Cortical Choline Acetyltransferase Activity Research Articles

Influence of chronic microglial activation and aging on the medial septal cholinergic cell number and morphology

Pathological ageing, involving chronic oxidative and inflammatory stress increases the risk of cognitive degenerative diseases such as dementia, including Alzheimer's disease (AD). Inflammatory stress contributes to unregulated aberrant activation of innate immune system leading to "neuroinflammation". Microglia, the resident immune cells of the CNS, can either trigger chronic neuroinflammatory pathways resulting in gradual neurodegeneration or promote neuroprotection and repair via down regulation of neuroinflammation. Augmented glial activation, accompanied by increased levels of pro-inflammatory cytokines such as IL-6 and TNF-α are biomarkers of ageing and age-related diseases, and shown to contribute to neurodegeneration. AD is characterized by a progressive downregulation of markers within cholinergic basal forebrain (BF) neurons, cholinergic cell loss and diminished cortical choline acetyltransferase (ChAT) activity associated with cognitive decline. While the underlying mechanism leading to cholinergic loss is still unknown, several studies suggest chronic inflammatory processes may contribute to the pathology. Within the BF, the medial septum (MS) provides most of the cholinergic innervations into the hippocampus and the cortex, hence this study investigated the effects of aging and chronic neuroinflammation on the septal cholinergic cell numbers and morphology. 12 and 24 months old GFAP-IL6 (presenting continuous IL6 expression under the GFAP promoter, contributing to chronic microglia activation) and ChATBAC -eGFP (cholinergic neurones tagged by GFP to represent the effect of healthy ageing) mice were used. Immunohistochemistry combined with unbiased stereology were used to estimate the number of Iba1+ microglia and ChAT+ cholinergic cells in the MS. The number of Iba1+ microglia in the MS showed a significant 1.2 and 1.4-fold increase in the GFAP-IL6 mice at 12 and 24 months respectively, compared to WT cohorts. Meanwhile, the number of ChAT+ cells in the MS are significantly reduced by 1.4 and 1.2-folds in the GFAP-IL6 mice at 12 and 24 months respectively, compared to WT cohorts. Differences in microglial and cholinergic cell morphology between GFAP-IL6 and WT was also investigated. These findings demonstrate a potential direct effect of chronic microglia activation on cholinergic cell number in the MS which can exacerbate throughout ageing leading to neurodegenerative impairments.

Chronic Microglial Activation and Ageing Reduces the Numbers of Medial Septal Cholinergic Cells and Alters Their Morphology

AbstractBackgroundPathological ageing is associated with brain inflammation that increases the risk of neurodegenerative disease like Alzheimer’s disease (AD). Chronic activation of microglia, the resident immune cells of the central nervous system (CNS), lead to “neuroinflammation” and result in gradual neurodegeneration. Pro‐inflammatory cytokine interleukin‐6 (IL‐6) is a common early inflammatory marker in AD. Medial septum (MS) of the basal forebrain (BF) provides most of the cholinergic innervations into the hippocampus and the cortex. While BF cholinergic cell loss and reduced cortical choline acetyltransferase (ChAT) activity is associated with cognitive decline, the underlying mechanism leading to cholinergic cell loss is still unknown.MethodAged, 12 (n=5) and 24 (n=5) months GFAP‐IL6 (presenting continuous IL‐6 expression under the control of GFAP promoter, contributing to chronic microglia activation), and aged, 12 (n=5) and 24 (n=5) months ChATBAC‐eGFP (GFP tagged cholinergic neurones to represent the effect of healthy ageing) mice were used. Immunohistochemistry combined with unbiased stereology and 3D morphology analysis were used to estimate the number and morphology of Iba1+ microglia and ChAT+ cholinergic cells, in the MS.ResultStereological estimation of MS microglia number displayed a significant 25% increase GFAP‐IL6 (12M) mice compared to control ChATBAC‐eGFP (12M) mice. A significant 30% increase was obtained for GFAP‐IL6 (24M) mice compared to control (24M) mice. Furthermore, a significant 23% increase in microglia was obtained for GFAP‐IL6 (24M) mice compared to the GFAP‐IL6 (12M) mice. Meanwhile, Cholinergic cell number displayed a significant 31% decrease in GFAP‐IL6 (12M) mice compared to control (12M) mice. While a significant 30% decrease was displayed in GFAP‐IL6 (24M) mice compared to control (24M) mice. There was a tendency for the cholinergic cell number to decrease with ageing among control and GFAP‐IL6 cohorts. A significant change in microglial morphology was also observed with ageing and neuroinflammation, that resembled reactive microglial morphology. Cholinergic cell morphology displayed visible effects of ageing and neuroinflammation with GFAP‐IL‐6 cohorts displaying patterns like neuronal degeneration.ConclusionThese findings demonstrate a potential direct effect of chronic microglia activation on cholinergic cell number in the MS which can exacerbate throughout ageing leading to neurodegenerative impairments.

