Comparative study of the expression of cholinergic system components in the CNS of experimental autoimmune encephalomyelitis mice: Acute vs remitting phase.

Acetylcholine (ACh) is involved in the modulation of the inflammatory response. ACh levels are regulated by its synthesizing enzyme, choline acetyltransferase (ChAT), and by its hydrolyzing enzymes, mainly acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). A more comprehensive understanding of the cholinergic system in experimental autoimmune encephalomyelitis (EAE) disease progression could pave the path for the development of therapies to ameliorate multiple sclerosis (MS). In this work, we analyzed possible alterations of the CNS cholinergic system in the neuroinflammation process by using a MOG-induced EAE mice model. MOG- and vehicle-treated animals were studied at acute and remitting phases. We examined neuropathology and analyzed mRNA expression of ChAT, AChE and the α7 subunit of the nicotinic acetylcholine receptor (α7nAChR), as well as AChE and BuChE enzyme activities, in brain and spinal cord sections during disease progression. The mRNA expression and enzyme activities of these cholinergic markers were up- or down-regulated in many cholinergic areas and other brain areas of EAE mice in the acute and remitting phases of the disease. BuChE was present in a higher proportion of astroglia and microglia/macrophage cells in the EAE remitting group. The observed changes in cholinergic markers expression and cellular localization in the CNS during EAE disease progression suggests their potential involvement in the development of the neuroinflammatory process and may lay the ground to consider cholinergic system components as putative anti-inflammatory therapeutic targets for MS.

Traced on the Timeline: Discovery of Acetylcholine and the Components of the Human Cholinergic System in a Primitive Unicellular Eukaryote Acanthamoeba spp.

Acetylcholine (ACh) is the neurotransmitter of cholinergic signal transduction that affects the target cells via muscarinic (mAChR) and nicotinic (nAChR) cholinergic receptors embedded in the cell membrane. Of the cholinergic receptors that bind to ACh, the mAChRs execute several cognitive and metabolic functions in the human central nervous system (CNS). Very little is known about the origins and autocrine/paracrine roles of the ACh in primitive life forms. With the recent report of the evidence of an ACh binding mAChR1 like receptor in Acanthamoeba spp., it was tempting to investigate the origin and functional roles of cholinergic G-Protein coupled receptors (GPCRs) in the biology of eukaryotes. We inferred the presence of ACh, its synthetic, degradation system, and a signal transduction pathway in an approximately ∼2.0 billion year old primitive eukaryotic cell Acanthamoeba castellanii. Bioinformatics analysis, ligand binding prediction, and docking methods were used to establish the origins of enzymes involved in the synthesis and degradation of ACh. Notably, we provide evidence of the presence of ACh in A. castellanii by colorimetric analysis, which to date is the only report of its presence in this primitive unicellular eukaryote. We show the evidence for the presence of homology of evolutionary conserved key enzymes of the cholinergic system like choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) in A. castellanii spp., which were found to be near identical to their human counterparts. Tracing the origin, functions of ACh, and primeval mAChRs in primitive eukaryotic cells has the potential of uncovering covert cholinergic pathways that can be extended to humans in order to understand the states of cholinergic deficiency in neurodegenerative diseases (ND).

Acute and long-term exposure to chlorpyrifos induces cell death of basal forebrain cholinergic neurons through AChE variants alteration.

Chlorpyrifos (CPF) is one of the most widely used organophosphates insecticides that has been reported to induce cognitive disorders both after acute and repeated administration similar to those induced in Alzheimer's disease (AD). However, the mechanisms through which it induces these effects are unknown. On the other hand, the cholinergic system, mainly basal forebrain cholinergic neurons, is involved in learning and memory regulation, and an alteration of cholinergic transmission or/and cholinergic cell loss could induce these effects. In this regard, it has been reported that CPF can affect cholinergic transmission, and alter AChE variants, which have been shown to be related with basal forebrain cholinergic neuronal loss. According to these data, we hypothesized that CPF could induce basal forebrain cholinergic neuronal loss through cholinergic transmission and AChE variants alteration. To prove this hypothesis, we evaluated in septal SN56 basal forebrain cholinergic neurons, the CPF toxic effects after 24h and 14 days exposure on neuronal viability and the cholinergic mechanisms related to it. This study shows that CPF impaired cholinergic transmission, induced AChE inhibition and, only after long-term exposure, increased CHT expression, which suggests that acetylcholine levels alteration could be mediated by these actions. Moreover, CPF induces, after acute and long-term exposure, cell death in cholinergic neurons in the basal forebrain and this effect is independent of AChE inhibition and acetylcholine alteration, but was mediated partially by AChE variants alteration. Our present results provide a new understanding of the mechanisms contributing to the harmful effects of CPF on neuronal function and viability, and the possible relevance of CPF in the pathogenesis of neurodegenerative diseases.

