Cortical Acetylcholinesterase Research Articles

Antioxidant and Acetylcholinesterase Response to Repeated Malathion Exposure in Rat Cerebral Cortex and Hippocampus

The wide use of the organophosphate insecticide malathion is accompanied by the risk of human exposure, especially in developing countries, which underlines the need of basic studies in this area. Some reports have shown that low doses of malathion, in a repeated treatment regimen, are unable to reduce acetylcholinesterase (AChE) activity in the rat brain, in contrast to the inhibitory effect in acute treatment. In order to investigate if AChE activity is affected by repeated low-level malathion administration, female Wistar rats were exposed to malathion (50 and 100 mg/kg, intraperitoneally) for 3 consecutive days. Exposure to malathion 50 mg/kg did not affect AChE activity, as previously observed. Contrary to expectation, 100 mg/kg malathion produced a significant increase in AChE activity in both cerebral cortex and hippocampus. Besides AChE inhibition, malathion may act as a pro-oxidative agent by interfering with antioxidant defences, as shown by a decrease of glutathione peroxidase and glutathione reductase activity in the cerebral cortex (100 mg/kg malathion). These effects are in contrast to response in the hippocampus where the increase in AChE activity correlates positively with the antioxidant defences, while the opposite was found in the cerebral cortex. These data indicate that, with low doses, and after a short period of exposure, malathion induces an up-regulation of AChE activity, a pattern similar to that found in the hippocampus for the antioxidant defences studied. The cerebral cortex was more vulnerable to malathion, as reflected in a decrease of two antioxidant enzymes. This study indicates that (i) alternatively to AChE inhibition, interference with the antioxidant defence system may be another important target for malathion toxicity; (ii) hippocampal and cortical AChE activity in rats can be increased after repeated low-dose malathion exposure. This response suggests the occurrence of a pathophysiological response in order to maintain the homeostasis of the cholinergic system in these cerebral structures.

Effects of tenuifolin extracted from radix polygalae on learning and memory: a behavioral and biochemical study on aged and amnesic mice.

Although normal cognitive changes take place when a person becomes older, aging in humans is generally associated with deterioration of cognitive performance and, in particular, of learning and memory. These cognitive deficits can cause debilitating consequences due to aging. There are a number of herbal medicines which are reported to improve brain function including intelligence. In the present study, improving effects of tenuifolin, extracted from Radix Polygalae (RP), on learning and memory in aged and dysmnesia mice were determined using step-down type passive avoidance test or Y type maze trial. Oral administration of tenuifolin (0.02, 0.04, 0.08 g/kgd(-1), for 15 d) evidently improved the latency and number of errors in aged and dysmnesia mice. The levels of cortical acetylcholine esterase (AChE) activity and hippocampal neurotransmitters in aged mice given tenuifolin (0.02, 0.04, 0.08 g/kgd(-1), for 15 d) were also investigated, and increased levels of norepinephrine (NE), dopamine (DA), decreased activity of AChE were found. However, serotonin (5-HT) had no significant difference from that of aged mice given distilled water. The evident improvement of learning and memory of aged mice is carried out by the effects of tenuifolin on the three stages of memory process, that is, acquisition, consolidation and retrieval. This may do so by relatively increasing the levels of NE, DA in the hippocampus and by decreasing the activity of AChE in the cortex.

Acetylcholine esterase activity in mild cognitive impairment and Alzheimer’s disease

Impairment of cholinergic neurotransmission is a well-established fact in Alzheimer's disease (AD), but there is controversy about its relevance at the early stages of the disease and in mild cognitive impairment (MCI). In vivo positron emission tomography imaging of cortical acetylcholine esterase (AChE) activity as a marker of cholinergic innervation that is expressed by cholinergic axons and cholinoceptive neurons has demonstrated a reduction of this enzyme activity in manifest AD. The technique is also useful to measure the inhibition of cerebral AChE induced by cholinesterase inhibitors for treatment of dementia symptoms. A reduction of cortical AchE activity was found consistently in all studies of AD and in few cases of MCI who later concerted to AD. The in vivo findings in MCI and very mild AD are still preliminary, and studies seem to suggest that cholinergic innervation and AChE as the main degrading enzyme are both reduced, which might result in partial compensation of their effect.

Effects of ibotenate and 192IgG-saporin lesions of the nucleus basalis magnocellularis/substantia innominata on spontaneous sleep and wake states and on recovery sleep after sleep deprivation in rats.

