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Nematode parasites of humans and livestock pose a significant burden to human health, economic development, and food security. Anthelmintic drug resistance is widespread among parasites of livestock and many nematode parasites of humans lack effective treatments. Here, we present a nitrophenyl-piperazine scaffold that induces motor defects rapidly in the model nematode Caenorhabditis elegans. We call this scaffold Nemacol and show that it inhibits the vesicular acetylcholine transporter (VAChT), a target recognized by commercial animal and crop health groups as a viable anthelmintic target. We demonstrate that it is possible to create Nemacol analogs that maintain potent in vivo activity whilst lowering their affinity to the mammalian VAChT 10-fold. We also show that Nemacol enhances the ability of the anthelmintic Ivermectin to paralyze C. elegans and the ruminant nematode parasite Haemonchus contortus. Hence, Nemacol represents a promising new anthelmintic scaffold that acts through a validated anthelmintic target.

International audience; Striatal cholinergic interneurons (CINs) use acetylcholine (ACh) and glutamate (Glut) to regulate the striatal network since they express vesicular transporters for ACh (VAChT) and Glut (VGLUT3). However, whether ACh and Glut are released simultaneously and/or independently from cholinergic varicosities is an open question. The answer to that question requires the multichannel detection of vesicular transporters at the level of single synaptic vesicle (SV). Here, we used super-resolution STimulated Emission Depletion microscopy (STED) to characterize and quantify the distribution of VAChT and VGLUT3 in CINs SVs. Nearest-neighbor distances analysis between VAChT and VGLUT3-immunofluorescent spots revealed that 34% of CINs SVs contain both VAChT and VGLUT3. In addition, 40% of SVs expressed only VAChT while 26% of SVs contain only VGLUT3. These results suggest that SVs from CINs have the potential to store simultaneously or independently ACh and/or Glut. Overall, these morphological findings support the notion that CINs varicosities can signal with either ACh or Glut or both with an unexpected level of complexity.

The expression and distribution of a protein can provide critical information about its function in a cell. For some neuronal proteins this information may include neurotransmitter (NT) usage and sites of NT release. However, visualizing the expression of a protein within a given neuron is often challenging because most neurons are intricately intermingled with numerous other neurons, making individual neuronal expression difficult to discern, especially since many neuronal genes are expressed at low levels. To overcome these difficulties for the Drosophila vesicular acetylcholine transporter (vAChT), attempts were made to generate conditional Drosophila vAChT alleles containing two tandem copies of epitope tags. In the course of these attempts, a strategy for multimerizing DNA repeats using the Gibson cloning reaction was serendipitously discovered. Attempts at optimization routinely yielded six or seven copies of MYC and OLLAS epitope tag coding sequences, but occasionally as many as 10 copies, thus potentially enhancing the sensitivity of protein detection up to an order of magnitude. As proof-of-principle of the method, conditionally expressible genome-edited 7XMYC-vAChT and 6XOLLAS-vAChT were developed and characterized for conditionality, synaptic vesicle specificity, and neurotransmitter specific-expression. The utility of these conditional vAChT variants was demonstrated for cholinergic neurotransmitter phenotyping and defining the polarity of cholinergic neurons, important information for understanding the functional role of neurons of interest in neural circuits and behavior. The repeat multimerization method is effective for DNA repeats of at least 56 bp and should be generally applicable to any species.

Ulcerative colitis is a chronic inflammatory condition of the gastrointestinal tract that can affect people of worldwide. In contrast with Crohn's disease, that can relate the entire thickness of the bowel wall, the inflammation of ulcerative colitis is limited to the colonic mucosa. Immune cells including activated T cells, plasma cells, mast cells, macrophages, and dendritic cells (DCs) trigger the inflammation. Furthermore, dendritic cells are antigen presenting cells involved in maintaining intestinal immune homeostasis. It has been described an increment of number in DCs colonic mucosa of patients with ulcerative colitis. The immune cells such as antigen-presenting cells can act as autocrine or paracrine modulators. Recent studies showed that dendritic cells synthetized and released classical neurotransmitters as glutamate, dopamine, acetylcholine, and serotonin. Paraformaldehyde-fixed intestinal tissues, obtained from the stricture sites of ten patients with ulcerative colitis were analyzed by immunostaining for Langerin/CD207, serotonin and vesicular acetylcholine transporter. As controls, unaffected (normal) portions of five patients were also investigated. Aim of this study was to characterize for the first time the human gut dendritic cells of ulcerative colitis patients, with Langerin/CD207 that is a c-type lectin expressed by different types of DCs and to colocalize in the same cells the expression of serotonin and vesicular acetylcholine transporter, showing the link between dendritic cells, gut enterochromaffin cells or autonomic nerves in immune activation and generation of intestinal inflammation.

