Enzyme ChE, cholinergic therapy and molecular docking: Significant considerations and future perspectives.

Enzyme Che plays an essential role in cholinergic and non-cholinergic functions. It is present in the fertilized/unfertilized eggs and sperm of different species. Inclusion criteria for data collection from electronic databases NCBI and Google Scholar are enzyme AChE/BChE, cholinergic therapy, genomic organization and gene transcription, enzyme structure, biogenesis, transport, processing and localization, molecular signaling and biological function, polymorphism and influencing factors. Enzyme Che acts as a signaling receptor during hematopoiesis, protein adhesion, amyloid fiber formation, neurite outgrowth, bone development, and maturation, explaining the activity out of synaptic neurotransmission. Polymorphism in the Che genes correlates to various diseases and diverse drug responses. In particular, change accompanies cancer, neurodegenerative, and cardiovascular disease. Literature knowledge indicates the importance of Che inhibitors that influence biochemical and molecular pathways in disease treatment, genomic organization, gene transcription, structure, biogenesis, transport, processing, and localization of Che enzyme. Enzyme Che polymorphism changes indicate the possibility of efficient and new inhibitor drug target mechanisms in diverse research areas.

The association between polymorphisms in miRNA and the cholinesterase activity of workers in an omethoate-exposed environment.

To explore the association between polymorphisms in microRNAs (miRNAs) and the cholinesterase (ChE) activity in omethoate-exposed workers, we recruited 180 omethoate-exposed workers and 115 controls to measure their ChE activity using acetylcholine and dithio-bis-(nitrobenzoic acid) and genotype susceptible SNPs in their miRNA by time-of-flight mass spectrometry. ChE activity in the exposure group was lower than that in the control group (P < 0.001). The analysis of covariance result showed that ChE activity was lower in the (- -/- T) genotype in miR-30a rs111456995 (1.97 ± 0.47) than in the TT genotype (2.23 ± 0.59) of the exposure group (P = 0.004). Multivariate linear regression was performed to find influencing factors on ChE activity, and variables kept in the model included omethoate exposure (b = -1.094, P < 0.001), gender (b = -0.381, P < 0.001), miR-30a rs111456995 (- -/- T)(b = -0.248, P < 0.001), and drinking (b = 0.258, P =0.019). The results suggest that individuals carrying a (- -/- T) genotype in miR-30a rs111456995 were more susceptible to damage in their cholinesterase induced by omethoate exposure.

Clinicopathological correlations and cholinesterase expression in early-onset familial Alzheimer's disease with the presenilin 1 mutation, Leu235Pro.

In sporadic Alzheimer's disease (SpAD), acetylcholinesterase and butyrylcholinesterase, co-regulators of acetylcholine, are associated with ß-amyloid plaques and tau neurofibrillary tangles in patterns suggesting a contribution to neurotoxicity. This association has not been explored in early-onset familial Alzheimer's disease (FAD). We investigated whether cholinesterases are observed in the neuropathological hallmarks in FAD expressing the presenilin 1 Leu235Pro mutation. Brain tissues from three FAD cases and one early-onset SpAD case were stained and analyzed for ß-amyloid, tau, α-synuclein, acetylcholinesterase and butyrylcholinesterase. AD pathology was prominent throughout the rostrocaudal extent of all 4 brains but α-synuclein-positive neurites were present in only one familial case. In FAD and SpAD cases, cholinergic activity was associated with plaques and tangles but not with α-synuclein pathology. Both cholinesterases showed similar or decreased plaque staining than detected with ß-amyloid immunostaining but greater plaque deposition than observed with thioflavin-S histofluorescence. Acetylcholinesterase and butyrylcholinesterase are highly associated with AD pathology in inherited disease and both may represent specific diagnostic and therapeutic targets for all AD forms.

Enzymes, Reacting with Organophosphorus Compounds as Detoxifiers: Diversity and Functions.