The basal forebrain (BF) is known for its role in cortical and behavioral activation, and has been postulated to have a role in compensatory mechanisms after sleep loss. However, specific neuronal phenotypes responsible for these roles are unclear. We investigated the effects of ibotenate (IBO) and 192IgG-saporin (SAP) lesions of the caudal BF on spontaneous sleep-waking and electroencephalogram (EEG), and recovery sleep and EEG after 6 h of sleep deprivation (SD). Relative to artificial CSF (ACSF) controls, IBO injections decreased parvalbumin and cholinergic neurons in the caudal BF by 43 and 21%, respectively, and cortical acetylcholinesterase staining by 41%. SAP injections nonsignificantly decreased parvalbumin neurons by 11%, but significantly decreased cholinergic neurons by 69% and cortical acetylcholinesterase by 84%. IBO lesions had no effect on sleep-wake states but increased baseline delta power in all states [up to 62% increase during non-rapid eye movement (NREM) sleep]. SAP lesions transiently increased NREM sleep by 13%, predominantly during the dark phase, with no effect on EEG. During the first 12 h after SD, animals with IBO and SAP lesions showed lesser rebound NREM sleep (32 and 77% less, respectively) and delta power (78 and 53% less) relative to ACSF controls. These results suggest that noncholinergic BF neurons promote cortical activation by inhibiting delta waves, whereas cholinergic BF neurons play a nonexclusive role in promoting wake. Intriguingly, these results also suggest that both types of BF neurons play important roles, probably through different mechanisms, in increased NREM sleep and EEG delta power after sleep loss.

Temporal effects of newly developed oximes (K027, K048) on malathion-induced acetylcholinesterase inhibition and lipid peroxidation in mouse prefrontal cortex

The potency of newly developed asymmetric bispyridinium oximes (K027, K048) in reactivating acetylcholinesterase and in eliminating oxidative stress induced by acute exposure to malathion was evaluated in mouse prefrontal cortex using in vivo methods. Malathion (1 g/kg, dissolved in saline) was administered subcutaneously. The asymmetric bispyridinium oximes K027 or K048 (1/4 of LD 50, dissolved in saline, i.p.) were administered immediately after malathion and atropine sulfate (20 mg/kg, dissolved in saline, i.p.). Control group received saline instead of malathion and antidotes. Acetylcholinesterase activity and biochemical parameters related to oxidative stress (glutathione levels, glutathione peroxidase and glutathione reductase activity and lipid peroxidation) were evaluated in mouse prefrontal cortex at two different time points (3 or 24 h after malathion poisoning). Malathion administration markedly inhibited cortical acetylcholinesterase activity (around 55%) at 3 h after malathion challenge and such inhibition was maintained till 24 h after poisoning. Although neither atropine sulfate nor oximes were able to eliminate cortical acetylcholinesterase inhibition at 3 h after malathion poisoning, K027 (in combination with atropine) completely eliminated the inhibitory effect of malathion exposure on cortical acetylcholinesterase activity at 24 h after malathion administration. K048 (in combination with atropine) significantly decreased acetylcholinesterase inhibition at 24 h after malathion poisoning. Even though glutathione levels and glutathione peroxidase and glutathione reductase activities were not affected, malathion administration markedly increased lipid peroxidation in the prefrontal cortex at 24 h after poisoning and the oxime K027 (in combination with atropine) was able to significantly decrease such phenomenon. Thus, our results clearly demonstrate that the newly developed asymmetric bispyridinium oximes K027 and K048 are able to reverse malathion-induced acetylcholinesterase inhibition in mouse prefrontal cortex. Moreover, the ameliorative effect of the oxime K027 on the increased lipid peroxidation observed at 24 h after malathion poisoning suggests a potential link between the hyperstimulation of cholinergic system and oxidative stress in the mouse prefrontal cortex after malathion exposure.