Sensory and motor systems in insects with hemimetabolous development must be ready to mediate adaptive behavior directly on hatching from the egg. For the desert locust S. gregaria, cholinergic transmission from antennal sensillae to olfactory or mechanosensory centers in the brain requires that choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter (vAChT) already be present in sensory cells in the first instar. In this study, we used immunolabeling to demonstrate that ChAT and vAChT are both expressed in sensory cells from identifiable sensilla types in the immature antennal nervous system. We observed ChAT expression in dendrites, neurites and somata of putative basiconic-type sensillae at the first instar stage. We also detected vAChT in the sensory axons of these sensillae in a major antennal nerve tract. We then examined whether evidence for cholinergic transmission is present during embryogenesis. Immunolabeling confirms that vAChT is expressed in somata typical of campaniform sensillae, as well as in small sensory cell clusters typically associated with either a large basiconic or coeloconic sensilla, at 99% of embryogenesis. The vAChT is also expressed in the somata of these sensilla types in multiple antennal regions at 90% of embryogenesis, but not at earlier (70%) embryonic stages. Neuromodulators are known to appear late in embryogenesis in neurons of the locust central complex, and the cholinergic system of the antenna may also only reach maturity shortly before hatching.

Acrylamide is found in food products manufactured with high-temperature processing, and exposure to acrylamide contained in food products may cause a potential risk to human health. The aim of this investigation was to demonstrate the changes in the population of CART-, nNOS-, and VAChT-immunoreactive enteric neurons in the porcine stomach in response to supplementation of low and high acrylamide doses. The study was carried out with 15 Danish landrace gilts divided into three experimental groups: the control group&mdash, animals were administered empty gelatine capsules, the low-dose group&mdash, animals were administrated a tolerable daily intake (TDI) dose (0.5 &micro, g/kg of body weight (b.w.)/day) of acrylamide capsules, and the high-dose group&mdash, animals were administrated high-dose (ten times higher than TDI: 5 &micro, g/kg b.w./day) acrylamide capsules for 28 days. Using the double immunofluorescence staining method, it was established that supplementation with low and high doses of acrylamide resulted in alterations of the porcine stomach neuron phenotype, which was reflected in an increased number of CART-, VAChT-, and nNOS-immunoreactive neurons. These changes were accompanied by an increased density of CART-, VAChT-, and nNOS-positive fibres. The results suggest that the enteric nervous system plays an important role in protecting the gastrointestinal tract during acrylamide intoxication.

Acetylcholine (ACh) has been suggested to facilitate plasticity and improve functional recovery after different types of brain lesions. Interestingly, numerous studies have shown that striatal cholinergic interneurons are relatively resistant to acute ischemic insults, but whether ACh released by these neurons enhances functional recovery after stroke is unknown. We investigated the role of endogenous striatal ACh in stroke lesion volume and functional outcomes following middle cerebral artery occlusion to induce focal ischemia in striatum-selective vesicular acetylcholine transporter-deficient mice (stVAChT-KO). As transporter expression is almost completely eliminated in the striatum of stVAChT-KO mice, ACh release is nearly abolished in this area. Conversely, in other brain areas, VAChT expression and ACh release are preserved. Our results demonstrate a larger infarct size after ischemic insult in stVAChT-KO mice, with more pronounced functional impairments and increased mortality than in littermate controls. These changes are associated with increased activation of GSK-3, decreased levels of β-catenin, and a higher permeability of the blood–brain barrier in mice with loss of VAChT in striatum neurons. These results support a framework in which endogenous ACh secretion originating from cholinergic interneurons in the striatum helps to protect brain tissue against ischemia-induced damage and facilitates brain recovery by supporting blood–brain barrier function.

The choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT) are fundamental to neurophysiological functions of the central cholinergic system. We confirmed and quantified the presence of extracellular ChAT protein in human plasma and also characterized ChAT and VAChT polymorphisms, protein and activity levels in plasma of Alzheimer's disease patients (AD; N = 112) and in cognitively healthy controls (EC; N = 118). We found no significant differences in plasma levels of ChAT activity and protein between AD and EC groups. Although no differences were observed in plasma ChAT activity and protein concentration among ChEI-treated and untreated AD patients, ChAT activity and protein levels variance in plasma were higher among the rivastigmine-treated group (ChAT protein: p = 0.005; ChAT activity: p = 0.0002). Moreover, AD patients homozygous for SNP rs1880676 A allele exhibited higher levels of ChAT activity. Considering this is the first study to report the influence of genetic variability of CHAT locus over ChAT activity in AD patients plasma, it opens a new set of important questions on peripheral cholinergic signaling in AD.

CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior O aumento da quantidade de glutamato por vesícula em Drosofila é compensado pela diminuição do número de vesículas liberadas por estimulação nervosa. Este mecanismo compensatório capaz de reestabelecer a homeostasia sináptica é chamado de depressão homeostática pré-sináptica (DHP). Nós hipotetizamos que o aumento crônico da liberação de acetilcolina (ACh) por vesícula na junção neuromuscular de camundongos (JNM) pode resultar em uma diminuição compensatória na liberação vesicular evocada. Para testar isto, nos utilizamos a linhagem de camundongos ChAT-ChR2-EYFP (Hyper VAChT) com 3 e 24 meses de idade que possuem um aumento dos níveis de expressão de VAChT, e seus controles selvagens (WT). Foram avaliados potenciais de placa espontâneos (MEPPs) e evocados (EPPs), em baixa (0.2 e 0.3Hz) e alta (20 e 30Hz) frequência de estimulação na junção neuromuscular do músculo diafragma. Nós também registramos, em Voltage Clamp, as correntes de placa em miniatura (MEPCs). Aos 3 meses de idade, os camundongos Hyper VAChT apresentaram aumento no tamanho e frequência dos quanta. O conteúdo quântico dos camundongos Hyper VAChT (29±2,5, média±EPM, n= 4 animais) não apresentou diferença significativa (p=0,44) em relação aos WT (32±2.2, média±EPM, n= 4 animais). A depressão sináptica foi 19% maior nos camundongos Hyper VAChT, e a PTP não foi estatisticamente diferente. Durante a estimulação tetânica, a área dos MEPPs dos camundongos Hyper VAChT foi menor que antes do trem de estimulação, enquanto que este parâmetro não foi alterado nos animais WT. Isto sugere que o aumento da depressão nos animais Hyper VAChT é devido ao menor efeito da liberação vesicular. Aos 24 meses de idade, os camundongos Hyper VAChT apresentaram uma diminuição significativa no CQ (WT= 41±3.5, média±EPM, n= 5 animais; HyperVAChT= 29±4.1, média±EPM, n= 3 animais; p=0.07). A depressão sináptica e a potenciação pós tetânica (PTP) não foram estatisticamente diferentes. Estes resultados mostram que os camundongos Hyper VAChT compensaram o aumento no tamanho dos quanta de duas maneiras diferentes: aos 3 meses, os EPPs em altas frequências de estimulação reduziram por aumento de dessensibilização ou diminuição da quantidade de ACh por vesícula. Enquanto que aos 24 meses observamos diminuição do CQ. Nossos dados mostram que o aumento crônico da liberação de ACh por vesícula sináptica aciona o mecanismo pré-sináptico de DHP, que se instala durante o envelhecimento dos animais, diminuindo o número de vesículas liberadas. Este mecanismo não é visto em camundongos jovens, que mantem as taxas de disparo pós-sináptico através de outros mecanismos. The increased amount of glutamate per vesicle in Drosophila is compensated by a decrease in the number of vesicles released by nerve stimulation. This compensatory mechanism capable of restoring synaptic homeostasis is called presynaptic homeostatic depression (PHD). We hypothesized that chronic increase in vesicular acetylcholine (ACh) release at the mouse neuromuscular junction (NMJ) may result in a compensatory decrease in evoked vesicular release. To test this, we used 3 and 24 months old ChAT-ChR2-EYFP (Hyper VAChT) mice that have increased levels of VAChT expression, and their wild-type controls (WT). All experimental procedures were approved by the Animal Ethics Committee (protocol number 106/2015 - CEUA). Spontaneous and evoked endplate potentials (EPPs) were evaluated at low (0.2 and 0.3Hz) and high (20 and 30Hz) frequency of stimulation at the NMJ of the diaphragm muscle. We also recorded, in voltage clamp, miniature plate currents (MEPCs). At 3 months of age, Hyper VAChT mice presented increased quantal size and frequency. The quantal content (QC) of Hyper VAChT mice (29 ± 2.5, mean ± SEM, n = 4 animals) showed no significant difference (p = 0.44) from the WT controls (31.5 ± 2.2, mean ± SEM, n = 4 animals). Synaptic depression was 19% higher in Hyper VAChT mice, and PTP was not statistically different. During tetanic stimulation, Hyper VAChT MEPPs areas were smaller than before the train of stimuli, while in the WT mice there was no change during high frequency stimulation. This suggests that the increased depression observed in Hyper VAChT mice is due a decrease effect of vesicular release. At 24 months of age, Hyper VAChT mice showed a significant decrease in QC (WT = 41 ± 3.5, mean ± SEM, n= 5 animals; Hyper VAChT = 29 ± 4.1, mean ± SEM, n = 3 animals; p = 0.07). Synaptic depression and postetanic potentiation (PTP) were not statistically different. These results show that Hyper VAChT mice compensated for the increase in quanta size in two different ways: at 3 months, EPPs at high frequency stimulation decreased by increased desensitization or decreased ACh per vesicle. While at 24 months, we observed a decreased in QC. Our data show that longterm increase in ACh per quantum triggers the pre-synaptic mechanism of DHP seeing as a decrease in the number of vesicles released. This mechanism is not seeing in young animals, which keep the same postsynaptic output through other mechanism.