Butyrylcholinesterase K and Apolipoprotein E-ɛ4 Reduce the Age of Onset of Alzheimer's Disease, Accelerate Cognitive Decline, and Modulate Donepezil Response in Mild Cognitively Impaired Subjects.

Genetic heterogeneity in amnestic mild cognitively impaired (aMCI) subjects could lead to variations in progression rates and response to cholinomimetic agents. Together with the apolipoprotein E4 (APOE-ɛ4) gene, butyrylcholinesterase (BCHE) has become recently one of the few Alzheimer's disease (AD) susceptibility genes with distinct pharmacogenomic properties. To validate candidate genes (APOE/BCHE) which display associations with age of onset of AD and donepezil efficacy in aMCI subjects. Using the Petersen et al. (2005) study on vitamin E and donepezil efficacy in aMCI, we contrasted the effects of BCHE and APOE variants on donepezil drug response using the Alzheimer's Disease Assessment Score-Cognition (ADAS-Cog) scale. Independently, we assessed the effects of APOE/BCHE genotypes on age of onset and cortical choline acetyltransferase activity in autopsy-confirmed AD and age-matched control subjects. Statistical analyses revealed a significant earlier age of onset in AD for APOE-ɛ4, BCHE-K*, and APOE-ɛ4/BCHE-K* carriers. Among the carriers of APOE-ɛ4 and BCHE-K*, the benefit of donepezil was evident at the end of the three-year follow-up. The responder's pharmacogenomic profile is consistent with reduced brain cholinergic activity measured in APOE-ɛ4 and BCHE-K* positive subjects. APOE-ɛ4 and BCHE-K* positive subjects display an earlier age of onset of AD, an accelerated cognitive decline and a greater cognitive benefits to donepezil therapy. These results clearly emphasize the necessity of monitoring potential pharmacogenomic effects in this population of subjects, and suggest enrichment strategies for secondary prevention trials involving prodromal AD subjects.

Development of NMR spectroscopic methods for dynamic detection of acetylcholine synthesis by choline acetyltransferase in hippocampal tissue

Choline acetyltransferase (ChAT) is the key enzyme for acetylcholine (ACh) synthesis and constitutes a reliable marker for the integrity of cholinergic neurons. Cortical ChAT activity is decreased in the brain of patients suffering from Alzheimer's and Parkinson's diseases. The standard method used to measure the activity of ChAT enzyme relies on a very sensitive radiometric assay, but can only be performed on post-mortem tissue samples. Here, we demonstrate the possibility to monitor ACh synthesis in rat brain homogenates in real time using NMR spectroscopy. First, the experimental conditions of the radiometric assay were carefully adjusted to produce maximum ACh levels. This was important for translating the assay to NMR, which has a low intrinsic sensitivity. We then used (15) N-choline and a pulse sequence designed to filter proton polarization by nitrogen coupling before (1) H-NMR detection. ACh signal was resolved from choline signal and therefore it was possible to monitor ChAT-mediated ACh synthesis selectively over time. We propose that the present approach using a labeled precursor to monitor the enzymatic synthesis of ACh in rat brain homogenates through real-time NMR represents a useful tool to detect neurotransmitter synthesis. This method may be adapted to assess the state of the cholinergic system in the brain in vivo in a non-invasive manner using NMR spectroscopic techniques.

