Elevated Acetylcholine Levels Research Articles

Neuro-intestinal acetylcholine signalling regulates the mitochondrial stress response in Caenorhabditis elegans

Neurons coordinate inter-tissue protein homeostasis to systemically manage cytotoxic stress. In response to neuronal mitochondrial stress, specific neuronal signals coordinate the systemic mitochondrial unfolded protein response (UPRmt) to promote organismal survival. Yet, whether chemical neurotransmitters are sufficient to control the UPRmt in physiological conditions is not well understood. Here, we show that gamma-aminobutyric acid (GABA) inhibits, and acetylcholine (ACh) promotes the UPRmt in the Caenorhabditis elegans intestine. GABA controls the UPRmt by regulating extra-synaptic ACh release through metabotropic GABAB receptors GBB-1/2. We find that elevated ACh levels in animals that are GABA-deficient or lack ACh-degradative enzymes induce the UPRmt through ACR-11, an intestinal nicotinic α7 receptor. This neuro-intestinal circuit is critical for non-autonomously regulating organismal survival of oxidative stress. These findings establish chemical neurotransmission as a crucial regulatory layer for nervous system control of systemic protein homeostasis and stress responses.

D-Serine Increases Release of Acetylcholine in Rat Submandibular Glands.

d-serine has been observed in submandibular gland tissue in rats, but its functions remain to be clarified. Oral administration of d-serine, but not l-serine, increased its concentrations in the submandibular gland and pilocarpine-induced salivary secretion. In vivo microdialysis was used to collect the d- and l-enantiomers of amino acids from local interstitial fluid in the rat submandibular gland. The proportion of the d-form of serine in interstitial fluid was higher than that in plasma or saliva. Perfusion of the rat submandibular gland with d-serine and l-glutamic acid via the submandibular gland artery resulted in a significant increase in salivary secretion after stimulation of muscarinic receptors with carbachol. In vivo microdialysis applied to the submandibular glands of rats showed that infusion of d-serine along with l-glutamate through the microdialysis probe significantly elevated acetylcholine levels in local interstitial fluids in the submandibular glands of anesthetized rats as compared to that with l-glutamate alone in an N-methyl-d-aspartate receptor glycine site antagonist-sensitive manner. These results indicate that d-serine augments salivary secretion by increasing acetylcholine release in the salivary glands.

Protective Role of Acetate Against Depressive-Like Behaviour Associated with Letrozole-Induced PCOS Rat Model: Involvement of HDAC2 and DNA Methylation.

Polycystic ovarian syndrome (PCOS) is the most common endocrine disorder amongst women of reproductive age. PCOS has been demonstrated to induce depressive-like behaviour. Epigenetic alterations such as histone deacetylation (HDAC) and DNA methylation have been suggested in major depression. However, their effects with respect to neuroinflammation are not clear. This study therefore investigated the pathogenic involvement of epigenetic changes in PCOS-associated depression and the protective role of HDACi, especially acetate. Virgin female Wistar rats (140 ± 10g) were assigned into four groups: the groups received vehicle (control), acetate (200mg/kg), letrozole (1mg/kg) and letrozole plus acetate, respectively. The administrations were done concomitantly by oral gavage for 21days. Treatment with letrozole caused hyperandrogenism, hypoestrogenism, hyperinsulinemia and multiple ovarian cysts/degenerated follicles. In addition, these animals showed depressive-like behaviours and increased expression of HDAC2 and DNA methyltransferase in PFC and hippocampal tissues. Biochemical analyses showed elevated levels of NF-κB, malondialdehyde and acetylcholine (ACH) with glutathione depletion in PFC and hippocampus as well as elevated plasma malondialdehyde and impaired anti-oxidant system in letrozole-treated animals. Histological analysis of PFC and hippocampus showed neurodegeneration in letrozole-treated animals compared with control. However, these alterations were attenuated when treated with acetate. The study demonstrates that PCOS-associated depression is characterised by neuroinflammation and elevated ACH levels, accompanied by increased expression of HDAC2/DNA methyltransferase in PFC and hippocampus. Besides, the study suggests that acetate protects against PCOS-associated depression through suppression of prefrontal and hippocampal DNA methylation and prefrontal but not hippocampal HDAC2 expression.