In addition to driving contraction of skeletal muscles, acetylcholine (ACh) acts as an anti-synaptogenic agent at neuromuscular junctions (NMJs). Previous studies suggest that aging is accompanied by increases in cholinergic activity at the NMJ, which may play a role in neuromuscular degeneration. In this study, we hypothesized that moderately and chronically reducing ACh could attenuate the deleterious effects of aging on NMJs and skeletal muscles. To test this hypothesis, we analyzed NMJs and muscle fibers from heterozygous transgenic mice with reduced expression of the vesicular ACh transporter (VAChT; VKDHet), which present with approximately 30% less synaptic ACh compared to control mice. Because ACh is constitutively decreased in VKDHet, we first analyzed developing NMJs and muscle fibers. We found no obvious morphological or molecular differences between NMJs and muscle fibers of VKDHet and control mice during development. In contrast, we found that moderately reducing ACh has various effects on adult NMJs and muscle fibers. VKDHet mice have significantly larger NMJs and muscle fibers compared to age-matched control mice. They also present with reduced expression of the pro-atrophy gene, Foxo1, and have more satellite cells in skeletal muscles. These molecular and cellular features may partially explain the increased size of NMJs and muscle fibers. Thus, moderately reducing ACh may be a therapeutic strategy to prevent the loss of skeletal muscle mass that occurs with advancing age. National Institute on Aging (NI) [R01AG05545, R56AG051501]; National Institute of Neurological Disorders and Stroke (NINDS)United States Department of Health & Human ServicesNational Institutes of Health (NIH) - USANIH National Institute of Neurological Disorders & Stroke (NINDS) [R21NS106313] Grants from the National Institute on Aging (NI grants R01AG05545 and R56AG051501) and the National Institute of Neurological Disorders and Stroke (NINDS grant R21NS106313) supported this study.

Rationale: Les électrodes implantables pour la création d’une interface neurotechnologique avec le système nerveux périphérique représentent une solution prometteuse pour pallier aux limitations causées par des blessures ou maladies nerveuses. Leur viabilité à long terme pourrait être améliorée en enrobant l’électrode d’une substance synthétique tolérable par l’hôte. La synaptogenèse sur des matériaux artificiels serait un marqueur indirect de la biocompatibilité, démontrant la reconnaissance de la substance comme étant une cible post-synaptique viable. Notre objectif est de démontrer la propriété synaptogénique de la poly-D-lysine (PDL) et d’un polyglycérol hyperbranché expérimental (dendrimère, DND) dans le système nerveux périphérique humain in vitro et murin in vivo. Ultimement, nous visons la création d’un enrobage facilitant la synaptogenèse sur un objet synthétique qui sera inséré dans un nerf périphérique dans le but d’augmenter la longévité de l’implant. Méthodes: In vitro, des neurones moteurs dérivés de cellules souches pluripotentes induites humaines ont été cultivés avec des microsphères (non-enrobé, enrobé de poly-D-lysine ou de dendrimère) pour 1, 3, 5, 7 ou 9 jours. In vivo, le nerf sciatique du rat a été écrasé avant l’injection de microsphères proximalement, distalement et au site de la blessure qui y demeureront pour 2, 7 ou 14 jours. Les cellules et les nerfs ont été traités à l’immunochimie pour les marqueurs présynaptiques synaptophysine et vesicular acetylcholine transporter (VAchT). Les images acquises à la microscopie confocale ont été analysées pour l’intensité de la fluorescence autour des microsphères sur le logiciel Fiji. Une imagerie en temps réel in vitro avec du FM-143 a été réalisée pour vérifier la présence d’exocytose près des sites de contact des microsphères. Résultats: In vitro, les résultats démontrent significativement plus de fluorescence pour la synaptophysine (p, Background: Implantable intraneural electrodes as a neurotechnological interface with the peripheral nervous system are a promising solution to overcome the limitations caused by injury or disease. Their long-term viability could be enhanced by coating electrodes with synthetic substances which are well tolerated by the host. Synaptogenesis onto artificial material can be seen as an indirect marker of biocompatibility as it demonstrates the recognition of the said substance as a viable post-synaptic target. Our objective is to demonstrate the synaptogenic properties of poly-D-lysine (PDL) and an experimental hyperbranched polyglycerol (dendrimer, DND) with human peripheral neurons in vitro and rat neurons in vivo. Ultimately, we aim to develop coatings that promote synaptogenesis onto a synthetic object embedded in a peripheral nerve with the objective of increasing its longevity. Methods: Uncoated (control) and coated (poly-D-lysine or dendrimer) microspheres were used. In vitro, motor neurons derived from human induced pluripotent stem cells (iPSCs) were grown with microspheres for 1, 3, 5, 7 or 9 days. In vivo, a nerve crush injury was introduced to adult rat sciatic nerve to induce a regenerative state before injecting microspheres proximally, distally and at the crush site, which were left for 2, 7 and 14 days. Cells and nerves were immunostained for synaptophysin and VAchT (presynaptic markers). All experiments were visualized using confocal microscopy and images were analyzed for staining intensity surrounding the microspheres using Fiji. Live cell imaging of human motor neurons with FM-143 was performed to verify the presence of exocytosis at contact sites with the microspheres. Results: In vitro, our results show significantly more staining for synaptophysin (p