PET imaging of the in vivo brain acetylcholinesterase activity and nicotine binding in galantamine-treated patients with AD

The effect of galantamine treatment on cortical acetylcholinesterase (AChE) activity and nicotinic receptor binding was investigated by positron emission tomography (PET) in 18 patients with mild Alzheimer's disease (AD) in relation to galantamine concentration and the patients’ cognitive performances. The first 3 months of the study was of a randomized double-blind placebo-controlled design, during which 12 patients received galantamine (16–24 mg/day) and 6 patients the placebo, and this was followed by 9 months’ galantamine treatment in all patients. The patients underwent PET examinations to measure cortical AChE activity ( 11C-PMP) and 11C-nicotine binding. Neuropsychological tests were performed throughout the study. Inhibition (30–40%) of cortical AChE activity was observed after 3 weeks to 12 months of galantamine treatment. No significant change in mean cortical 11C-nicotine binding was observed during the study. 11C-Nicotine binding, however, positively correlated with plasma galantamine concentration. Both the changes of AChE activity and 11C-nicotine binding correlated positively with the results of a cognitive test of attention. In conclusion, galantamine caused sustained AChE inhibition for up to 12 months. At the individual level, the in vivo cortical AChE inhibition and 11C-nicotine binding were associated with changes in the attention domain of cognition rather than episodic memory.

Optic nerve transection affects development and use-dependent plasticity in neocortex of the rat: Quantitative acetylcholinesterase imaging

We investigated the effects of neonatal optic nerve transection on cortical acetylcholinesterase (AChE) activity in hooded rats during postnatal development and following behavioral manipulation after weaning. AChE reaction product was quantified on digitized images of histochemically stained sections in layer IV of primary somatic sensory, primary visual and visual association cortex. Rats with optic nerve transection were compared to sham-operated littermates. In all cortical regions of both types of animal, AChE reaction product was increased to peak 2 weeks after birth and decreased thereafter, reaching adult levels at the end of the third postnatal week. During postnatal development, reaction product in primary visual cortex was lower in rats deprived of retinal input than in sham-operated littermates and the area delineated by reaction product was smaller. However, optic nerve transection did not modify the time course of postnatal development or statistically significantly diminish adult levels of AChE activity. Behavioral manipulations after weaning statistically significantly increased enzyme activity in sham-operated rats in all cortical areas examined. Compared with cage rearing, training in a discrimination task with food reward had a greater impact than environmental enrichment. By contrast, in the rats with optic nerve transection enrichment and training resulted in statistically significantly increased AChE activity only in lateral visual association cortex. Our findings provide evidence for intra- and supramodal influences of the neonatal removal of retinal input on neural activity- and use-dependent modifications of cortical AChE activity. The laminar distribution of the AChE reaction product suggests that the observed changes in AChE activity were mainly related to cholinergic basal forebrain afferents. These afferents may facilitate the stabilization of transient connections between the somatic sensory and the visual pathway.

Cortical acetylcholine esterase activity and ApoE4-genotype in Alzheimer disease

Positron emission tomography (PET) studies have demonstrated reduced acetylcholine esterase (AChE) activity as an indicator of cholinergic impairment, which is a main pathogenic process in Alzheimer's disease (AD). The E4 allele of apolipoprotein ɛ (ApoE4) is a major risk factor for AD. We examined the relation between ApoE-genotype and cortical AChE activity. In 19 patients (mean age 64) with mild to moderate AD (mean MMSE 22) PET with C-11-labeled N-methyl-4-piperidyl-acetate (MP4A) was performed and the ApoE4-genotype was determined. Parametric images of AChE hydrolysis were generated using a non-invasive technique and analysed globally and regionally. A neuropsychological battery testing memory, attention, executive functions, visuoconstruction, and language was administered. The mean cortical AChE activity was reduced significantly in both groups compared to reference values. The ApoE4 positive subjects (two homozygotes, nine heterozygotes) had significantly higher ( p < 0.05) AChE levels (MP4A hydrolysis rate 0.0753 ± 0.0088 min −1) than the ApoE4 negative subjects ( n = 8, 0.0654 ± 0.0090 min −1). Both groups were comparable with regard to age (63 versus 65) and dementia severity (MMSE 20 versus MMSE 22). AChE-impairment correlated significantly with the word fluency task ( r = 0.041, p < 0.05) in the ApoE4 negative group only. These results indicate that cortical AChE activity is relatively well preserved in ApoE4 carriers, either by preservation of its cellular expression or as AChE activity in amyloid plaques.