Rac1b Increases with Progressive Tau Pathology within Cholinergic Nucleus Basalis Neurons in Alzheimer's Disease

Cholinergic basal forebrain (CBF) nucleus basalis (NB) neurons display neurofibrillary tangles (NFTs) during Alzheimer's disease (AD) progression, yet the mechanisms underlying this selective vulnerability are currently unclear. Rac1, a member of the Rho family of GTPases, may interact with the proapoptotic pan-neurotrophin receptor p75(NTR) to induce neuronal cytoskeletal abnormalities in AD NB neurons. Herein, we examined the expression of Rac1b, a constitutively active splice variant of Rac1, in NB cholinergic neurons during AD progression. CBF tissues harvested from people who died with a clinical diagnosis of no cognitive impairment (NCI), mild cognitive impairment, or AD were immunolabeled for both p75(NTR) and Rac1b. Rac1b appeared as cytoplasmic diffuse granules, loosely aggregated filaments, or compact spheres in p75(NTR)-positive NB neurons. Although Rac1b colocalized with tau cytoskeletal markers, the percentage of p75(NTR)-immunoreactive neurons expressing Rac1b was significantly increased only in AD compared with both mild cognitive impairment and NCI. Furthermore, single-cell gene expression profiling with custom-designed microarrays showed down-regulation of caveolin 2, GNB4, and lipase A in AD Rac1b-positive/p75(NTR)-labeled NB neurons compared with Rac1b-negative/p75(NTR)-positive perikarya in NCI. These proteins are involved in Rac1 pathway/cell cycle progression and lipid metabolism. These data suggest that Rac1b expression acts as a modulator or transducer of various signaling pathways that lead to NFT formation and membrane dysfunction in a subgroup of CBF NB neurons in AD.

Pre- and post-synaptic cortical cholinergic deficits are proportional to amyloid plaque presence and density at preclinical stages of Alzheimer’s disease

Amyloid imaging has identified cognitively normal older people with plaques as a group possibly at increased risk for developing Alzheimer's disease-related dementia. It is important to begin to thoroughly characterize this group so that preventative therapies might be tested. Existing cholinotropic agents are a logical choice for preventative therapy as experimental evidence suggests that they are anti-amyloidogenic and clinical trials have shown that they delay progression of mild cognitive impairment to dementia. A detailed understanding of the status of the cortical cholinergic system in preclinical AD is still lacking, however. For more than 30years, depletion of the cortical cholinergic system has been known to be one of the characteristic features of AD. Reports to date have suggested that some cholinergic markers are altered prior to cognitive impairment while others may show changes only at later stages of dementia. These studies have generally been limited by relatively small sample sizes, long postmortem intervals and insufficient definition of control and AD subjects by the defining histopathology. We, therefore, examined pre- and post-synaptic elements of the cortical cholinergic system in frontal and parietal cortex in 87 deceased subjects, including non-demented elderly with and without amyloid plaques as well as demented persons with neuropathologically confirmed AD. Choline acetyltransferase (ChAT) activity was used as a presynaptic marker while displacement of (3)H-pirenzepine binding by oxotremorine-M in the presence and absence of GppNHp was used to assess postsynaptic M1 receptor coupling. The results indicate that cortical ChAT activity as well as M1 receptor coupling are both significantly decreased in non-demented elderly subjects with amyloid plaques and are more pronounced in subjects with AD and dementia. These findings confirm that cortical cholinergic dysfunction in AD begins at the preclinical stage of disease and suggest that cholinotropic agents currently used for AD treatment are a logical choice for preventative therapy.

Cholinergic system during the progression of Alzheimer’s disease: therapeutic implications

Alzheimer’s disease (AD) is characterized by a progressive phenotypic downregulation of markers within cholinergic basal forebrain (CBF) neurons, frank CBF cell loss and reduced cortical choline acetyltransferase activity associated with cognitive decline. Delaying CBF neurodegeneration or minimizing its consequences is the mechanism of action for most currently available drug treatments for cognitive dysfunction in AD. Growing evidence suggests that imbalances in the expression of NGF, its precursor proNGF and the high (TrkA) and low (p75NTR) affinity NGF receptors are crucial factors underlying CBF dysfunction in AD. Drugs that maintain a homeostatic balance between TrkA and p75NTR may slow the onset of AD. A NGF gene therapy trial reduced cognitive decline and stimulated cholinergic fiber growth in humans with mild AD. Drugs treating the multiple pathologies and clinical symptoms in AD (e.g., M1 cholinoceptor and/or galaninergic drugs) should be considered for a more comprehensive treatment approach for cholinergic dysfunction.