The effects of postnatal alcohol exposure and galantamine on the context pre-exposure facilitation effect and acetylcholine efflux using in vivo microdialysis.

Fetal alcohol spectrum disorders (FASD) are characterized by damage to multiple brain regions, including the hippocampus, which is involved in learning and memory. The acetylcholine neurotransmitter system provides major input to the hippocampus and is a possible target of developmental alcohol exposure. Alcohol (3.0 g/kg/day) was administered via intubation to male rat pups (postnatal day [PD] 2-10; ethanol-treated [ET]). Controls received a sham intubation (IC) or no treatment (NC). Acetylcholine efflux was measured using in vivo microdialysis (PD 32-35). ET animals were not different at baseline, but had decreased K(+)/Ca(2+)-induced acetylcholine efflux compared to NC animals and an enhanced acetylcholine response to galantamine (acetylcholinesterase inhibitor; 2.0 mg/kg) compared to both control groups. A separate cohort of animals was tested in the context pre-exposure facilitation effect task (CPFE; PD 30-32) following postnatal alcohol exposure and administration of galantamine (2.0 mg/kg; PD 11-30). Neither chronic galantamine nor postnatal alcohol exposure influenced performance in the CPFE task. Using immunohistochemistry, we found that neither alcohol exposure nor behavioral testing significantly altered the density of vesicular acetylcholine transporter or alpha7 nicotinic acetylcholine receptor in the ventral hippocampus (CA1). In the medial septum, the average number of choline acetyltransferase (ChAT+) cells was increased in ET animals that displayed the context-shock association; there were no changes in IC and NC animals that learned the context-shock association or in any animals that were in the control task that entailed no learning. Taken together, these results indicate that the hippocampal acetylcholine system is significantly disrupted under conditions of pharmacological manipulations (e.g., galantamine) in alcohol-exposed animals. Furthermore, ChAT was up­regulated in ET animals that learned the CPFE, which may account for their ability to perform this task. In sum, developmental alcohol exposure may disrupt learning and memory in adolescence via a cholinergic mechanism.

Lack of synaptic vesicle protein SV2B protects against amyloid-ß25₋35-induced oxidative stress, cholinergic deficit and cognitive impairment in mice.

SV2B is a synaptic protein widely distributed throughout the brain, which is part of the complex vesicle protein machinery involved in the regulation of synaptic vesicle endocytosis and exocytosis, and therefore in neurotransmitters release. The aims of the present work were twofold: (1) phenotype SV2B knockout mice (SV2B KO) in a battery of cognitive tests; and (2) examine their vulnerability to amyloid-ß25-35 (Aß25-35) peptide-induced toxicity. SV2B KO mice showed normal learning and memory abilities in absence of Aß25-35 injection. SV2B KO mice were protected against the learning deficits induced after icv injection of an oligomeric preparation of amyloid-ß25-35 peptide, as compared to wild-type littermates (SV2B WT). These mice failed to show Aß25-35-induced impairments in a number of cognitive domains: working memory measured by a spontaneous alternation procedure, recognition memory measured by a novel object recognition task, spatial reference memory assessed in a Morris water-maze, and long-term contextual memory assessed in a inhibitory avoidance task. In addition, SV2B KO mice were protected against Aß25-35-induced oxidative stress and decrease in ChAT activity in the hippocampus. These data suggest that SV2B could be a key modulator of amyloid toxicity at the synaptic site.

Cholinergic degeneration is associated with increased plaque deposition and cognitive impairment in APPswe/PS1dE9 mice.