Effects of Artemisia macrocephala Jacquem on Memory Deficits and Brain Oxidative Stress in Streptozotocin-Induced Diabetic Mice.

Different species of Artemisia have been reported to have therapeutic potential in treating various health disorders, including diabetes and memory dysfunction. The present study was planned to evaluate the effects of Artemisia macrocephala Jacquem crude extract and its subfractions as antiamnesic agents in streptozotocin-induced (STZ) diabetic mice. The in vivo behavioral studies were performed using the Y Maze test and novel object recognition test (NORT) test at doses of 100 and 200 mg/kg of crude extract and 75 and 150 mg/kg of fractions. The in vitro and ex vivo anticholinesterase activities, along with biochemical parameters (superoxide dismutase, catalase, glutathione and lipid peroxidation) in the brain, were evaluated. Blood glucose levels were monitored with a glucometer; crude extract and fractions reduced the glucose level considerably, with some differences in the extent of their efficacies. The crude extract and fractions demonstrated significant inhibitory activity against cholinesterases (AChE and BuChE) in vitro. Crude, chloroform and ethyl acetate extract were found to be more potent than the other fractions, with IC50 of Crd-Am = 116.36 ± 1.48 and 240.52 ± 1.35 µg/mL, Chl-Am = 52.68 ± 1.09 and 57.45 ± 1.39 µg/mL and Et-Am = 75.19 ± 1.02 and 116.58 ± 1.09 µg/mL, respectively. Oxidative stress biomarkers like superoxide dismutase, catalase and glutathione levels were elevated, whereas MDA levels were reduced by crude extract and all fractions with little difference in their respective values. The Y-maze test and novel object recognition test demonstrated declines in memory impairment in groups (n = 6) treated with crude extract and fractions as compared to STZ diabetic (amnesic) group. The most active fraction, Chl-Am, was also subjected to isolation of bioactive compounds; three compounds were obtained in pure state and designated as AB-I, AB-II and AB-III. Overall, the results of the study showed that Artemisia macrocephala Jacquem enhanced the memory impairment associated with diabetes, elevated acetylcholine levels and ameliorated oxidative stress. Further studies are needed to explore the beneficial role of the secondary metabolites isolated in the present study as memory enhancers. Toxicological aspects of the extracts are also important and need to be evaluated in other animal models.

Design, Synthesis and Biological Evaluation of New Cycloalkyl Fused Quinolines Tethered to Isatin Schiff Bases as Cholinesterase Inhibitors.

Alzheimer's disease is now a most prevalent neurodegenerative disease of central nervous system leading to dementia in elderly population. Numerous pathological changes have been associated in the progression of Alzheimer's disease. One of such pathological hypotheses is declined cholinergic activity which eventually leads to cognitive and memory deficits. Inhibition o f cholinesterases will apparently elevate acetyl choline levels which is benefactor on cognitive symptoms of the disease. This manuscript describes the new tacrine derivatives tethered to isatin Schiff bases through alkanoyl linker and screened for cholinesterase inhibitory activity. Tacrine and two more cycloalkyl ring fused quinolones were synthesized and converted to N-cycloalkyl fused quinoline chloroamides. Isatin Schiff bases were also synthesized by the reaction between isatin and substituted aromatic anilines and in subsequent reaction, isatin Schiff bases were reacted with cycloalkyl fused quinolones to afford anticipated compounds 10a-i, 11a-i and 12a-i. All the compounds have been screened for acetyl- and butyrylcholinesterase inhibitory activity and in vivo behavioral studies. Binding interactions of the desired compounds have also been studied by docking them in active site of both cholinesterases. Three compounds 12d, 12e and 12h with propionyl and butyroyl linker between amine and isatin Schiff base scaffold have shown potent acetyl- and butyrylcholinesterase inhibitory activity. However most potent cholinesterase inhibitor was 13d with IC50 value of 0.71±0.004 and 1.08±0.02 μM against acetyl- and butyrylcholinesterases respectively. The hepatotoxicity of potent compounds revealed that the tested compounds were less hepatotoxic than tacrine and also exhibited encouraging in vivo behavioral studies in test animals. Docking studies of all the molecules disclosed close hydrogen bond interactions within the binding site of both cholinesterases. New cycloalkyl fused quinolones tethered with alkanoyl linker to isatin Schiff bases endowed significant and potent cholinesterase inhibitory activities. Few of the compounds have also exhibited lesser hepatotoxicity and all the synthesized compounds were good in behavioral studies. Molecular docking studies also indicated close interactions in active site of cholinesterases.