Superior Frontal Cortex Cholinergic Axon Density in Mild Cognitive Impairment and Early Alzheimer Disease

Loss of cortical choline acetyltransferase (ChAT) activity contributes to end-stage Alzheimer disease (AD) dementia. In general, ChAT activity levels are stable in the neocortex in mild to moderate AD (mAD) and there is a selective up-regulation in the superior frontal cortex (SFC) in mild cognitive impairment (MCI), indicating a transient, region-specific cholinergic neuroplastic response. To assess whether a proliferation of cholinergic axons underlies increased ChAT activity levels in the SFC in subjects with MCI. Stereologic principles were applied to assess the density of ChAT-immunoreactive fibers and axon varicosities in SFC tissue obtained postmortem from subjects with no cognitive impairment, MCI, and mAD. Thirty-six subjects enrolled in the Religious Orders Study, with records of annual clinical evaluation for frontal lobe specific and global cognitive functions. Compared with the group with no cognitive impairment, SFC ChAT-immunoreactive fiber and axon varicosity densities were not altered in the MCI group but were significantly reduced in the group with mAD and correlated with impaired frontal lobe and global cognitive function. The lack of an increase in cholinergic axonal innervation of the SFC in MCI suggests that structural reorganization of cholinergic profiles is not the mechanism underlying the transient cholinergic plasticity reported in this region. Furthermore, the stability of cholinergic enzyme activity in mAD is likely the result of a biochemical up-regulation of ChAT protein or enzyme activity levels in the SFC, compensating for decreased regional cholinergic fibers and axon varicosities.

Brain Infusion of Lipopolysaccharide Increases Uterine Growth as a Function of Estrogen Replacement Regimen: Suppression of Uterine Estrogen Receptor-α by Constant, But Not Pulsed, Estrogen Replacement

The effects of estrogen therapy can differ depending on the regimen of estrogen administration. In addition, estrogen can modulate the effects of stressors. To examine the interaction between these systems, we infused adult female rats with lipopolysaccharide (LPS) into the fourth ventricle of the brain for 6 d and compared the effects of constant and pulsed estrogen replacement. Constant, but not pulsed, estrogen treatment reduced estrogen receptor-alpha (ERalpha) protein by 90% in the uterus and increased heat-shock proteins 70 and 90 by 74 and 48%, respectively, whereas progesterone receptor levels increased in all ovariectomized rats receiving estrogen replacement. In contrast to the uterine decline in ERalpha, no changes in ERalpha were observed in the hypothalamus or hippocampus, and ERbeta levels were unchanged in all regions tested. Brain infusion of LPS did not alter these proteins but increased the number of activated microglia in the thalamus and reduced body weight in all rats as well as activated the hypothalamic-pituitary-adrenal axis in ovariectomized rats, as determined by elevations in circulating corticosterone and progesterone. Estrogen treatments did not alter these markers, and no differences were observed in cortical choline acetyltransferase activity or nitrotyrosine for any of the treatment groups. The current study found an unexpected increase in uterine weight in lipopolysaccharide-infused rats treated with constant, but not pulsed, estrogen. This report suggests that constant and pulsed regimens of estrogen administration produce different effects and that stress may be an important factor in the postmenopausal intervention with estrogen.

Galanin Fiber Hypertrophy within the Cholinergic Nucleus Basalis during the Progression of Alzheimer’s Disease

Galanin (GAL)-containing fibers enlarge and hyperinnervate remaining cholinergic basal forebrain (CBF) neurons within the anterior nucleus basalis (NB) in late-stage Alzheimer’s disease (AD). Whether GAL hypertrophy occurs in the CBF in the prodromal or early stages of AD remains unknown. The present study used GAL immunohistochemistry and an unbiased semiquantitative scoring method to evaluate GAL innervation in the anterior NB of subjects clinically diagnosed as having no cognitive impairment, mild cognitive impairment or early-stage (mild/moderate) AD. There was no difference in GAL fiber staining within the anterior NB across the three clinical groups examined. Furthermore, GAL fiber innervation was not correlated with the number of NB neurons expressing the nerve growth factor receptors p75^NTR or TrkA or with cortical choline acetyltransferase activity in the same cases. Single-cell gene expression analysis demonstrated that cholinergic NB neurons express mRNA for the GAL receptors GALR1, GALR2 and GALR3, yet the levels of these mRNAs were unchanged across the three diagnostic groups. These observations indicate that GAL hypertrophy within the anterior NB subfield is a late-stage AD response, which may play a role in regulating the cholinergic tone of remaining basocortical projection neurons.