© FASEB The pedunculopontine tegmental nucleus (PPT) and laterodorsal tegmental nucleus (LDT) are heterogeneous brainstem structures that contain cholinergic, glutamatergic, and GABAergic neurons. PPT/LDT neurons are suggested to modulate both cognitive and noncognitive functions, yet the extent to which acetylcholine (ACh) signaling from the PPT/LDT is necessary for normal behavior remains uncertain. We addressed this issue by using a mouse model in which PPT/LDT cholinergic signaling is highly decreased by selective deletion of the vesicular ACh transporter (VAChT) gene. This approach interferes exclusively with ACh signaling, leaving signaling by other neurotransmitters from PPT/LDT cholinergic neurons intact and sparing other cells. VAChT mutants were examined on different PPT/LDT-associated cognitive domains. Interestingly, VAChT mutants showed no attentional deficits and only minor cognitive flexibility impairments while presenting large deficiencies in both spatial and cued Morris water maze (MWM) tasks. Conversely, working spatial memory determined with the Y-maze and spatial memory measured with the Barnes maze were not affected, suggesting that deficits in MWM were unrelated to spatial memory abnormalities. Supporting this interpretation, VAChT mutants exhibited alterations in anxiety-like behavior and increased corticosterone levels after exposure to the MWM, suggesting altered stress response. Thus, PPT/LDT VAChT-mutant mice present little cognitive impairment per se, yet they exhibit increased susceptibility to stress, which may lead to performance deficits in more stressful conditions.—Janickova, H., Kljakic, O., Rosborough, K., Raulic, S., Matovic, S., Gros, R., Saksida, L. M., Bussey, T. J., Inoue, W., Prado, V. F., Prado, M. A. M. Selective decrease of cholinergic signaling from pedunculopontine and laterodorsal tegmental nuclei has little impact on cognition but markedly increases susceptibility to stress. FASEB J. 33, 7018–7036 (2019). www.fasebj.org.

Endogenous acetylcholine (ACh), which depends of the levels of vesicular ACh transport (VAChT) to be released, is the central mediator of the cholinergic anti-inflammatory system. ACh controls the release of cytokine in different models of inflammation. Diesel exhaust particles (DEP) are one of the major environmental pollutants produced in large quantity by automotive engines in urban center. DEP bind the lung parenchyma and induce inflammation. We evaluated whether cholinergic dysfunction worsens DEP-induced lung inflammation. Male mice with decreased ACh release due to reduced expression of VAChT (VAChT-KD mice) were submitted to DEP exposure for 30 days (3 mg/mL of DEP, once a day, five days a week) or saline. Pulmonary function and inflammation as well as extracellular matrix fiber deposition were evaluated. Additionally, airway and nasal epithelial mucus production were quantified. We found that DEP instillation worsened lung function and increased lung inflammation. Higher levels of mononuclear cells were observed in the peripheral blood of both wild-type (WT) and VAChT-KD mice. Also, both wild-type (WT) and VAChT-KD mice showed an increase in macrophages in bronchoalveolar lavage fluid (BALF) as well as increased expression of IL-4, IL-6, IL-13, TNF-alpha, and NF-kappa B in lung cells. The collagen fiber content in alveolar septa was also increased in both genotypes. On the other hand, we observed that granulocytes were increased only in VAChT-KD peripheral blood. Likewise, increased BALF lymphocytes and neutrophils as well as increased elastic fibers in alveolar septa, airway neutral mucus, and nasal epithelia acid mucus were observed only in VAChT-KD mice. The cytokines IL-4 and TNF-alpha were also higher in VAChT-KD mice compared with WT mice. In conclusion, decreased ability to release ACh exacerbates some of the lung alterations induced by DEP in mice, suggesting that VAChT-KD animals are more vulnerable to the effects of DEP in the lung.