Potentially inappropriate prescriptions of anticholinergics drugs in Alzheimer's disease patients.

Alzheimer's disease is a common cause of dementia, and is usually treated with medications that elevate acetylcholine levels. The objective of the present study was to identify drugs with anticholinergic properties prescribed to patients diagnosed with Alzheimer's disease in Colombia. A cross-sectional study was carried out in outpatients diagnosed with Alzheimer's disease who were identified from a population database from Colombia, and had been treated with cholinesterase inhibitors and glutamate N-methyl-D-aspartate receptor antagonists. The anticholinergic burden was evaluated using the Anticholinergic Cognitive Burden scale, and patients were classified on a scale of 0-3 points according to anticholinergic potential, and were grouped into those with mild-to-moderate (1-2 points) or high (≥3 points) anticholinergic load. The study included 4134 Alzheimer's disease patients. The mean age was 81.50 ± 8.16 years, and 67.8% were women. At least 22.9% of patients took anticholinergic drugs. Of these, the most frequently prescribed medication was quetiapine (8.6%). Age >85 years was associated with a high risk of having an anticholinergic burden ≥3 points (OR 2.19, 95%CI 1.159-4.162). Potential interactions between cholinesterase inhibitors and anticholinergic drugs were identified in 7.8% of patients. The majority of patients who were prescribed anticholinergic drugs were older women, had a significant total anticholinergic burden and had frequent pharmacological interactions with cholinesterase inhibitors. The use of anticholinergics reduces the clinical effectiveness of antidementia drugs and increases the risk of adverse reactions. Geriatr Gerontol Int 2019; 19: 913-917.

PND50 PRESCRIPTION PATTERN OF ANTICHOLINERGIC DRUGS IN ALZHEIMER'S DISEASE

Alzheimer's disease is a common cause of dementia and is usually treated with medications that elevate acetylcholine levels. The objective of this study was to identify drugs with anticholinergic properties prescribed to patients diagnosed with Alzheimer's disease in Colombia. Design: A cross-sectional study. Setting and participants: the study was conducted in outpatients diagnosed with Alzheimer's disease who were identified from a population database from Colombia and had been treated with cholinesterase inhibitors and glutamate NMDA receptor antagonists. Measures: The anticholinergic burden was evaluated using the Anticholinergic Cognitive Burden (ACB) scale, and patients were classified on a scale of 0-3 points according to anticholinergic potential. The study included 4134 Alzheimer's disease patients. The mean age was 81.50±8.16 years, and 67.8% were women. At least 22.9% of patients took anticholinergic drugs, including 32 different drugs. Of these, the most frequently prescribed medication was quetiapine (8.6%). A total of 78.4% of patients were receiving pharmacological treatment for Alzheimer's disease. Cholinesterase inhibitors were prescribed to 45.9% of patients, while memantine was prescribed for 27.4% of patients. The concomitant use of the two drugs was observed in 5.2% of cases. Age greater than 85 years was associated with a 119% risk of having an anticholinergic burden ≥3 points (OR 2.197, 95% CI: 1.159-4.162, p: 0.016). Potential interactions between cholinesterase inhibitors and anticholinergic drugs were identified in 7.8% of patients. The majority of patients who were prescribed anticholinergic drugs were elderly women, had a significant total anticholinergic burden and had frequent pharmacological interactions with cholinesterase inhibitors. The use of anticholinergics reduces the clinical effectiveness of antidementia drugs and increases the risk of adverse reactions.