The presence of the app swe mutation in mice does not increase the vulnerability of cholinergic basal forebrain neurons to neuroinflammation

Neuroinflammation, and elevated levels of inflammatory proteins, such as tumor necrosis factor-α, and the deposition of β-amyloid may interact to contribute to the pathogenesis of Alzheimer's disease. We reproduced a component of the neuroinflammatory state within the basal forebrain cholinergic system, a region that is vulnerable to degeneration in Alzheimer's disease, of transgenic Tg2576 mice that express the Swedish double mutation of the human amyloid precursor protein (APPswe). We have previously shown that basal forebrain cholinergic neurons are selectively vulnerable to the consequences of neuroinflammation. In the current study, tumor necrosis factor-α was infused into the basal forebrain region of APPswe and nontransgenic control mice for 20 days with the expectation that the presence of the transgene would enhance the loss of cholinergic neurons. Chronic infusion of tumor necrosis factor-α significantly decreased cortical choline acetyltransferase activity, reduced the number of choline acetyltransferase-immunoreactive cells and increased the number of activated astrocytes and microglia within the basal forebrain. The presence of the APPswe gene did not enhance the vulnerability of forebrain cholinergic neurons to the chronic neuroinflammation. Furthermore, combined treatment of these mice with memantine demonstrated that the neurotoxic effects of tumor necrosis factor-α upon cholinergic cells did not require the activation of the N-methyl- d-aspartate receptors. In contrast, we have previously shown that memantine was able to provide neuroprotection to cholinergic forebrain neurons from the consequences of exposure to the inflammogen lipopolysaccharide. These results provide insight into the mechanism by which neuroinflammation may selectively target specific neural systems during the progression of Alzheimer's disease.

The toxicity of tumor necrosis factor-α upon cholinergic neurons within the nucleus basalis and the role of norepinephrine in the regulation of inflammation: implications for alzheimer's disease

Inflammation and reduced forebrain norepinephrine are features of Alzheimer's disease that may interact to contribute to the degeneration of specific neural systems. We reproduced these conditions within the basal forebrain cholinergic system, a region that is vulnerable to degeneration in Alzheimer's disease. Tumor necrosis factor-α was infused into the basal forebrain of young mice pretreated with a norepinephrine neuronal toxin, N-(2-chloroethyl)- N-ethyl-2 bromobenzylamine (DSP4), with the expectation that the loss of noradrenergic input would enhance the loss of cholinergic neurons. The results indicate that chronic infusion of tumor necrosis factor-α alone significantly decreased cortical choline acetyltransferase activity and increased the number of activated microglia and astrocytes within the basal forebrain. The loss of forebrain norepinephrine following systemic treatment with DSP4 did not alter the level of cortical choline acetyltransferase activity or activate microglia but significantly activated astrocytes within the basal forebrain. Infusion of tumor necrosis factor-α into DSP4-pretreated mice also reduced cortical choline acetyltransferase activity on the side of the infusion; however, the decline was not significantly greater than that produced by the infusion of tumor necrosis factor-α alone. The neurodegeneration seen may be indirect since a double-immunofluorescence investigation did not find evidence for the co-existence of tumor necrosis factor-α type I receptors on choline acetyltransferase-positive cells in the basal forebrain. The results suggest that noradrenergic cell loss in Alzheimer's disease does not augment the consequences of the chronic neuroinflammation and does not enhance neurodegeneration of forebrain cholinergic neurons.