Cholinergic dysfunction and deposition of plaques containing amyloid ß-peptides (Aß) are two of the characteristics of Alzheimer's disease. Here, we combine APPswe/PS1dE9 (APP/PS1) mice with the cholinergic immunotoxin mu p75-saporin (SAP) to integrate partial basal forebrain cholinergic degeneration and the neuropathology of APP/PS1 mice. By 6 months of age, APP/PS1 mice and wild type littermates (Wt) received intracerebroventricular injection of 0.6 µg SAP (lesion) or PBS (sham). Two months following surgery, APP/PS1 mice treated with SAP were significantly impaired compared to sham treated APP/PS1 mice in a behavioural paradigm addressing working memory. Conversely, the performance of Wt mice was unaffected by SAP treatment. Choline acetyltransferase activity was reduced in the hippocampus and frontal cortex following SAP treatment. The selective effect of a mild SAP lesion in APP/PS1 mice was not due to a more extensive cholinergic degeneration since the reduction in choline acetyltransferase activity was similar following SAP treatment in APP/PS1 mice and Wt. Interestingly, plaque load was significantly increased in SAP treated APP/PS1 mice relative to sham lesioned APP/PS1 mice. Additionally, APP/PS1 mice treated with SAP showed a tendency towards an increased level of soluble and insoluble Aß1-40 and Aß1-42 measured in brain tissue homogenate. Our results suggest that the combination of cholinergic degeneration and Aß overexpression in the APP/PS1 mouse model results in cognitive decline and accelerated plaque burden. SAP treated APP/PS1 mice might thus constitute an improved model of Alzheimer's disease-like neuropathology and cognitive deficits compared to the conventional APP/PS1 model without selective removal of basal forebrain cholinergic neurons.

Treatment with a coinducer of the heat shock response delays muscle denervation in the SOD1-G93A mouse model of amyotrophic lateral sclerosis.

We undertook a longitudinal study of the histological and biochemical changes at the neuromuscular junction (NMJ) in muscles of SOD1-G93A mice. We also assessed these functions in mice treated with a known heat shock protein inducer, arimoclomol. Tissue samples of treated and untreated mSOD mice were analysed for AChE and ChAT enzyme activities as markers of neuromuscular function. Sections of hindlimb muscles (TA, EDL and soleus) were also stained for succinate dehydrogenase and silver cholinesterase activities as well as for immunohistochemistry. Hsp70 levels were also measured from muscle samples using ELISA. Results showed that denervation and nerve sprouting were present at symptom onset in fast muscles, although slow muscles remained fully innervated. Cholinergic enzyme activities were reduced prior to denervation and declined further with disease progression. Reduction of endplate size, a slow to fast shift in muscle phenotype was also observed. Treatment with arimoclomol delayed the appearance of these changes, increased innervation, cholinergic enzyme activities and endplate size and reversed muscle fibre transformation. These beneficial effects of arimoclomol in muscles were accompanied by an increase in Hsp70 expression. In conclusion, our results indicate that pharmacological targeting of muscles at early stages of disease may be a successful strategy to ameliorate disease progression in ALS.

Choline acetyltransferase of the common type immunoreactivity in the rat brain following different heroin treatments: a pilot study.

Previous studies suggest that behavioral consequences of heroin treatment depend on the drug history of the animals and that cholinergic neurotransmission is involved in both behavioral and motor sensitization induced by heroin and other drugs of abuse. Immunohistochemistry, using a recently developed antiserum, specific for choline acetyl-transferase of the common type (cChAT), was applied to four different groups of rats, differing in drug regimens. Two groups of rats were submitted to the same schedule of heroin sensitization and then challenged for vehicle or heroin before sacrifice, obtaining two distinct groups, namely heroin-vehicle (HV) and heroin-heroin (HH). The same challenge was applied to another group of rats, previously submitted to a treatment with vehicle, obtaining other two groups, vehicle-vehicle (VV) and vehicle-heroin (VH), respectively. The number of cChAT-positive neurons is significantly increased (p<0.05) in the diagonal band nuclei (with a consequent increase of cChAT positive fibers in the dentate gyrus) and notably, even not significantly (p>0.05), increased in the nucleus accumbens core of heroin-sensitized rats (HV, HH). Instead, acute heroin treatment significantly increase (p<0.05) the number of cChAT-positive cells in the nucleus accumbens shell of both heroin-naïve (VH) and heroin-sensitized (HH) rats. In heroin-sensitized rats (HV, HH), moreover, staining intensity of cChAT-positive fibers is significantly increased in the dorsal striatum, and basolateral amygdala (p<0.05). Unlikely, cChAT positive fibers in the central amygdala are significantly increased (p<0.05) by acute heroin treatments (VH, HH). The increase of cholinergic fibers in the dentate gyrus of the heroin sensitized rats (HV, HH) seems accompanied by a evident reduction in calretinin immunoreactive neurons in the same area. Our results, in a small group of animals, support the view that cholinergic mechanisms are intimately associated with the development of addictive phenotype. Furthermore, they suggest that cholinergic system is differentially engaged, following different heroin treatments.