A Novel Nitric Oxide Donor, S-Nitroso-NPivaloyl-D-Penicillamine, Activates a Non-Neuronal Cardiac Cholinergic System to Synthesize Acetylcholine and Augments Cardiac Function.

In a previous study, we reported that cardiomyocytes were equipped with non-neuronal cardiac cholinergic system (NNCCS) to synthesize acetylcholine (ACh), which is indispensable for maintaining the basic physiological cardiac functions. The aim of this study was to identify and characterize a pharmacological inducer of NNCCS. To identify a pharmacological inducer of NNCCS, we screened several chemical compounds with chemical structures similar to the structure of S-nitroso-N-acetyl-DL-penicillamine (SNAP). Preliminary investigation revealed that SNAP is an inducer of non-neuronal ACh synthesis. We screened potential pharmacological inducers in H9c2 and HEK293 cells using western blot analysis, luciferase assay, and measurements of intracellular cGMP, NO₂ and ACh levels. The effects of the screened compound on cardiac function of male C57BL6 mice were also evaluated using cardiac catheter system. Among the tested compounds, we selected S-nitroso-Npivaloyl-D-penicillamine (SNPiP), which gradually elevated the intracellular cGMP levels and nitric oxide (NO) levels in H9c2 and HEK293 cells. These elevated levels resulted in the gradual transactivation and translation of the choline acetyltransferase gene. Additionally, in vitro and in vivo SNPiP treatment elevated ACh levels for 72 h. SNPiP-treated mice upregulated their cardiac function without tachycardia but with enhanced diastolic function resulting in improved cardiac output. The effect of SNPiP was dependent on SNPiP nitroso group as verified by the ineffectiveness of N-pivaloyl-D-penicillamine (PiP), which lacks the nitroso group. SNPiP is identified to be one of the important pharmacological candidates for induction of NNCCS.

Neuroprotective effects of 20(S)-protopanaxatriol (PPT) on scopolamine-induced cognitive deficits in mice.

20(S)-protopanaxatriol (PPT), one of the ginsenosides from Panax ginseng, has been reported to have neuroprotective effects and to improve memory. The present study was designed to investigate the protective effect of PPT on scopolamine-induced cognitive deficits in mice. Male Institute of Cancer Research mice were pretreated with 2 different doses of PPT (20 and 40μmol/kg) for 27days by intraperitoneal injection, and scopolamine (0.75mg/kg) was injected intraperitoneally for 9days to induce memory impairment. Thirty minutes after the last pretreatment, the locomotor activity was firstly examined to evaluate the motor function of mice. Then, memory-related behaviors were evaluated, and the related mechanism was further researched. It was founded that PPT treatment significantly reversed scopolamine-induced cognitive impairment in the object location recognition experiment, the Morris water maze test, and the passive avoidance task, showing memory-improving effects. PPT also significantly improved cholinergic system reactivity and suppressed oxidative stress, indicated by inhibition of acetylcholinesterase activity, elevation of acetylcholine levels, increasing superoxide dismutase activity and lowering levels of malondialdehyde in the hippocampus. In addition, the expression levels of Egr-1, c-Jun, and cAMP responsive element binding in the hippocampus were significantly elevated by PPT administration. These results suggest that PPT may be a potential drug candidate for the treatment of cognitive deficit in Alzheimer's disease.

Cholinergic Modulation of Cortical Microcircuits Is Layer-Specific: Evidence from Rodent, Monkey and Human Brain.