Organophosphorus compounds (OPCs) are able to interact with various biological targets in living organisms, including enzymes. The binding of OPCs to enzymes does not always lead to negative consequences for the body itself, since there are a lot of natural biocatalysts that can catalyze the chemical transformations of the OPCs via hydrolysis or oxidation/reduction and thereby provide their detoxification. Some of these enzymes, their structural differences and identity, mechanisms, and specificity of catalytic action are discussed in this work, including results of computational modeling. Phylogenetic analysis of these diverse enzymes was specially realized for this review to emphasize a great area for future development(s) and applications.

Identification, characterization and expression analyses of cholinesterases genes in Yesso scallop (Patinopecten yessoensis) reveal molecular function allocation in responses to ocean acidification.

Cholinesterases are key enzymes in central and peripheral cholinergic nerve system functioning on nerve impulse transmission in animals. Though cholinesterases have been identified in most vertebrates, the knowledge about the variable numbers and multiple functions of the genes is still quite meagre in invertebrates, especially in scallops. In this study, the complete cholinesterase (ChE) family members have been systematically characterized in Yesso scallop (Patinopecten yessoensis) via whole-genome scanning through in silico analysis. Ten ChE family members in the genome of Yesso scallop (designated PyChEs) were identified and potentially acted to be the largest number of ChE in the reported species to date. Phylogenetic and protein structural analyses were performed to determine the identities and evolutionary relationships of these genes. The expression profiles of PyChEs were determined in all developmental stages, in healthy adult tissues, and in mantles under low pH stress (pH 6.5 and 7.5). Spatiotemporal expression suggested the ubiquitous functional roles of PyChEs in all stages of development, as well as general and tissue-specific functions in scallop tissues. Regulation expressions revealed diverse up- and down-regulated expression patterns at most time points, suggesting different functional specialization of gene superfamily members in response to ocean acidification (OA). Evidences in gene number, phylogenetic relationships and expression patterns of PyChEs revealed that functional innovations and differentiations after gene duplication may result in altered functional constraints among PyChEs gene clusters. Collectively, our results provide the potential clues that the selection pressures coming from the environment were the potential inducement leading to function allocation of ChE family members in scallop.

Comparative mapping of selected structural determinants on the extracellular domains of cholinesterase-like cell-adhesion molecules.

Cell adhesion generally involves formation of homophilic or heterophilic protein complexes between two cells to form transcellular junctions. Neural cell-adhesion members of the α/ß-hydrolase fold superfamily of proteins use their extracellular or soluble cholinesterase-like domain to bind cognate partners across cell membranes, as illustrated by the neuroligins. These cell-adhesion molecules currently comprise the synaptic organizers neuroligins found in all animal phyla, along with three proteins found only in invertebrates: the guidance molecule neurotactin, the glia-specific gliotactin, and the basement membrane protein glutactin. Although these proteins share a cholinesterase-like fold, they lack one or more residues composing the catalytic triad responsible for the enzymatic activity of the cholinesterases. Conversely, they are found in various subcellular localisations and display specific disulfide bonding and N-glycosylation patterns, along with individual surface determinants possibly associated with recognition and binding of protein partners. Formation of non-covalent dimers typical of the cholinesterases is documented for mammalian neuroligins, yet whether invertebrate neuroligins and their neurotactin, gliotactin and glutactin relatives also form dimers in physiological conditions is unknown. Here we provide a brief overview of the localization, function, evolution, and conserved versus individual structural determinants of these cholinesterase-like cell-adhesion proteins. This article is part of the special issue entitled 'Acetylcholinesterase Inhibitors: From Bench to Bedside to Battlefield'.

Enzymatic decontamination of paraoxon-ethyl limits long-term effects in planarians.