Comparative studies of huperzine A, donepezil, and rivastigmine on brain acetylcholine, dopamine, norepinephrine, and 5-hydroxytryptamine levels in freely-moving rats

To assess the effects of cholinesterase inhibitors huperzine A, donepezil and rivastigmine on cerebral neurotransmitters in the cortex and hippocampus in freely-moving rats. Double-probe cerebral microdialysis and HPLC with electrochemical detection were used to detect neurotransmitters. Our results showed that huperzine A (0.25, 0.5, and 0.75 micromol/kg, po) dose-dependently elevated extracellular acetylcholine (ACh) levels in the medial prefrontal cortex (mPFC) and hippocampus. Oral administration of donepezil (5.4 micromol/kg) or rivastigmine (1 micromol/kg) also elicited significant increases in ACh in the mPFC and hippocampus. The time course of cortical acetylcholinesterase (AChE) inhibition with the 3 inhibitors mirrored the increases of ACh at the same dose. The marked elevation of ACh after oral administration of huperzine A (0.5 micromol/kg) and donepezil (5.4 micromol/kg) was associated with a significantly increased release of dopamine (DA) in the mPFC or hippocampus. None of the 3 inhibitors affected norepinephrine (NE) and 5-hydroxytryptamine (5-HT) levels in the mPFC and hippocampus. The effects of huperzine A and rivastigmine did not depend on the route of administration, but donepezil was less efficacious by the oral route than by ip injection. The ability of huperzine A to increase ACh levels was unchanged when tests were performed after multiple oral administration of the drug at 0.5 micromol/kg, once per day for 30 d. The present findings showed that, in molar terms, huperzine A had similar potency on increasing mPFC ACh and DA levels as compared to the 11- and 2-fold dosages of donepezil and rivastigmine, respectively, and had longer lasting effects after oral dosing.

P1-198: Cortical acetylcholine esterase activity, glucose metabolism, and ApoE4-genotype in Alzheimer disease: A PET study

P1-198: Cortical acetylcholine esterase activity, glucose metabolism, and ApoE4-genotype in Alzheimer disease: A PET study

Measuring Cortical Acetylcholine Esterase Activity by PET in Dementia: Clinical Correlates

The acetylcholine analogue C-11- N -methyl-4-piperidinyl-acetate (MP4A) is a tracer for positron emission tomography (PET) to investigate the integrity of the cerebral cholinergic system. It is a substrate of acetylcholine esterase (AChE), which is associated with cholinergic axons in human cortex. We used this technique to determine whether cortical reductions of AChE activity in dementia are associated with clinical symptoms and progression. We examined the following age-matched subjects: 12 normal controls, 8 mild cognitive impairment (MCI, MMSE 27 ± 2), 10 mild Alzheimer’s disease (AD, MMSE 21 ± 4), 10 Parkinson’s disease dementia (PDD, MMSE 21 ± 6), and 10 non-demented PD (MMSE 27 ± 2). Cortical AChE activity was reduced significantly in all demented subjects and in those four MCI patients, who progressed to dementia within 18 months. The most severe reduction (by 30 ± 5%) was seen in PDD, followed by AD (23 ± 2%) and nondemented PD (12 ± 2%, p p We conclude that a reduction of cortical AChE activity is a common feature of Alzheimer’s and Parkinson’s dementia and appears to be related mainly to impairment of attention. It is present very early during the course of the disease, even at a predementia stage. It is likely to indicate functional impairment of the synapses or of axonal transport of ascending cholinergic neurons.

White matter hyperintensities and cortical acetylcholinesterase activity in parkinsonian dementia

To investigate the relationship between the severity of white matter hyperintensities (WMH) and cortical acetylcholinesterase (AChE) activity in parkinsonian dementia (PDem). PDem (n = 11) and control subjects (n = 14) underwent [11C]methyl-4-piperidinyl propionate (11C-PMP) AChE brain positron emission tomography and magnetic resonance (MR) imaging. Presence of WMH on proton density and T2 MR images was scored using a modified version of the semi-quantitative rating scale by Scheltens et al. [J Neurol Sci114 (1993)]. Analysis demonstrated significantly lower mean cortical (11)C-PMP k3 hydrolysis rates in PDem (-19.9%) when compared with control subjects (P < 0.0001). PDem subjects had higher mean severity of WMH (+20.1%) when compared with control subjects (P < 0.05). When WMH severity was entered into the analysis of variance model, there was no significant co-variate effect on cortical AChE activity (F = 0.24, ns). The concomitant presence of mild to moderate WMH in patients with PDem does not have a significant effect on cortical AChE activity.

Cerebral acetylcholine esterase activity in mild cognitive impairment

Mild cognitive impairment may be an early clinical manifestation of Alzheimer's disease, but there are also patients who remain stable or remit. In-vivo measurements of cortical acetylcholine esterase activity by positron emission tomography have shown that it is reduced in Alzheimer's disease, and we investigated whether there is also a reduction in mild cognitive impairment. A significant reduction was observed in three of eight patients, and a significant association was found with progression to Alzheimer's disease within 18 months. These results suggest that low cortical acetylcholine esterase activity may be an indicator of impending dementia in patients with mild cognitive impairment.