Acetylcholine (ACh) signaling shapes neuronal circuit development and underlies specific aspects of cognitive functions and behaviors, including attention, learning, memory and motivation. During behavior, activation of muscarinic and nicotinic acetylcholine receptors (mAChRs and nAChRs) by ACh alters the activation state of neurons, and neuronal circuits most likely process information differently with elevated levels of ACh. In several brain regions, ACh has been shown to alter synaptic strength as well. By changing the rules for synaptic plasticity, ACh can have prolonged effects on and rearrange connectivity between neurons that outlasts its presence. From recent discoveries in the mouse, rat, monkey and human brain, a picture emerges in which the basal forebrain (BF) cholinergic system targets the neocortex with much more spatial and temporal detail than previously considered. Fast cholinergic synapses acting on a millisecond time scale are abundant in the mammalian cerebral cortex, and provide BF cholinergic neurons with the possibility to rapidly alter information flow in cortical microcircuits. Finally, recent studies have outlined novel mechanisms of how cholinergic projections from the BF affect synaptic strength in several brain areas of the rodent brain, with behavioral consequences. This review highlights these exciting developments and discusses how these findings translate to human brain circuitries.

'Hot' vs. 'cold' behavioural-cognitive styles: motivational-dopaminergic vs. cognitive-cholinergic processing of a Pavlovian cocaine cue in sign- and goal-tracking rats.

Discrete Pavlovian reward cues acquire more potent incentive motivational properties (incentive salience) in some animals (sign-trackers; STs) compared to others (goal-trackers; GTs). Conversely, GTs appear to be better than STs in processing more complex contextual cues, perhaps reflecting their relatively greater bias for goal-directed cue processing. Here, we investigated the activity of two major prefrontal neuromodulatory input systems, dopamine (DA) and acetylcholine (ACh), in response to a discrete Pavlovian cue that was previously paired with cocaine administration in STs and GTs. Rats underwent Pavlovian training in which light cue presentations were either paired or unpaired with an intravenous cocaine infusion. Following a 10-day abstinence period, prefrontal dialysates were collected in STs and GTs during cue presentations in the absence of cocaine. In STs, the cue previously paired with cocaine significantly increased prefrontal DA levels. DA levels remained elevated over baseline across multiple cue presentation blocks, and DA levels and approaches to the cue were significantly correlated. In STs, ACh levels were unaffected by cue presentations. In contrast, in GTs, presentations of the cocaine cue increased prefrontal ACh, but not DA, levels. GTs oriented towards the cue at rates similar to STs, but they did not approach it and elevated ACh levels did not correlate with conditioned orientation. The results indicate a double dissociation between the role of prefrontal DA and ACh in STs and GTs, and suggest that these phenotypes will be useful for studying the role of neuromodulator systems in mediating opponent behavioural-cognitive styles.

Non-neuronal cholinergic activity is potentiated in myasthenia gravis

BackgroundNon–neuronal acetylcholine (ACh) restricts autoimmune responses and attenuates inflammation by cholinergic anti-inflammation pathway. To date, the implication of ACh in myasthenia gravis (MG) remained unexplored. This study aimed to investigate the possible relationship between ACh levels, anti–muscle-specific tyrosine kinase (MuSK) antibody titers, main clinical features and outcomes of MG patients.MethodsWe successfully measured ACh levels in human peripheral blood mononuclear cells (PBMCs) from 125 MG patients and 50 matched healthy controls by using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). We assessed the quantitative MG (QMG) scores for each patient and titered anti-MuSK antibody.ResultsWe found that PBMC-derived ACh level was significantly higher in MG patients, especially in patients of class III, IV-V, compared with that in controls (0.142 ± 0.108 vs. 0.075 ± 0.014 ng/million cells, p = 0.0003) according to the Myasthenia Gravis Foundation of America clinical classification. Importantly, we also found that ACh levels were positively correlated with QMG scores (r = 0.83, p < 0.0001) and anti–MuSK Ab levels (r = 0.85, p < 0.0001).ConclusionsOur demonstration of elevated ACh levels in PBMCs of MG patients foreshadows potential new avenues for MG research and treatment.