Organophosphorus compounds (OP) are highly toxic molecules used as insecticides that inhibit cholinesterase enzymes involved in neuronal transmission. The intensive use of OP for vector control and agriculture has led to environmental pollutions responsible for severe intoxications and putative long-term effects on humans and wild animals. Many in vivo models were studied over the years to assess OP acute toxicity, but the long-term effects are poorly documented. Planarian, a freshwater flatworm having a cholinergic system, has emerged as a new original model for addressing both toxicity and developmental perturbations. We used Schmidtea mediterranea planarians to evaluate long-term effects of paraoxon-ethyl at two sublethal concentrations over three generations. Toxicity, developmental perturbations and disruption of behavior were rapidly observed and higher sensitivity to paraoxon-ethyl of next generations was noticed suggesting that low insecticide doses can induce transgenerational effects. With the view of limiting OP poisoning, SsoPox, an hyperthermostable enzyme issued from the archaea Saccharolobus solfataricus, was used to degrade paraoxon-ethyl prior to planarian exposure. The degradation products, although not lethal to the worms, were found to decrease cholinesterase activities for the last generation of planarians and to induce abnormalities albeit in lower proportion than insecticides.

Molecular Insight into the Therapeutic Promise of Flavonoids against Alzheimer's Disease.

Alzheimer's disease (AD) is one of the utmost chronic neurodegenerative disorders, which is characterized from a neuropathological point of view by the aggregates of amyloid beta (Aß) peptides that are deposited as senile plaques and tau proteins which form neurofibrillary tangles (NFTs). Even though advancement has been observed in order to understand AD pathogenesis, currently available therapeutic methods can only deliver modest symptomatic relief. Interestingly, naturally occurring dietary flavonoids have gained substantial attention due to their antioxidative, anti-inflammatory, and anti-amyloidogenic properties as alternative candidates for AD therapy. Experimental proof provides support to the idea that some flavonoids might protect AD by interfering with the production and aggregation of Aß peptides and/or decreasing the aggregation of tau. Flavonoids have the ability to promote clearance of Aß peptides and inhibit tau phosphorylation by the mTOR/autophagy signaling pathway. Moreover, due to their cholinesterase inhibitory potential, flavonoids can represent promising symptomatic anti-Alzheimer agents. Several processes have been suggested for the aptitude of flavonoids to slow down the advancement or to avert the onset of Alzheimer's pathogenesis. To enhance cognitive performance and to prevent the onset and progress of AD, the interaction of flavonoids with various signaling pathways is proposed to exert their therapeutic potential. Therefore, this review elaborates on the probable therapeutic approaches of flavonoids aimed at averting or slowing the progression of the AD pathogenesis.

A novel understanding of postoperative complications: In vitro study of the impact of propofol on epigenetic modifications in cholinergic genes.

BACKGROUND: Propofol is a widely used anaesthetic drug with advantageous operating conditions and recovery profile. However, propofol could have long term effects on neuronal cells and is associated with post-operative delirium (POD). In this context, one of the contributing factors to the pathogenesis of POD is a reduction of cholinesterase activity. Accordingly, we investigated the effects of propofol on the methylation, expression and activity of cholinergic genes and proteins in an in-vitro model. RESULTS: We found that propofol indeed reduced the activity of AChE / BChE in our in-vitro model, without affecting the protein levels. Furthermore, we could show that propofol reduced the methylation of a repressor region of the CHRNA7 gene without changing the secretion of pro-or anti-inflammatory cytokines. Lastly, propofol changed the expression patterns of genes responsible for maintaining the epigenetic status of the cell and accordingly reduced the tri-methylation of H3 K27. CONCLUSION: In conclusion we found a possible functional link between propofol treatment and POD, due to a reduced cholinergic activity. In addition to this, propofol changed the expression of different maintenance genes of the epigenome that also affected histone methylation. Thus, propofol treatment may also induce strong, long lasting changes in the brain by potentially altering the epigenetic landscape.

An evolutionary perspective on the first disulfide bond in members of the cholinesterase-carboxylesterase (COesterase) family: Possible outcomes for cholinesterase expression in prokaryotes.