Aluminum hydroxide injections lead to motor deficits and motor neuron degeneration.

Gulf War Syndrome is a multi-system disorder afflicting many veterans of Western armies in the 1990-1991 Gulf War. A number of those afflicted may show neurological deficits including various cognitive dysfunctions and motor neuron disease, the latter expression virtually indistinguishable from classical amyotrophic lateral sclerosis (ALS) except for the age of onset. This ALS "cluster" represents the second such ALS cluster described in the literature to date. Possible causes of GWS include several of the adjuvants in the anthrax vaccine and others. The most likely culprit appears to be aluminum hydroxide. In an initial series of experiments, we examined the potential toxicity of aluminum hydroxide in male, outbred CD-1 mice injected subcutaneously in two equivalent-to-human doses. After sacrifice, spinal cord and motor cortex samples were examined by immunohistochemistry. Aluminum-treated mice showed significantly increased apoptosis of motor neurons and increases in reactive astrocytes and microglial proliferation within the spinal cord and cortex. Morin stain detected the presence of aluminum in the cytoplasm of motor neurons with some neurons also testing positive for the presence of hyper-phosphorylated tau protein, a pathological hallmark of various neurological diseases, including Alzheimer's disease and frontotemporal dementia. A second series of experiments was conducted on mice injected with six doses of aluminum hydroxide. Behavioural analyses in these mice revealed significant impairments in a number of motor functions as well as diminished spatial memory capacity. The demonstrated neurotoxicity of aluminum hydroxide and its relative ubiquity as an adjuvant suggest that greater scrutiny by the scientific community is warranted.

Eye drop NGF administration promotes the recovery of chemically injured cholinergic neurons of adult mouse forebrain.

We have recently shown that conjunctivally applied nerve growth factor (NGF) in rats can reach the retina, the optic nerve and the CNS. In the present study, we investigated whether NGF application as collyrium can promote the recovery of chemically injured basal forebrain cholinergic neurons. NGF was administered on the eye of adult male mice previously treated i.c.v. with ibotenic acid to impair cholinergic pathways. Mice were tested in the passive avoidance test, and after 2 weeks of NGF administration were killed and the brains used for structural, biochemical and molecular analyses. The results showed that application of NGF on the eye surface protected choline acetyl transferase levels. These findings strengthen the hypothesis that application of NGF on the eye can represent an alternative delivery route to promote the recovery of brain cells during degeneration, including neurons involved in learning and memory activities.

Altered cholinergic metabolism and muscarinic receptor linked second messenger pathways after chronic exposure to dichlorvos in rat brain.

Chronic dichlorvos exposure (6 mg/kg b.wt/day) for a period of 8 weeks resulted in significant reduction in body weight gain of the male Wistar rats. However, the dietary intake remained unchanged in experimental animals following dichlorvos treatment. Activity of the synthesizing enzyme of acetylcholine (ACh) ie, choline acetyltransferase, was found to be significantly increased and the activity of hydrolyzing enzyme, acetyl cholinesterase (AChE), was inhibited in all the three brain regions studied. Chronic dichlorvos treatment also caused significant reduction in both high affinity (HA) and low affinity (LA) choline uptake (CU), with maximal effect being observed in the brain stem followed by cerebellum and cerebrum. Muscarinic receptor binding was significantly decreased in brain stem and cerebellum as reflected in the decreased receptor number (B(max)), without any change in the binding affinity (K(d)) of the receptors. Dichlorvos treatment caused marked inhibition in cAMP synthesis as indicated by decreased adenylate cyclase activity as well as cAMP levels in cerebrum, cerebellum and brain stem. Our study shows that organophosphates may interact with muscarinic receptor-linked second messenger system and this could be a potential mechanism for the neurotoxic effects observed after repeated exposure to low levels of organophosphates, which are unexplainable on the basis of cholinergic hyperactivity.