Neuroprotective effects of polygalacic acid on scopolamine-induced memory deficits in mice

BackgroundPolygala tenuifolia Willd is a Traditional Chinese Medicine used for the treatment of learning and memory deficits. Triterpenoid saponins, the main bioactive compounds of Polygala tenuifolia Willd, are easily hydrolyzed to polygalacic acid (PA). PurposeThe present study was undertaken to investigate the neuroprotective effects of PA on scopolamine-induced cognitive dysfunction and to elucidate its underlying mechanisms of action. MethodsPA (3, 6, and 12 mg/kg) was administered orally to mice for fourteen days, and scopolamine (1 mg/kg) was injected intraperitoneally for fourteen days to induce memory impairment. Memory-related behaviors were evaluated using the Morris water maze. Cholinergic and neuroinflammatory activities were measured in brain tissue. Superoxide dismutase activities, malondialdehyde and reduced glutathione contents were also measured in the brains. ResultsTreatment with scopolamine significantly increased the escape latency time, decreased the number of crossings, and shortened the time spent in the target quadrant, while PA reversed these scopolamine-induced effects. PA significantly improved cholinergic system reactivity, as indicated by decreased acetylcholinesterase (AChE) activity, increased choline acetyltransferase (ChAT) activity, and elevated levels of acetylcholine (ACh) in the hippocampus and frontal cortex. PA also significantly ameliorated neuroinflammation and oxidative stress in mice. ConclusionThese results suggest that PA might exert a significant neuroprotective effect on cognitive impairment, driven in part by the modulation of cholinergic activity and neuroinflammation.

Neuroanatomical basis for cholinergic modulation of locomotor networks by sacral relay neurons with ascending lumbar projections.

Synaptic excitation by sacrocaudal afferent (SCA) input of sacral relay neurons projecting rostrally through the ventral white matter funiculi (VF neurons) is a potent activator of the hindlimb central pattern generators (CPGs) in rodent spinal cords lacking descending supraspinal control. Using electrophysiological recordings from the sacral and lumbar spinal segments, we show that the motor output of the lumbar segments produced by SCA stimulation is enhanced by exposing the sacral segments of the neonatal rat spinal cord to the acetylcholinesterase inhibitor edrophonium (EDR). Histochemical and immunostaining of the sacral cord reveals expression of acetylcholinesterase activity, ability to synthesize acetylcholine, and/or innervation by cholinergic synaptic inputs in significant proportions of fluorescently back-labeled sacral VF neurons. Moreover, the majority of the VF neurons express M2 muscarinic receptors, raising the possibility that the elevated acetylcholine levels resulting from inhibition of acetylcholinesterase act on such receptors. Indeed, sacral application of atropine or the M2 -type receptor antagonist methoctramine was found to reverse the effects of EDR. We suggest that variations in the sacral level of acetylcholine modulate the SCA-induced locomotor rhythm via muscarinic receptor-dependent mechanisms and that the modified activity of sacral VF neurons in the presence of an acetylcholinesterase inhibitor can be partially ascribed to the cholinergic components associated with them. Thus, pharmacological manipulations of the sacral cholinergic system may be used to modulate the locomotor-related motor output in the absence of descending supraspinal control.

Human neural stem cells over-expressing choline acetyltransferase restore cognition in rat model of cognitive dysfunction

A human neural stem cell (NSC) line over-expressing human choline acetyltransferase (ChAT) gene was generated and these F3.ChAT NSCs were transplanted into the brain of rat Alzheimer disease (AD) model which was induced by application of ethylcholine mustard aziridinium ion (AF64A) that specifically denatures cholinergic nerves and thereby leads to memory deficit as a salient feature of AD. Transplantation of F3.ChAT human NSCs fully recovered the learning and memory function of AF64A animals, and induced elevated levels of acetylcholine (ACh) in cerebrospinal fluid (CSF). Transplanted F3.ChAT human NSCs were found to migrate to various brain regions including cerebral cortex, hippocampus, striatum and septum, and differentiated into neurons and astrocytes. The present study demonstrates that brain transplantation of human NSCs over-expressing ChAT ameliorates complex learning and memory deficits in AF64A-cholinotoxin-induced AD rat model.