Within the alpha/beta hydrolase fold superfamily of proteins, the COesterase group (carboxylesterase type B, block C, cholinesterases …) diverged from the other groups through simultaneous integration of an N-terminal, first disulfide bond and a significant increase in the protein mean size. This first disulfide bond ties a large Cys loop, which in the cholinesterases is named the omega loop and forms the upper part of the active center gorge, essential for the high catalytic activity of these enzymes. In some non-catalytic members of the family, the loop may be necessary for heterologous partner recognition. Reshuffling of this protein portion occurred at the time of emergence of the fungi/metazoan lineage. Homologous proteins with this first disulfide bond are absent in plants but they are found in a limited number of bacterial genomes. In prokaryotes, the genes coding for such homologous proteins may have been acquired by horizontal transfer. However, the cysteines of the first disulfide bond are often lost in bacteria. Natural expression in bacteria of CO-esterases comprising this disulfide bond may have required compensatory mutations or expression of new chaperones. This disulfide bond may also challenge expression of the eukaryote-specific cholinesterases in prokaryotic cells. Yet recently, catalytically active human cholinesterase variants with enhanced thermostability were successfully expressed in E. coli. The key was the use of a peptidic sequence optimized through the Protein Repair One Stop Shop process, an automated structure- and sequence-based algorithm for expression of properly folded, soluble and stable eukaryotic proteins. Surprisingly however, crystal structures of the optimized cholinesterase variants expressed in bacteria revealed co-existing formed and unformed states of the first disulfide bond. Whether the bond never formed, or whether it properly formed then broke during the production/analysis process, cannot be inferred from the structural data. Yet, these features suggest that the recently acquired first disulfide bond is difficult to maintain in E. coli-expressed cholinesterases. To explore the fate of the first disulfide bond throughout the cholinesterase relatives, we reanalyzed the crystal structures of representative COesterases members from natural prokaryotic or eukaryotic sources or produced as recombinant proteins in E. coli. We found that in most cases this bond is absent.

First evidence of cholinesterase-like activity in Basidiomycota.

Cholinesterases (ChE), the enzymes whose primary function is the hydrolysis of choline esters, are widely expressed throughout the nature. Although they have already been found in plants and microorganisms, including ascomycete fungi, this study is the first report of ChE-like activity in fungi of the phylum Basidiomycota. This activity was detected in almost a quarter of the 45 tested aqueous fungal extracts. The ability of these extracts to hydrolyse acetylthiocholine was about ten times stronger than the hydrolytic activity towards butyrylthiocholine and propionylthiocholine. In-gel detection of ChE-like activity with acetylthiocholine indicated a great variability in the characteristics of these enzymes which are not characterized as vertebrate-like based on (i) differences in inhibition by excess substrate, (ii) susceptibility to different vertebrate acetylcholinesterase and butyrylcholinesterase inhibitors, and (iii) a lack of orthologs using phylogenetic analysis. Limited inhibition by single inhibitors and multiple activity bands using in-gel detection indicate the presence of several ChE-like enzymes in these aqueous extracts. We also observed inhibitory activity of the same aqueous mushroom extracts against insect acetylcholinesterase in 10 of the 45 samples tested; activity was independent of the presence of ChE-like activity in extracts. Both ChE-like activities with different substrates and the ability of extracts to inhibit insect acetylcholinesterase were not restricted to any fungal family but were rather present across all included Basidiomycota families. This study can serve as a platform for further research regarding ChE activity in mushrooms.

Genome-wide association studies reveal genetic loci associated with plasma cholinesterase activity in ducks.

Plasma cholinesterase (PCHE) activity is an important auxiliary test in human clinical medicine. It can distinguish liver diseases from non-liver diseases and help detect organophosphorus poisoning. Animal experiments have confirmed that PCHE activity is associated with obesity and hypertension and changes with physiological changes in an animal's body. The objective of this study was to locate the genetic loci responsible for PCHE activity variation in ducks. PCHE activity of Pekin duck × mallard F2 ducks at 3 and 8 weeks of age were analyzed, and genome-wide association studies were conducted. A region of about 1.5 Mb (21.8-23.3 Mb) on duck chromosome 9 was found to be associated with PCHE activity at both 3 and 8 weeks of age. The top SNP, g.22643979C>T in the butyrylcholinesterase (BCHE) gene, was most highly associated with PCHE activity at 3 weeks (-logP = 21.45) and 8 weeks (-logP = 27.60) of age. For the top SNP, the strong associations of CC and CT genotypes with low PCHE activity and the TT genotype with high PCHE activity indicates the dominant inheritance of low PCHE activity. Problems with block inheritance or linkage exist in this region. This study supports that BCHE is a functional gene for determining PCHE levels in ducks and that the genetic variations around this gene can cause phenotypic variations of PCHE activity.