Permanent, sex-selective effects of prenatal or adolescent nicotine exposure, separately or sequentially, in rat brain regions: indices of cholinergic and serotonergic synaptic function, cell signaling, and neural cell number and size at 6 months of age.

Nicotine is a neuroteratogen that disrupts neurodevelopment and synaptic function, with vulnerability extending into adolescence. We assessed the permanence of effects in rats on indices of neural cell number and size, and on acetylcholine and serotonin (5HT) systems, conducting assessments at 6 months of age, after prenatal nicotine exposure, adolescent exposure, or sequential exposure in both periods. For prenatal nicotine, indices of cell number and size showed few abnormalities by 6 months, but there were persistent deficits in cerebrocortical choline acetyltransferase activity and hemicholinium-3 binding to the presynaptic choline transporter, a pattern consistent with cholinergic hypoactivity; these effects were more prominent in males than females. The expression of 5HT receptors also showed permanent effects in males, with suppression of the 5HT(1A) subtype and upregulation of 5HT(2) receptors. In addition, cell signaling through adenylyl cyclase showed heterologous uncoupling of neurotransmitter responses. Nicotine exposure in adolescence produced lasting effects that were similar to those of prenatal nicotine. However, when animals were exposed to prenatal nicotine and received nicotine subsequently in adolescence, the adverse effects then extended to females, whereas the net effect in males was similar to that of prenatal nicotine by itself. Our results indicate that prenatal or adolescent nicotine exposure evoke permanent changes in synaptic function that transcend the recovery of less-sensitive indices of structural damage; further, prenatal exposure sensitizes females to the subsequent adverse effects of adolescent nicotine, thus creating a population that may be especially vulnerable to the lasting behavioral consequences of nicotine intake in adolescence.

Nerve growth factor eye drop administrated on the ocular surface of rodents affects the nucleus basalis and septum: biochemical and structural evidence.

It has been shown that conjunctivally applied NGF in rats can reach the retina and optic nerve. Whether topical eye NGF application reaches the central nervous system is not known. In the present study, we have addressed this question. It was found that topical eye NGF application affects brain cells. Time-course studies revealed that repeated NGF application leads to high concentration of this neurotrophins after 6 h and normal levels after 24 h. Our results also showed that topical eye application of NGF causes an enhanced expression of NGF receptors and ChAT immunoreactivity in forebrain cholinergic neurons, suggesting that ocular NGF application could have a functional role on damaged brain cells. The present findings suggest that eye NGF application can represent an alternative route to prevent degeneration of NGF-receptive neurons involved in disorders such as Alzheimer and Parkinson.

Effects of Yukmijihwang-tang derivatives (YMJd) on ibotenic acid-induced amnesia in the rat.

The present study investigates the effects of Yukmijihwang-tang Derivatives (YMJd) on learning and memory through the Morris water maze task and the central cholinergic system of rats with excitotoxic medial septum (MS) lesion. In the water maze test, the animals were trained to find a platform in a fixed position for 6 d and then received a 60-s probe trial in which the platform was removed from the pool on the 7th day. Ibotenic lesion of the MS showed the impaired performance in the Morris water maze test and severe cell losses in the MS, as indicated by decreased choline acetyltransferase-immunoreactivity in the medial septum. Daily administrations of YMJd (100 mg/kg, i.p.) for 21 consecutive days produced significant reversals of ibotenic acid-induced deficit in learning and memory. These treatments also reduced the loss of choline acetyltransferase (ChAT) immunoreactivity in the MS induced by ibotenic acid. These results suggest that impairments of spatial learning and memory might be attributable to the degeneration of septohippocampal cholinergic (SHC) neurons and that YMJd treatment ameliorated learning and memory deficits partly due through neuroprotective effects on the central acetylcholine system. Our studies suggest that YMJd might be useful in the treatment of Alzheimer's disease.