Human Neural Stem Cells Overexpressing Choline Acetyltransferase Restore Cognitive Function of Kainic Acid-Induced Learning and Memory Deficit Animals

Alzheimer disease (AD) is a progressive neurodegenerative disease, which is characterized by loss of memory and cognitive function. In AD patients dysfunction of the cholinergic system is the main cause of cognitive disorders, and decreased activity of choline acetyltransferase (ChAT), an enzyme responsible for acetylcholine (ACh) synthesis, is observed. In the present study we investigated if brain transplantation of human neural stem cells (NSCs) genetically modified to encode ChAT gene improves cognitive function of kainic acid (KA)-induced learning deficit rats. Intrahippocampal injection of KA to hippocampal CA3 region caused severe neuronal loss, resulting in profound learning and memory deficit. F3.ChAT human NSCs transplanted intracerebroventricularly improved fully the learning and memory function of KA-induced learning deficit animals, in parallel with the elevation of ACh levels in cerebrospinal fluid. F3.ChAT human NSCs migrated to the KA-induced injury site (CA3) and differentiated into neurons and astrocytes. The present study demonstrates that human NSCs expressing ChAT have lesion-tropic property and improve cognitive function of learning deficit model rats with hippocampal injury by increasing ACh level.

Reduced striatal acetylcholine efflux in the R6/2 mouse model of Huntington's disease: An examination of the role of altered inhibitory and excitatory mechanisms

Huntington's disease (HD) is a genetic neurodegenerative disorder that is characterized by the progressive onset of cognitive, psychiatric, and motor symptoms. In parallel, the neuropathology of HD is characterized by progressive loss of projection neurons in cortex and striatum; striatal cholinergic interneurons are relatively spared. Nonetheless, there is evidence that striatal acetylcholine (ACh) function is altered in HD. The present study is the first to examine striatal ACh function in awake, behaving animals, using the R6/2 mouse model of HD, which is transgenic for exon 1 of the mutant huntingtin gene. Physiological levels of extracellular striatal ACh were monitored in R6/2 mice and wild type controls using in vivo microdialysis. Results indicate that spontaneous ACh release is reduced in R6/2 mice relative to controls. Intrastriatal application of the GABAA antagonist bicuculline methiodide (10.0μM) significantly elevated ACh levels in both R6/2 mice and wild type controls, while overall ACh levels were reduced in the R6/2 mice compared to the wild type group. In contrast, systemic administration of the D1 dopamine receptor partial agonist, SKF-38393 (10.0mg/kg, IP), elevated ACh levels in control animals, but not R6/2 mice. Taken together, the present results suggest that GABA-mediated inhibition of striatal ACh release is intact in R6/2 mice, further demonstrating that cholinergic interneurons are capable of increased ACh release, whereas D1 receptor-dependent activation of excitatory inputs to striatal cholinergic interneurons is dysfunctional in R6/2 mice. Reduced levels of extracellular striatal ACh in HD may reflect abnormalities in the excitatory innervation of cholinergic interneurons, which may have implications ACh-dependent processes that are altered in HD, including corticostriatal plasticity.

Compensatory responses to age-related decline in odor quality acuity: Cholinergic neuromodulation and olfactory enrichment