Acetylcholine (ACh) has a crucial role in the peripheral and central nervous systems. The enzyme choline acetyltransferase (ChAT) is responsible for synthesizing ACh from acetyl-CoA and choline in the cytoplasm and the vesicular acetylcholine transporter (VAChT) uptakes the neurotransmitter into synaptic vesicles. Following depolarization, ACh undergoes exocytosis reaching the synaptic cleft, where it can bind its receptors, including muscarinic and nicotinic receptors. ACh present at the synaptic cleft is promptly hydrolyzed by the enzyme acetylcholinesterase (AChE), forming acetate and choline, which is recycled into the presynaptic nerve terminal by the high-affinity choline transporter (CHT1). Cholinergic neurons located in the basal forebrain, including the neurons that form the nucleus basalis of Meynert, are severely lost in Alzheimer's disease (AD). AD is the most ordinary cause of dementia affecting 25 million people worldwide. The hallmarks of the disease are the accumulation of neurofibrillary tangles and amyloid plaques. However, there is no real correlation between levels of cortical plaques and AD-related cognitive impairment. Nevertheless, synaptic loss is the principal correlate of disease progression and loss of cholinergic neurons contributes to memory and attention deficits. Thus, drugs that act on the cholinergic system represent a promising option to treat AD patients.

Memory extinction is referred to as a learning process in which a conditioned response (CR) progressively reduces over time as an animal learns to uncouple a response from a stimulus. Extinction occurs when the rat is placed into a context without shock after training. Docosahexaenoic acid (DHA, C22:6, n-3) is implicated in memory formation in mammalian brains. In a two-way active shuttle-avoidance apparatus, we examined whether DHA affects the extinction memory and the expression of brain cognition-related proteins, including gastrin-releasing peptide receptor (GRPR), brain-derived neurotrophic factor receptor (BDNFR) tyrosine kinase receptor B (TrKB), and N-methyl-d-aspartate receptor (NMDAR) subunits NR2A and NR2B. Also, the protein levels of GRP, BDNF, postsynaptic density protein-95 (PSD-95), and vesicular acetylcholine transporter (VAChT), and the antioxidative potentials, in terms of lipid peroxide (LPO) and reactive oxygen species (ROS), were examined in the hippocampus. During the acquisition phase, the rats received a conditioned stimulus (CS-tone) paired with an unconditioned stimulus (UCS foot shock) for three consecutive days (Sessions S1, S2, and S3, each consisting of 30-trials) after 12 weeks of oral administration of DHA. After a three-day interval, the rats were re-subjected to two extinction sessions (S4, S5), each comprising 30 trials of CS alone. During the acquisition training in S1, the shock-related avoidance frequency (acquisition memory) was significantly higher in the DHA-administered rats compared with the control rats. The avoidance frequency, however, decreased with successive acquisition trainings in sessions S2 and S3. When the rats were subjected to the extinction sessions after a break for consolidation, the conditioned response (CR) was also significantly higher in the DHA-administered rats. Interestingly, the freezing responses (frequency and time) also significantly decreased in the DHA-administered rats, thus suggesting that a higher coping capacity was present during fear stress in the DHA-administered rats. DHA treatments increased the mRNA levels of GRPR, BDNF receptor TrKB, and NMDAR subunit NR2B. DHA also increased the protein levels of GRP, BDNF, PSD-95, and VAChT, and the antioxidative potentials in the hippocampus. These results suggest the usefulness of DHA for treating stress disorders.

Nine fluorine-containing vesicular acetylcholine transporter (VAChT) inhibitors were synthesized and screened as potential PET tracers for imaging the VAChT. Compound 18a was one of the most promising carbonyl-containing benzovesamicol analogs; the minus enantiomer, (−)- 18a displayed high potency (VAChT K i = 0.59 ± 0.06 nM) and high selectivity for VAChT versus σ receptors (>10,000-fold). The radiosynthesis of (−)-[ 18 F] 18a was accomplished by a two-step procedure with 30–40% radiochemical yield. Preliminary biodistribution studies of (−)-[ 18 F] 18a in adult male Sprague-Dawley rats at 5, 30, 60 and 120 min post-injection (p.i.) were promising. The total brain uptake of (−)-[ 18 F] 18a was 0.684 %ID/g at 5 min p.i. and by 120 min p.i. slowly washed out to 0.409 %ID/g; evaluation of regional brain uptake showed stable levels of ∼0.800 %ID/g from 5 to 120 min p.i in the VAChT-enriched striatal tissue of rats, indicating the tracer had crossed the blood brain barrier and was retained in the striatum. Subsequent microPET brain imaging studies of (−)-[ 18 F] 18a in nonhuman primates (NHPs) showed high striatal accumulation in the NHP brain; the standardized uptake value (SUV) for striatum reached a maximum value of 5.1 at 15 min p.i. The time–activity curve for the target striatal region displayed a slow and gradual decreasing trend 15 min after injection, while clearance of the radioactivity from the cerebellar reference region was much more rapid. Pretreatment of NHPs with 0.25 mg/kg of the VAChT inhibitor (−)-vesamicol resulted in a ∼90% decrease of striatal uptake compared to baseline studies. HPLC metabolite analysis of NHP plasma revealed that (−)-[ 18 F] 18a had a good in vivo stability. Together, these preliminary results suggest (−)-[ 18 F] 18a is a promising PET tracer candidate for imaging VAChT in the brain of living subjects.