Rivastigmine antagonizes deficits in prepulse inhibition induced by selective immunolesioning of cholinergic neurons in nucleus basalis magnocellularis

Impairments of cortical cholinergic inputs from the nucleus basalis magnocellularis fundamentally alter information processing and attentional function, thereby advancing the severity of psychopathology in major neuropsychiatric disorders. It was previously shown in adult rats that bilateral 192 IgG saporin-induced selective immunolesioning of the cholinergic neurons in the nucleus basalis produces pronounced and long-lasting deficits in sensorimotor gating measured by prepulse inhibition of the startle reflex. This behavioral paradigm is considered a valid model of sensorimotor gating deficits in the psychotic spectrum and efforts to analyze the significance of the cholinergic basal forebrain in this context are of great interest. In the present study the predictive value of the selective cholinergic immunolesioning model was tested by examining the ability of the cholinesterase inhibitor rivastigmine to restore prepulse inhibition in immunolesioned rats. We report here a pronounced restoring effect of acute (0.75 or 1.5 mg/kg s.c.) as well as repeated (0.75 mg/kg s.c. b.i.d., for 10 days) treatment with rivastigmine in this model of disrupted prepulse inhibition. Intra-nucleus basalis magnocellularis infusions of 192 IgG saporin resulted in extensive loss of basal–cortical cholinergic neurons as shown by the marked decrease in basal telencephalic choline acetyltransferase immunopositive neurons and cortical choline acetyltransferase activity. In this condition, rivastigmine was found to significantly increase cortical acetylcholine extracellular levels in lesioned animals measured by in vivo microdialysis. Taken together, our results strengthen the proposal that the nucleus basalis represents a critical station of the startle gating circuitry. In addition, our findings strongly indicate that even after dramatic decrease of cholinergic neurons, inhibition of acetylcholinesterase restores the cholinergic synaptic function to a point approaching normalization of experimentally induced psychopathology.

Visual hallucinations are associated with lower αbungarotoxin binding in dementia with Lewy bodies

Patients with dementia with Lewy bodies (DLB) commonly experience psychotic symptoms, most notably visual hallucinations. Previously, it has been shown that visual hallucinations in DLB are associated with reduced cortical choline acetyltransferase activity, a marker of cholinergic innervation, but not with predominantly postsynaptic muscarinic M1 receptor binding. In the present investigation, nicotinic acetylcholine receptor (nAChR) levels in the temporal cortex (Brodmann's areas [BA] 20 and 36) were measured in a group of 24 prospectively assessed DLB patients; comparisons were made between groups with or without visual and auditory hallucinations and delusional misidentification. Visual hallucinations and delusional misidentification were associated with lower [ 125I]αbungarotoxin binding in areas 36 and 20 ( P<.05), but not with changes in [ 3H]epibatidine binding. There were no significant associations with auditory hallucinations. [ 3H]epibatidine, but not [ 125I]αbungarotoxin, binding for all DLB cases was reduced compared to controls ( P<.001). Loss of cortical α7 nicotinic receptors may contribute to hallucinations and delusional misidentification in DLB, with implications for treatment and understanding the mechanisms of psychotic symptoms in dementia.

Locomotor and sensorimotor performance deficit in rats following exposure to pyridostigmine bromide, DEET, and permethrin, alone and in combination.