The perceptual differentiation of odors can be measured behaviorally using generalization gradients. The steepness of these gradients defines a form of olfactory acuity for odor quality that depends on neural circuitry within the olfactory bulb and is regulated by cholinergic activity therein as well as by associative learning. Using this system as a reduced model for age-related cognitive decline, we show that aged mice, while maintaining almost the same baseline behavioral performance as younger mice, are insensitive to the effects of acutely elevated acetylcholine, which sharpens generalization gradients in young adult mice. Moreover, older mice exhibit evidence of chronically elevated acetylcholine levels in the olfactory bulb, suggesting that their insensitivity to further elevated levels of acetylcholine may arise because the maximum capacity of the system to respond to acetylcholine has already been reached. We propose a model in which an underlying, age-related, progressive deficit is mitigated by a compensatory cholinergic feedback loop that acts to retard the behavioral effects of what would otherwise be a substantial age-related decline in olfactory plasticity. We also treated mice with 10-day regimens of olfactory environmental enrichment and/or repeated systemic injections of the acetylcholinesterase inhibitor physostigmine. Each treatment alone sharpened odor quality acuity, but administering both treatments together had no greater effect than either alone. Age was not a significant main effect in this study, suggesting that some capacity for acetylcholine-dependent plasticity is still present in aged mice despite their sharply reduced ability to respond to acute increases in acetylcholine levels. These results suggest a dynamical framework for understanding age-related decline in neural circuit processing in which the direct effects of aging can be mitigated, at least temporarily, by systemic compensatory responses. In particular, a decline in cholinergic efficacy can precede any breakdown in cholinergic production, which may help explain the limited effectiveness of cholinergic replacement therapies in combating cognitive decline.

Adaptation to excess acetylcholine by downregulation of adrenoceptors and muscarinic receptors in lungs of acetylcholinesterase knockout mice

The acetylcholinesterase knockout mouse has elevated acetylcholine levels due to the complete absence of acetylcholinesterase. Our goal was to determine the adaptive changes in lung receptors that allow these animals to tolerate excess neurotransmitter. The hypothesis was tested that not only muscarinic receptors but also alpha(1)-adrenoceptors and beta-adrenoceptors are downregulated, thus maintaining a proper balance of receptors and accounting for lung function in these animals. The quantity of alpha(1A), alpha(1B), alpha(1D), beta(1), and beta(2)-adrenoceptors and muscarinic receptors was determined by binding of radioligands. G-protein coupling was assessed using pseudo-competition with agonists. Phospholipase C activity was measured by an enzymatic assay. Cyclic AMP (cAMP) content was measured by immunoassay. Muscarinic receptors were decreased to 50%, alpha(1)-adrenoceptors to 23%, and beta-adrenoceptors to about 50% of control. Changes were subtype specific, as alpha(1A), alpha(1B), and beta(2)-adrenoceptors, but not alpha(1D)-adrenoceptor, were decreased. In contrast, receptor signaling into the cell as measured by coupling to G proteins, cAMP content, and PI-phospholipase C activity was the same as in control. This shows that the nearly normal lung function of these animals was explained by maintenance of a correct balance of adrenoceptors and muscarinic receptors. In conclusion, knockout mice have adapted to high concentrations of acetylcholine by downregulating receptors that bind acetylcholine, as well as by downregulating receptors that oppose the action of muscarinic receptors. Tolerance to excess acetylcholine is achieved by reducing the levels of muscarinic receptors and adrenoceptors.

Comparison of 1-(2-naphthyloxymethylcarbonyl) pyrrolidine, PP-20/DPJ, newly synthesized compound with the nootropic drug piracetam in normal and aluminum-induced neurotoxic state

Nootropic drugs like piracetam, oxiracetam, and nifiracetam are used as memory enhancers. They are thought to directly influence the energetic processes in the brain and produce elevated acetylcholine levels, but they lack protecting therapeutic potentials. Thus, there is a continuing effort directed towards developing a new cognition-enhancing agent, which would be more effective than the currently available drugs, and 2-Naphthyloxy derivatives (PP-20/DPJ) were consequently developed. This in vivo study was designed to compare the memory enhancing potential of PP-20/DPJ with the known nootropic agent, piracetam, in aluminum (Al)-induced neurotoxic model. Results indicated that PP-20/DPJ improved the short-term memory and cognitive behaviors similar to piracetam. Further, both the compounds were equally effective in elevating the acetylcholinesterase and Mg+-ATPase enzyme activity in both the brain regions after Al treatment. Thus the current study suggests, that PP-20/DPJ acts as both a cognition-enhancing agent and as a metabolic enhancer.