It is demonstrated that AChE is a highly selective and reliable interspecific marker for all divisions of the superficial pretectum, the nucleus corticalis, the posterior pretectal nucleus (or nucleus glomerulosus) and portions of the inferior lobe. Therefore, the histochemical data support the hypothesis of a homology between the three patterns of pretectal organization in teleosts. Furthermore, the present data provide a basis for more specific investigations regarding the involvement of acetylcholine as a neurotransmitter within the visual pathways to the hypothalamus in teleosts.

Acetylcholinesterase in the human frontal associative cortex during the period of cognitive development: early laminar shifts and late innervation of pyramidal neurons.

Laminar preferences in fibrillar acetylcholinesterase (AChE) staining change dramatically in the human frontal cortex during the first postnatal year and perikaryal reactivity is found only in non-pyramidal neurons. The AChE reactivity of layer III pyramidal cell bodies and surrounding fibrillar network begins to develop after the first postnatal year, increases gradually and reaches its peak intensity in young adults, displaying a cluster-like arrangement. These data suggest that AChE-rich elements participate in the innervation of cortical associative neurons and layers during the cognitive development in man.

Rat serum contains a developmentally regulated cholinergic inducing activity.

Sympathetic neurons cultured in defined medium do not develop the ability to produce acetylcholine, as do neurons grown with serum supplementation (lacovitti, L., M. I. Johnson, T. H. Joh, and R. P. Bunge (1982) Neuroscience 7:2225-2239; Wolinsky, E. J., S. C. Landis, and P. H. Patterson (1985) J. Neurosci. 5: 1497-1508). The implication that rat serum contains cholinergic inducing activity is further explored here. Dependence of cholinergic induction on serum concentration is demonstrated, and the activity is shown to reside in a macromolecular fraction. Very little cholinergic inducing activity is present in serum obtained from animals younger than 9 postnatal days. This age dependence correlates with the time of transition from noradrenergic to cholinergic transmitter status by the sympathetic innervation of the rat sweat gland in vivo (Landis, S. C., and D. Keefe (1983) Dev. Biol. 98: 349-372).