Cognitive correlates of cortical cholinergic denervation in Parkinson’s disease and parkinsonian dementia

We recently reported findings that loss of cortical acetylcholinesterase (AChE) activity is greater in parkinsonian dementia than in Alzheimer's disease (AD). In this study we determined cognitive correlates of in vivo cortical AChE activity in patients with parkinsonian dementia (PDem, n = 11), Parkinson's disease without dementia (PD, n = 13), and in normal controls (NC, n = 14) using N-[(11)C]methyl-piperidin-4-yl propionate ([(11)C]PMP) AChE positron emission tomography (PET). Cortical AChE activity was significantly reduced in the PDem (-20.9%) and PD (-12.7 %) subjects (P < 0.001) when compared with the control subjects. Analysis of the cognitive data within the patient groups demonstrated that scores on the WAIS-III Digit Span, a test of working memory and attention, had most robust correlation with cortical AChE activity (R = 0.61, p < 0.005). There were also significant correlations between cortical AChE activity and other tests of attentional and executive functions, such as the Trail Making and Stroop Color Word tests. There was no significant correlation between cortical AChE activity and duration of motor disease (R = -0.01, ns) or severity of parkinsonian motor symptoms (R = 0.14, ns). We conclude that cortical cholinergic denervation in PD and parkinsonian dementia is associated with decreased performance on tests of attentional and executive functioning.

Cholinergic parameters and the retrieval of learned and re-learned spatial information: A study using a model of Wernicke–Korsakoff Syndrome

This is a factorial (2 × 2 × 2) spatial memory and cholinergic parameters study in which the factors are chronic ethanol, thiamine deficiency and naivety in Morris water maze task. Both learning and retention of the spatial version of the water maze were assessed. To assess retrograde retention of spatial information, half of the rats were pre-trained on the maze before the treatment manipulations of pyrithiamine (PT)-induced thiamine deficiency and post-tested after treatment (pre-trained group). The other half of the animals was only trained after treatment to assess anterograde amnesia (post-trained group). Thiamine deficiency, associated to chronic ethanol treatment, had a significant deleterious effect on spatial memory performance of post-trained animals. The biochemical data revealed that chronic ethanol treatment reduced acetylcholinesterase (AChE) activity in the hippocampus while leaving the neocortex unchanged, whereas thiamine deficiency reduced both cortical and hippocampal AChE activity. Regarding basal and stimulated cortical acetylcholine (ACh) release, both chronic ethanol and thiamine deficiency treatments had significant main effects. Significant correlations were found between both cortical and hippocampal AChE activity and behaviour parameters for pre-trained but not for post-trained animals. Also for ACh release, the correlation found was significant only for pre-trained animals. These biochemical parameters were decreased by thiamine deficiency and chronic ethanol treatment, both in pre-trained and post-trained animals. But the correlation with the behavioural parameters was observed only for pre-trained animals, that is, those that were retrained and assessed for retrograde retention.

Degree of inhibition of cortical acetylcholinesterase activity and cognitive effects by donepezil treatment in Alzheimer’s disease

Objectives: To determine in vivo cortical acetylcholinesterase (AChE) activity and cognitive effects in subjects with mild Alzheimer’s disease (AD, n = 14) prior to and after 12 weeks of donepezil...

Cognitive correlates of alterations in acetylcholinesterase in Alzheimer's disease

We recently reported findings of modest loss of cortical acetylcholinesterase (AChE) activity in patients with overall mild Alzheimer's disease (AD) using N-[ 11C]methyl-pi-peridin-4-yl propionate ([ 11C]PMP) AChE positron emission tomography (PET). To determine cognitive correlates of in vivo cortical AChE activity in patients with mild to moderate AD ( n = 15), and in normal controls (NC, n = 12) using [ 11C]PMP AChE PET imaging. Mean cortical AChE activity in the AD subjects was mildly reduced (−11.1%) compared to the control subjects ( P < 0.05). Analysis of the cognitive data showed that mean cortical AChE activity was significantly associated with performance on a test of attention and working memory (WAIS-III Digit Span, R = 0.46, P = 0.01) but not with tests of delayed short or long-term memory functions. Similar findings were present when the analysis was limited to the temporal cortex. Cortical AChE activity is more robustly associated with functions of attention and working memory compared to performance on primary memory tests in AD.