To verify vesamicol as lead structure in the development of radioligands for imaging of VAChT in the brain by PET, we systematically modified this molecule and investigated five different groups of derivatives. Structural changes were conducted in all three ring systems A, B, and C resulting in a library of 67 different vesamicol analogs. Based on their in vitro binding affinity toward VAChT as well as σ1 and σ2 receptors, we performed an extensive structure-affinity relationship (SAR) study regarding both affinity and selectivity. The compounds possessed VAChT affinities in the range of 1.32 nM (benzovesamicol) to > 10 µM and selectivity factors from 0.1 to 73 regarding σ1 and σ2 receptors, respectively. We could confirm the exceptional position of benzovesamicols as most affine VAChT ligands. However, we also observed that most of the compounds with high VAChT affinity demonstrated considerable affinity in particular to the σ1 receptor. Finally, none of the various vesamicol analogs in all five groups showed an in vitro binding profile suitable for specific VAChT imaging in the brain.

The identification of neurotransmitter type used by a neuron is important for the functional dissection of neuronal circuits. In the model organism Drosophila melanogaster, several methods for discerning the neurotransmitter systems are available. Here, we expanded the toolbox for the identification of cholinergic neurons by generating a new line FRT-STOP-FRT-VAChT::HA that is a conditional tagged knock-in of the vesicular acetylcholine transporter (VAChT) gene in its endogenous locus. Importantly, in comparison to already available tools for the detection of cholinergic neurons, the FRT-STOP-FRT-VAChT::HA allele also allows for identification of the subcellular localization of the cholinergic presynaptic release sites in a cell-specific manner. We used the newly generated FRT-STOP-FRT-VAChT::HA line to characterize the Mi1 and Tm3 neurons in the fly visual system and found that VAChT is present in the axons of both cell types, suggesting that Mi1 and Tm3 neurons provide cholinergic input to the elementary motion detectors, the T4 neurons., Summary: A new transgenic Drosophila melanogaster line for the cell-type-specific identification of cholinergic release sites expands the available methods toolbox for discerning the neurotransmitter systems in the fly nervous system.

The vesicular acetylcholine transporter (VAChT) is an important target for in vivo imaging of neurodegenerative processes using positron emission tomography (PET). So far the development of VAChT PET radioligands is based on the single known lead compound vesamicol. In this study we investigated a recently published spiroindoline based compound class (Sluder et al., 2012), which was suggested to have potential in the development of VAChT ligands. Therefore, we synthesized a small series of N,N-substituted spiro[indoline-3,4'-piperidine] derivatives and determined their in vitro binding affinities toward the VAChT. In order to investigate the selectivity, the off-target binding toward σ1 and σ2 receptors was determined. The compounds possessed VAChT affinities with Ki values in the range of 39-376nM. Binding affinities toward the σ1 and σ2 receptors are in a similar range indicating that the strong structural difference between the spiroindolines and vesamicol did not improve the selectivity. The observed potential to additionally bind to σ receptors let us assume that the herein investigated spiroindolines are not suitable to replace vesamicol as lead compound for the development of VAChT ligands.

Herein, new ligands for the vesicular acetylcholine transporter (VAChT), based on a benzovesamicol scaffold, are presented. VAChT is acknowledged as a marker for cholinergic neurons and a positron emission tomography tracer for VAChT could serve as a tool for quantitative analysis of cholinergic neuronal density. With an easily accessible triflate precursor, aminocarbonylations were utilized to evaluate the chemical space around the C5 position on the tetrahydronaphthol ring. Synthesized ligands were evaluated for their affinity and selectivity for VAChT. Small, preferably aromatic, N-substituents proved to be more potent than larger substituents. Of the fifteen compounds synthesized, benzyl derivatives (±)-7i and (±)-7l had the highest affinities for VAChT. Compound (±)-7i was chosen to investigate the importance of stereochemistry for binding to VAChT and selectivity toward the σ1 and σ2 receptors. Enantiomeric resolution gave (+)-7i and (-)-7i, and the eutomer showed seven times better affinity. Although racemate (±)-7i was initially promising, the affinity of (-)-7i for VAChT was not better than 56.7nM which precludes further preclinical evaluation. However, the nanomolar binding together with the ready synthesis of [11C]-(±)-7i shows that (-)-7i can serve as a scaffold for future optimizations to provide improved 11C-labelled VAChT PET tracers.