Molecular and biochemical characterization of the bed bug salivary gland cholinesterase as an acetylcholine-sequestering enzyme.

The common bed bug, Cimex lectularius, possesses a cholinesterase expressed exclusively in the salivary gland (ClSChE). In this study, we investigated the molecular forms, tissue distribution patterns and biochemical properties of ClSChE and showed that ClSChE exists as a soluble monomeric form or a soluble dimeric form connected by a disulfide bridge. Immunohistochemical analysis confirmed that ClSChE was expressed in the epithelial cells of both the salivary gland and the duct. In addition, the secretion of monomeric ClSChE through the proboscis during feeding was confirmed by western blotting using a ClSChE-specific antibody. To predict the role of ClSChE injected into the tissue of an animal host, we analyzed the extent of hydrolysis of acetylcholine (ACh) by ClSChE by ultra-performance liquid chromatography-tandem mass spectrometry. ClSChE binding to ACh was not clearly resolved in the binding assay format used in this study, probably due to the weak but detectable ACh-hydrolytic activity of ClSChE. Nevertheless, kinetic analysis revealed that ClSChE possesses extremely low Km (high affinity to ACh) and Vmax values. These findings suggest that ClSChE functions virtually as an ACh-sequestering protein by having a very strong affinity to ACh but an extremely long turnover time. Given that ACh regulates a wide variety of host physiologies, we discuss the tentative roles of ClSChE in blood vessel constriction and itch/pain regulation in the host.

Planarian cholinesterase: molecular and functional characterization of an evolutionarily ancient enzyme to study organophosphorus pesticide toxicity.

The asexual freshwater planarian Dugesia japonica has emerged as a medium-throughput alternative animal model for neurotoxicology. We have previously shown that D. japonica are sensitive to organophosphorus pesticides (OPs) and characterized the in vitro inhibition profile of planarian cholinesterase (DjChE) activity using irreversible and reversible inhibitors. We found that DjChE has intermediate features of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Here, we identify two candidate genes (Djche1 and Djche2) responsible for DjChE activity. Sequence alignment and structural homology modeling with representative vertebrate AChE and BChE sequences confirmed our structural predictions, and show that both DjChE enzymes have intermediate sized catalytic gorges and disrupted peripheral binding sites. Djche1 and Djche2 were both expressed in the planarian nervous system, as anticipated from previous activity staining, but with distinct expression profiles. To dissect how DjChE inhibition affects planarian behavior, we acutely inhibited DjChE activity by exposing animals to either an OP (diazinon) or carbamate (physostigmine) at 1 µM for 4 days. Both inhibitors delayed the reaction of planarians to heat stress. Simultaneous knockdown of both Djche genes by RNAi similarly resulted in a delayed heat stress response. Furthermore, chemical inhibition of DjChE activity increased the worms' ability to adhere to a substrate. However, increased substrate adhesion was not observed in Djche1/Djche2 (RNAi) animals or in inhibitor-treated day 11 regenerates, suggesting this phenotype may be modulated by other mechanisms besides ChE inhibition. Together, our study characterizes DjChE expression and function, providing the basis for future studies in this system to dissect alternative mechanisms of OP toxicity.

RNA interference of acetylcholinesterase in the Asian citrus psyllid, Diaphorina citri, increases its susceptibility to carbamate and organophosphate insecticides.