Since their return from Persian Gulf War (PGW), many veterans have complained of symptoms including muscle and joint pain, ataxia, chronic fatigue, headache, and difficulty with concentration. The causes of the symptoms remain unknown. Because these veterans were exposed to a combination of chemicals including pyridostigmine bromide (PB), DEET, and permethrin, we investigated the effects of these agents, alone and in combination, on the sensorimotor behavior and central cholinergic system of rats. Male Sprague-Dawley rats (200-250 gm) were treated with DEET (40 mg/kg, dermal) or permethrin (0.13 mg/kg, dermal), alone and in combination with PB (1.3 mg/kg, oral, last 15 days only), for 45 days. Sensorimotor ability was assessed by a battery of behavioral tests that included beam-walk score, beam-walk time, incline plane performance, and forepaw grip on days 30 and 45 following the treatment. On day 45 the animals were sacrificed, and plasma and CNS cholinesterase, and brain choline acetyl transferase, muscarinic and nicotinic acetylcholine receptors were evaluated. Animals treated with PB, alone or in combination with DEET and permethrin, showed a significant deficit in beam-walk score as well as beam-walk time as compared with controls. Treatment with either DEET or permethrin, alone or in combination with each other, did not have a significant effect on beam-walk score. All chemicals, alone or in combination, resulted in a significant impairment in incline plane testing on days 30 and 45 following treatment. Treatment with PB, DEET, or permethrin alone did not have any inhibitory effect on plasma or brain cholinesterase activities, except that PB alone caused moderate inhibition in midbrain acetylcholinesterase (AChE) activity. Treatment with permethrin alone caused significant increase in cortical and cerebellar AChE activity. A combination of DEET and permethrin or PB and DEET led to significant decrease in AChE activity in brainstem and midbrain and brainstem, respectively. A significant decrease in brainstem AChE activity was observed following combined exposure to PB and permethrin. Coexposure with PB, DEET, and permethrin resulted in significant inhibition in AChE in brainstem and midbrain. No effect was observed on choline acetyl transferase activity in brainstem or cortex, except combined exposure to PB, DEET, and permethrin caused a slight but significant increase in cortical choline acetyltransferase activity. Treatment with PB, DEET, and permethrin alone caused a significant increase in ligand binding for m2 muscarinic acetylcholine receptor (mAChR) in the cortex. Coexposure to PB, DEET, and permethrin did not have any effect over that of PB-induced increase in ligand binding. There was no significant change in ligand binding for nicotinic acetylcholine receptor (nAChR) associated with treatment with the chemical alone; a combination of PB and DEET or coexposure with PB, DEET, and permethrin caused a significant increase in nAChR ligand binding in the cortex. Thus, these results suggest that exposure to physiologically relevant doses of PB, DEET, and permethrin, alone or in combination, leads to neurobehavioral deficits and region-specific alterations in AChE and acetylcholine receptors.

The cytotoxicity of chronic neuroinflammation upon basal forebrain cholinergic neurons of rats can be attenuated by glutamatergic antagonism or cyclooxygenase-2 inhibition.

The proinflammagen lipopolysaccharide (LPS) was infused chronically (37 days) into the basal forebrain of rats. The current study determined whether the chronic administration of either a non-competitive N-methyl-D-aspartate- (NMDA-) sensitive receptor antagonist, memantine, or a selective cyclooxygenase-2 (COX2)/lipoxygenase inhibitor, CI987, could provide significant neuroprotection from the cytotoxic effects of LPS-induced neuroinflammation. Chronic LPS infusions decreased cortical choline acetyltransferase activity, which paralleled a decline in the number of choline-acetyltransferase-immunoreactive-cells within the basal forebrain as well as the number of activated resident microglia. The infusions appeared to be selective for cholinergic neurons. Peripheral administration of memantine (i.p.) or CI987 (s.c.) significantly attenuated the cytotoxic effects of the chronic inflammatory processes upon cholinergic cells within the basal forebrain. However, only CI987 attenuated the neuroinflammation produced by LPS and the subsequent changes in microglial activation. These results indicate that the cytotoxic effects of chronic neuroinflammation may involve prostanoid synthesis and may operate through NMDA receptors, and that the effects of prostaglandins occur upstream to NMDA-receptor activation.

Apolipoprotein E-deficient mice are not more susceptible to the biochemical and memory deficits induced by nucleus basalis lesion

We investigated whether the nucleus basalis lesion induced by quisqualic acid was associated with a more severe impairment of spatial navigation in a water maze, a greater reduction in frontal choline acetyltransferase activity and decrease in the number of choline acetyltransferase-positive neurons in the nucleus basalis in apolipoprotein E-deficient mice than in control mice. We also studied the effect of ageing on water maze spatial navigation and cortical choline acetyltransferase activity in 16-month-old control and apolipoprotein E-deficient mice. We found that the lesion decreased choline acetyltransferase-positive neurons in the nucleus basalis and frontal choline acetyltransferase activity equally in control and apolipoprotein E-deficient mice. The nucleus basalis lesion had no effect on the initial acquisition in the water maze in control and apolipoprotein E-deficient mice after 25 or 106 days of recovery. However, the nucleus basalis lesion impaired the reversal learning in the water maze similarly in both strains after 25 days of recovery, but had no effect after 106 days of recovery. Finally, water maze spatial navigation and cortical choline acetyltransferase activity were similar in old control and apolipoprotein E-deficient mice. These results suggest that young and old apolipoprotein E-deficient mice do not have impairments in cholinergic activity or spatial navigation. Furthermore, apolipoprotein E deficiency does not increase the sensitivity to cholinergic and spatial navigation deficits induced by lesioning of the nucleus basalis with an excitatory amino acid and does not slow down the behavioral recovery.