Simvastatin regulates non-neuronal cholinergic activity in T lymphocytes via CD11a-mediated pathways.

Lymphocyte function associated antigen-1 (LFA-1; CD11a/CD18) is an important mediator of leukocyte migration and T cell activation. We previously showed that antithymocyte globulin stimulates an independent, non-neuronal cholinergic system in T cells via LFA-1-mediated pathways, as evidenced by increases in acetylcholine (ACh) synthesis and choline acetyltransferase (ChAT) mRNA expression. The cholesterol-lowering drug simvastatin inhibits LFA-1 signaling by binding to an allosteric site on CD11a (LFA-1 alpha chain), which leads to immunomodulation. In the present study, we investigated whether simvastatin modulates lymphocytic cholinergic activity in T cells. We found that anti-CD11a monoclonal antibody (mAb) increased ChAT activity, ACh synthesis and release, and expression of ChAT and M5 muscarinic ACh receptor mRNA in MOLT-3 cells, a human leukemic T cell line. Simvastatin abolished these anti-CD11a mAb-induced increases in lymphocytic cholinergic activity in a manner independent of its cholesterol-lowering activity. These results indicate that LFA-1 contributes to the regulation of lymphocytic cholinergic activity via CD11a-mediated pathways, and suggest that simvastatin exerts its immunosuppressive effects in part via modification of lymphocytic cholinergic activity.

Neuroprotective effect of citicoline against KA-induced neurotoxicity in the rat retina.

We examined whether citicoline has neuroprotective effect on kainic acid (KA)-induced retinal damage. KA (6 nmol) was injected into the vitreous of rat eyes. Rats were injected intraperitoneally with citicoline (500 mgkg-1, i.p.) twice (09:00 and 21:00) daily for 1, 3 and 7 days after KA-injection. The neuroprotective effects of citicoline were estimated by measuring the thickness of the various retinal layers. In addition, immunohistochemistry was conducted to elucidate the expression of choline acetyltransferase (ChAT) and tyrosine hydroxylase (TH). Morphometric analysis of retinal damage in KA-injected eyes showed a significant cell loss in the inner nuclear layer (INL) and inner plexiform layer (IPL) of the retinas at the 1, 3 and 7 days after KA injection, but not in the outer nuclear layers (ONL). At 1 and 3 days after citicoline treatment, no significant changes were detected in the retinal thickness and immunoreactivities of ChAT and TH. The immunoreactivities of ChAT and TH had almost disappeared in the retina after 7 days of KA injection. However, prolonged citicoline treatment for 7 days significantly attenuated the reduction of retinal thickness and immunoreactivities of ChAT and TH. The present study suggests that treatment with citicoline has neuroprotective effect on the retinal damage due to KA-induced neurotoxicity.

Implanted cannula-mediated repetitive administration of Abeta25-35 into the mouse cerebral ventricle effectively impairs spatial working memory.

Amyloid beta (Abeta) is closely related to the onset of Alzheimer's disease (AD). To construct AD animal models, a bolus administration of a large dose of toxic Abeta into the cerebral ventricles of rodents has been performed in earlier studies. In parallel, a continuous infusion system via an osmotic pump into the cerebral ventricle has been developed to make a rat AD model. In this study, we developed a mouse AD model by repetitive administration of Abeta25-35 via a cannula implanted into the cerebral ventricle. Using this administration system, we reproducibly constructed a mouse with impaired spatial working memory. In accordance with the occurrence of the abnormal mouse behavior, we found that the number of choline acetyltransferase (ChAT)-positive neurons was reduced in paraventricular regions of brains of Abeta25-35-administered mice in a dose-dependent manner. Considering that the repetitive administration of a small dose of toxic Abeta via an implanted cannula leads to a brain status more resembling that of the AD patients than a bolus injection of a large dose of Abeta, and therapeutic as well as toxic agents are able to be repeatedly and reliably administered via an implanted cannula, we concluded that the implanted cannula-bearing AD mouse model is useful for development of new AD therapy.

Galantamine-induced behavioral recovery after sublethal excitotoxic lesions to the rat medial septum.