The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Lividae) transmits the Candidatus Liberibacter asiaticus, which causes citrus greening disease or Huanglongbing, (HLB). To date, there is no efficient cure for HLB disease and the control of D. citri using insecticides became the most important tools for the management of HLB. However, the extensive use of insecticides could increase D. citri resistance to these insecticides. The objective of this study was to investigate the effect of RNA interference of acetylcholinesterase (AChE) on the mortality and susceptibility of D. citri to the four major insecticides used in Florida. In this study, we used a consensus sequence derived from the two AChE genes and cholinesterase 2-like (ChE-2-like) gene to target all of the three genes. Treatment with dsRNA-AChE increased the mortality percentages of both nymphs and adults of D. citri. The mortality percentage increased with the increase in the concentration of applied dsRNA-AChE, and the highest mortality (> 60%) was observed at the highest applied concentration (125ng/µl). Treatments of nymphs or adults with dsRNA-AChE down-regulated the expression of the three targeted genes of D. citri. Silencing of AChE and ChE in D. citri nymphs increased the susceptibility of emerged adults to chlorpyrifos and carbaryl, which act as AChE inhibitors. However, treatment with dsRNA-AChE did not increase the susceptibility of emerged adults to imidacloprid, which acts as an agonist of nicotinic acetylcholine receptors. In the same manner, treatment of adults with dsRNA-AChE increased their susceptibility to chlorpyrifos and carbaryl, but did not affect their susceptibility to imidacloprid. The ANOVA did not show any significant increase in susceptibility of D. citri adults to fenpropathrin after treatment with dsRNA-AChE, either as nymphs or as adults. However, simple linear regression showed that treatment with dsRNA-AChE increased D. citri susceptibility to fenpropathrin, which indicated that AChE could be involved in the metabolism of fenpropathrin. Our results indicated that silencing of AChE and ChE genes in D. citri to increase its susceptibility to insecticides could be a promising tool for the control of this important vector.

Mapping insecticide resistance and characterization of resistance mechanisms in Anopheles arabiensis (Diptera: Culicidae) in Ethiopia.

BACKGROUND: The emergence and spread of insecticide resistance in the major African malaria vectors Anopheles gambiae (s.s.) and An. arabiensis may compromise the current vector control interventions and threatens the global malaria control and elimination efforts. METHODS: Insecticide resistance was monitored in several study sites in Ethiopia from 2013 to 2015 using papers impregnated with discriminating concentrations of DDT, deltamethrin, bendiocarb, propoxur, malathion, fenitrothion and pirimiphos-methyl, following the WHO insecticide susceptibility test procedure. Mosquitoes sampled from different localities for WHO bioassay were morphologically identified as An. gambiae (s.l.) using standard taxonomic keys. Samples were identified to species using species-specific polymerase chain reaction (PCR) and screened for the presence of target site mutations L1014F, L1014S and N1575Y in the voltage gated sodium channel (VGSC) gene and G119S in the acethylcholinesterase (AChE) gene using allele-specific PCR. Biochemical assays were performed to assess elevated levels of acetylcholinesterases, carboxylcholinesterases, glutathione-S-transferases (GSTs) and cytochrome P450s monooxygenases in wild populations of An. arabiensis, compared to the fully susceptible Sekoru An. arabiensis laboratory strain. RESULTS: Populations of An. arabiensis were resistant to DDT and deltamethrin but were susceptible to fenitrothion in all the study sites. Reduced susceptibility to malathion, pirimiphos-methyl, propoxur and bendiocarb was observed in some of the study sites. Knockdown resistance (kdr L1014F) was detected in all mosquito populations with allele frequency ranging from 42 to 91%. Elevated levels of glutathione-S-transferases (GSTs) were detected in some of the mosquito populations. However, no elevated levels of monooxygenases and esterases were detected in any of the populations assessed. CONCLUSIONS: Anopheles arabiensis populations from all surveyed sites in Ethiopia exhibited resistance against DDT and pyrethroids. Moreover, some mosquito populations exhibited resistance to propoxur and possible resistance to bendiocarb. Target site mutation kdr L1014F was detected in all mosquito populations while elevated levels of glutathione-S-transferases (GSTs) was detected in some mosquito populations. The reduced susceptibility of An. arabiensis to propoxur and bendiocarb, which are currently used for indoor residual spraying (IRS) in Ethiopia, calls for continuous resistance monitoring, in order to plan and implement evidence based insecticide resistance management.