Comparative effects of huperzine A, donepezil and rivastigmine on cortical acetylcholine level and acetylcholinesterase activity in rats*1

The cholinesterase inhibitors huperzine A, donepezil and rivastigmine were compared for their effects on extracellular acetylcholine concentration and acetylcholinesterase activity in the rat cortex. After i.p. injection, huperzine A (0.25–0.75 μmol/kg), donepezil (2–6 μmol/kg) and rivastigmine (0.75–1.5 μmol/kg) dose-dependently elevated the concentration of acetylcholine. The duration of huperzine A was longest. The time courses of cortical acetylcholinesterase inhibition with middle doses of these agents mirrored the increases of acetylcholine at the same doses. However, acetylcholinesterase inhibition was disproportionately greater after middle dose of rivastigmine than doses of huperzine A and donepezil that increased acetylcholine to a similar extent. Muscle fasciculation appeared only after donepezil with a dose-dependent incidence and intensity. In molar terms, huperzine A was 8- and 2-fold more potent than donepezil and rivastigmine, respectively, in increasing cortical acetylcholine levels, with a longer-lasting effect.

Comparative effects of huperzine A, donepezil and rivastigmine on cortical acetylcholine level and acetylcholinesterase activity in rats

The cholinesterase inhibitors huperzine A, donepezil and rivastigmine were compared for their effects on extracellular acetylcholine concentration and acetylcholinesterase activity in the rat cortex. After i.p. injection, huperzine A (0.25–0.75 μmol/kg), donepezil (2–6 μmol/kg) and rivastigmine (0.75–1.5 μmol/kg) dose-dependently elevated the concentration of acetylcholine. The duration of huperzine A was longest. The time courses of cortical acetylcholinesterase inhibition with middle doses of these agents mirrored the increases of acetylcholine at the same doses. However, acetylcholinesterase inhibition was disproportionately greater after middle dose of rivastigmine than doses of huperzine A and donepezil that increased acetylcholine to a similar extent. Muscle fasciculation appeared only after donepezil with a dose-dependent incidence and intensity. In molar terms, huperzine A was 8- and 2-fold more potent than donepezil and rivastigmine, respectively, in increasing cortical acetylcholine levels, with a longer-lasting effect.

Neurological Deficits Induced by Malathion, Deet, and Permethrin, Alone or in Combination in Adult Rats

Malathion (OO-dimethyl-S-[1,2-carbethoxyethyl]phosphorodithionate), DEET (NN-diethyl-m-toluamide), and permethrin [(±)-cis/trans-3-(2,2-dichloroethenyl)-2,2-dimethylcyclopropane carboxylic acid (3-phenoxyphenyl) methyl ester] are commonly used pesticides. To determine the effects of the dermal application of these chemicals, alone or in combination, the sensorimotor behavior, central cholinergic system, and histopathological alterations were studied in adult male Sprague-Dawley rats following a daily dermal dose of 44.4 mg/kg malathion, 40 mg/kg DEET, and 0.13 mg/kg permethrin, alone and in combination for 30 d. Neurobehavioral evaluations of sensorimotor functions included beam-walking score, beam walk time, inclined plane, and grip response assessments. Twenty-four hours after the last treatment with each chemical alone or in combination all behavioral measures were impaired. The combination of DEET and permethrin, malathion and permethrin, or the three chemicals together resulted in greater impairments in inclined performance than permethrin alone. Only animals treated with a combination of DEET and malathion or with DEET and permethrin exhibited significant increases in plasma butyrlcholinesterase (BChE) activity. Treatment with DEET or permethrin alone, malathion and permethrin, or DEET and permethrin produced significant increases in cortical acetylcholinesterase (AChE) activity. Combinations of malathion and permethrin or of DEET and permethrin produced significant decreases in midbrain AChE activity. Animals treated with DEET alone exhibited a significant increase in cortical m2 muscarinic ACh receptor binding. Quantification of neuron density in the dentate gyrus, CA1 and CA3 subfields of the hippocampus, midbrain, brainstem, and cerebellum revealed significant reductions in the density of surviving neurons with various treatments. These results suggest that exposure to real-life doses of malathion, DEET, and permethrin, alone or in combination, produce no overt signs of neurotoxicity but induce significant neurobehavioral deficits and neuronal degeneration in brain.