Clinical trials show beneficial effects of acetylcholinesterase (AChE) inhibitors, including galantamine, on cognitive functions in patients with mild to moderate Alzheimer's disease. Galantamine shows a dual action profile by also acting as an allosteric modulator of nicotinic acetylcholine receptors. Nevertheless, its in vivo mechanism of action is only partly understood. Here, we first established a novel lesion model provoking significant functional impairment of the septo-hippocampal projection system without triggering massive neuronal death in the rat medial septum. Next, we studied whether galantamine, administered in doses of 1 and 3mg/kg post-lesion, promotes functional recovery of spatial navigation behaviors, and affects the output of septal cholinergic projections. Infusion of N-methyl-d-aspartate (NMDA; 30nmol/1microl) in the medial septum resulted in spatial learning deficits associated with significant shrinkage of cholinergic neurons and reduced AChE activity in the hippocampus at 7 days post-lesion. Galantamine treatment alone significantly increased the hippocampal acetylcholine concentration and attenuated the NMDA-induced spatial learning impairment. Galantamine post-treatment also affected NMDA-induced changes in AChE and choline-acetyltransferase activities. In conclusion, our data show that galantamine attenuates experimentally-induced cognitive impairments underscored by mild neuronal damage.

Methanesulfonyl fluoride, an acetylcholinesterase inhibitor, attenuates simple learning and memory deficits in ischemic rats.

Methanesulfonyl fluoride (MSF), a highly selective CNS inhibitor of acetylcholinesterase, has been recently demonstrated to promote improvement in cognitive performance in patients with senile dementia of Alzheimer type. Because a similar cognitive impairment may accompany stroke, we investigated in the present study whether treatment with MSF could produce beneficial effects in adult rats subjected to an experimental stroke model. Sprague-Dawley rats received transient 60 min intraluminal occlusion of the right middle cerebral artery (MCAo) and were given i.p. injections of either MSF (1 mg/kg at 24 and 48 h post-MCAo and 0.3 mg/kg thereafter every other day) or the vehicle, peanut oil, for 4 weeks. Behavioral tests and biochemical assays were performed at 28 days post-surgery. MSF treatment produced about 90% inhibition of acetylcholinesterase in the brain. Ischemic animals that received the vehicle displayed significant elevated body swing biased activity (84.8 +/- 10%) and significantly prolonged acquisition (398 +/- 62 s) and shortened retention (79 +/- 26 s) of the passive avoidance task. Interestingly, while the ischemic animals that received the MSF exhibited elevated body swing biased activity (87.7 +/- 8%), they performed significantly better in the passive avoidance task (255 +/- 36 s and 145 +/- 18 s in acquisition and retention) than the vehicle-treated animals. Moreover, whereas brains from both groups of animals revealed similar extent and degree of cerebral infarction, the MSF-treated ischemic animals showed more intense immunoreactivity, as well as a significantly higher number (10-15% increase) of septal choline acetyltransferase-positive cells than the vehicle-treated ischemic animals. These results show that MSF, possibly by preserving a functional cholinergic system, attenuated stroke-induced deficits in a simple learning and memory task.

Urea enhances the nerve growth factor-induced neuroprotective effect on cholinergic neurons in organotypic rat brain slices.

Cholinergic neurons degenerate in Alzheimer's disease and dementia and neuroprotective substances are of high interest to counteract this cell death. The aim of the present study was to test the effect of urea and the nitric oxide synthetase inhibitor l-thiocitrulline on the survival of cholinergic neurons. Organotypic brain slices of the basal nucleus of Meynert were cultured for 2 weeks in the presence of 1-100 microM urea with or without NGF or other growth factors or with or without 1-10 microM of the NOS inhibitor L-thiocitrulline. A high number of cholinergic neurons survived in the presence of 0.1-100 ng/ml NGF. Urea or L-thiocitrulline alone did not exhibit neuroprotective activity; however, when brain slices were incubated with urea or L-thiocitrulline together with NGF there was a significant potentiating survival effect. Incubation of brain slices with NGF + urea + L-thiocitrulline did not further enhance the number of cholinergic neurons. NGF as well as urea did not stimulate expression of the enzyme choline acetyltransferase pointing to survival promoting effects. Urea did not modulate the NGF binding in PC12 cells indicating that this effect was indirect. It is concluded that urea may play a role as an indirect survival promoting molecule possibly involving the nitric oxide pathway.