Genetic Landscape of Congenital Myasthenic Syndromes From Turkey: Novel Mutations and Clinical Insights.

Congenital myasthenic syndromes are clinically and genetically heterogeneous disorders of neuromuscular transmission. Most are treatable, but certain subtypes worsen with cholinesterase inhibitors. This underlines the importance of genetic diagnosis. Here, the authors report on cases with genetically proven congenital myasthenic syndromes from Turkey. The authors retrospectively reviewed their experience of all patients with congenital myasthenic syndromes, referred over a 5-year period (2011-2016) to the Child Neurology Department of Dokuz Eylül University, Izmir, Turkey. In addition, PubMed was searched for published cases of genetically proven congenital myasthenic syndromes originating from Turkey. In total, the authors identified 43 (8 new patients, 35 recently published patients) cases. Defects in the acetylcholine receptor (n = 15; 35%) were the most common type, followed by synaptic basal-lamina associated (n = 14; 33%) and presynaptic syndromes (n = 10; 23%). The authors had only 3 cases (7%) who had defects in endplate development. One patient had mutation GFPT1 gene (n = 1; 2%). Knowledge on congenital myasthenic syndromes and related genes in Turkey will lead to prompt diagnosis and treatment of these rare neuromuscular disorders.

Natural genomic amplification of cholinesterase genes in animals.

Tight control of the concentration of acetylcholine at cholinergic synapses requires precise regulation of the number and state of the acetylcholine receptors, and of the synthesis and degradation of the neurotransmitter. In particular, the cholinesterase activity has to be controlled exquisitely. In the genome of the first experimental models used (man, mouse, zebrafish and drosophila), there are only one or two genes coding for cholinesterases, whereas there are more genes for their closest relatives the carboxylesterases. Natural amplification of cholinesterase genes was first found to occur in some cancer cells and in insect species subjected to evolutionary pressure by insecticides. Analysis of the complete genome sequences of numerous representatives of the various metazoan phyla show that moderate amplification of cholinesterase genes is not uncommon in molluscs, echinoderms, hemichordates, prochordates or lepidosauria. Amplification of acetylcholinesterase genes is also a feature of parasitic nematodes or ticks. In these parasites, over-production of cholinesterase-like proteins in secreted products and the saliva are presumed to have effector roles related to host infection. These amplification events raise questions about the role of the amplified gene products, and the adaptation processes necessary to preserve efficient cholinergic transmission. This is an article for the special issue XVth International Symposium on Cholinergic Mechanisms.

Specific connections of the interpeduncular subnuclei reveal distinct components of the habenulopeduncular pathway.

The habenulopeduncular pathway consists of the medial habenula (MHb), its output tract, the fasciculus retroflexus, and its principal target, the interpeduncular nucleus (IP). Several IP subnuclei have been described, but their specific projections and relationship to habenula inputs are not well understood. Here we have used viral, transgenic, and conventional anterograde and retrograde tract-tracing methods to better define the relationship between the dorsal and ventral MHb, the IP, and the secondary efferent targets of this system. Although prior studies have reported that the IP has ascending projections to ventral forebrain structures, we find that these projections originate almost entirely in the apical subnucleus, which may be more appropriately described as part of the median raphe system. The laterodorsal tegmental nucleus receives inhibitory inputs from the contralateral dorsolateral IP, and mainly excitatory inputs from the ipsilateral rostrolateral IP subnucleus. The midline central gray of the pons and nucleus incertus receive input from the rostral IP, which contains a mix of inhibitory and excitatory neurons, and the dorsomedial IP, which is exclusively inhibitory. The lateral central gray of the pons receives bilateral input from the lateral IP, which in turn receives bilateral input from the dorsal MHb. Taken together with prior studies of IP projections to the raphe, these results form an emerging map of the habenulopeduncular system that has significant implications for the proposed function of the IP in a variety of behaviors, including models of mood disorders and behavioral responses to nicotine.