Synergistic effects of tetrahydroaminoacridine and lithium on cholinergic function after excitotoxic basal forebrain lesions in rat.

Effects of lithium and tetrahydroaminoacridine (THA), either alone or in combination, were tested in an animal model of excitotoxic cholinergic deafferentation of the cerebral cortex. Rats received ibotenic acid lesions of cholinergic basal forebrain nuclei resulting in a 30% to 40% depletion of both cortical choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activity. Lithium as well as THA, given separately either prior or subsequently to the development of the lesion, had small but significant effects on the recovery of cortical ChAT and AChE activity. Applied in combination, these drugs clearly showed synergistic effects. These potentiating actions might be due to neuroprotective/ neurotrophic mechanisms as well as to effects on acetylcholine turnover and muscarinic receptor-coupled phosphoinositide turnover. Similar approaches of combination therapy might prove useful for the management of mental disorders associated with cholinergic dysfunction.

Effect of 192IgG-saporin injections into the nucleus basalis magnocellularis on acquisition and performance of a go/no-go procedure in the rat

The effects of injections of low (105 ng) and high (210 ng) doses of the cholinergic-selective neurotoxin 192IgG-saporin (SAP) into the nucleus basalis magnocellularis on both the acquisition and performance of a go/no-go operant task in Long-Evans rats were examined and were correlated with regional changes in choline acetyltransferase (ChAT) activity. The go/no-go procedure rewards a rat for pressing a lever on alternate trials, with intertrial intervals (i.e., delays) of 5, 20, and 40 sec. Over a period of 15 days, normal rats rapidly become proficient in this task, so that the ratio of latency to the first press on the rewarded go trial to latency to press on the nonrewarded no-go trial is significantly less than 1. With injections made prior to acquisition, there was a significant disruptive effect of both doses of SAP that was not delay dependent. In previously trained rats, SAP disrupted performance during a 2-week training period, with the high dose of SAP having a more pronounced effect. A uniform 60% reduction of cortical ChAT activity relative to control was found after either high- or low-SAP injections; however, there was a dose-dependent reduction in hippocampal ChAT activity with SAP treatment (low dose [20%–29%] and high dose [39%–44%]). Thus, the dose-dependent disruption in rats trained prior to injection may be related to combined cortical and hippocampal cholinergic deficits. The presence of an impairment at even the shortest delay suggests that impaired working memory may play only a minor role in the go/no-go deficit produced by disruption of the basal forebrain cholinergic system and that impaired attention may be more important.

Pathological and biochemical consequences of acute and chronic neuroinflammation within the basal forebrain cholinergic system of rats

Inflammatory processes may play a critical role in the degeneration of basal forebrain cholinergic cells that underlies some of the cognitive impairments associated with Alzheimer's disease. In the present study, the proinflammagen lipopolysaccharide, from the cell wall of Gram-negative bacteria, was used to produce inflammation within the basal forebrain of rats. The effects of acute, high-dose injections of lipopolysaccharide (2, 20 or 40 μg) upon basal forebrain chemistry and neuronal integrity were compared with the effects of chronic, low-dose lipopolysaccharide infusions (0.18, 0.25, 1.8 or 5.0 μg/h) for either 14, 37, 74 or 112 days. Acute exposure to lipopolysaccharide decreased cortical choline acetyltransferase activity and the number of immunoreactive choline acetyltransferase-positive cells within a small region of the basal forebrain. Regional levels of five different neuropeptides were unchanged by acute, high-dose lipopolysaccharide injections. Chronic lipopolysaccharide infusions produced (i) a time-dependent, but not dose-dependent, decrease in cortical choline acetyltransferase activity that paralleled a decline in the number of choline acetyltransferase- and p75-immunoreactive cells within the basal forebrain, and (ii) a dense distribution of reactive astrocytes and microglia within the basal forebrain. Chronic neuroinflammation might underlie the genesis of some neuropathological changes associated with normal ageing or Alzheimer's disease.