The model genetic organism Drosophila melanogaster, commonly known as the fruit fly, uses many of the same neurotransmitters as mammals and very similar mechanisms of neurotransmitter storage, release and recycling. This system offers a variety of powerful molecular-genetic methods for the study of transporters, many of which would be difficult in mammalian models. We review here progress made using Drosophila to understand the function and regulation of neurotransmitter transporters and discuss future directions for its use.

PurposeThe vesicular acetylcholine transporter (VAChT) is a specific biomarker for imaging presynaptic cholinergic neurons. Herein, two potent and selective (11)C-labeled VAChT inhibitors were evaluated in rodents and nonhuman primates for imaging VAChT in vivo.ProceduresFor both (-)-[(11)C]2 and (-)-[(11)C]6, biodistribution, autoradiography, and metabolism studies were performed in male Sprague Dawley rats. Positron emission tomography (PET) brain studies with (-)-[(11)C]2 were performed in adult male cynomolgus macaques; 2 h dynamic data was acquired, and the regions of interest were drawn by co-registration of the PET images with the MRI.ResultsThe resolved enantiomers (-)-2 and (-)-6 were very potent and selective for VAChT in vitro (K i 35-fold selectivity for VAChT vs. σ receptors); both radioligands, (-)-[(11)C]2 and (-)-[(11)C]6, demonstrated high accumulation in the VAChT-enriched striatum of rats. (-)-[(11)C]2 had a higher striatum to cerebellum ratio of 2.4-fold at 60 min; at 30 min, striatal uptake reached 0.550 ± 0.086 %ID/g. Uptake was also specific and selective; following pretreatment with (±)-2, striatal uptake of (-)-[(11)C]2 in rats at 30 min decreased by 50 %, while pretreatment with a potent sigma ligand had no significant effect on striatal uptake in rats. In addition, (-)-[(11)C]2 displayed favorable in vivo stability in rat blood and brain. PET studies of (-)-[(11)C]2 in nonhuman primates indicate that it readily crosses the blood-brain barrier (BBB) and provides clear visualization of the striatum; striatal uptake reaches the maximum at 60 min, at which time the target to nontarget ratio reached ~2-fold.ConclusionsThe radioligand (-)-[(11)C]2 has high potential to be a suitable PET radioligand for imaging VAChT in the brain of living subjects.

Cholinergic innervation is extensive throughout the central and peripheral nervous systems. Among its many roles, the neurotransmitter acetylcholine (ACh) contributes to the regulation of motor function, locomotion, and exploration. Cholinergic deficits and replacement strategies have been investigated in neurodegenerative disorders, particularly in cases of Alzheimer's disease (AD). Focus has been on blocking acetylcholinesterase (AChE) and enhancing ACh synthesis to improve cholinergic neurotransmission. As a first step in evaluating the physiological effects of enhanced cholinergic function through the upregulation of the vesicular acetylcholine transporter (VAChT), we used the hypercholinergic B6eGFPChAT congenic mouse model that has been shown to contain multiple VAChT gene copies. Analysis of biochemical and behavioral paradigms suggest that modest increases in VAChT expression can have a significant effect on spontaneous locomotion, reaction to novel stimuli, and the adaptation to novel environments. These observations support the potential of VAChT as a therapeutic target to enhance cholinergic tone, thereby decreasing spontaneous hyperactivity and increasing exploration in novel environments.

Acetylcholine (ACh) is important for different cognitive functions such as learning, memory and attention. The release of ACh depends on its vesicular loading by the vesicular acetylcholine transporter (VAChT). It has been demonstrated that VAChT expression can modulate object recognition memory. However, the role of VAChT expression on object recognition memory persistence still remains to be understood. To address this question we used distinct mouse lines with reduced expression of VAChT, as well as pharmacological manipulations of the cholinergic system. We showed that reduction of cholinergic tone impairs object recognition memory measured at 24h. Surprisingly, object recognition memory, measured at 4 days after training, was impaired by substantial, but not moderate, reduction in VAChT expression. Our results suggest that levels of acetylcholine release strongly modulate object recognition memory consolidation and appear to be of particular importance for memory persistence 4 days after training.

Although there are number of studies showing that phosphodiesterase (PDE) 4 and 5 inhibitors affect different kinds of memory, their effects on spatial memory consolidation in conjunction with the cholinergic activity in the hippocampus have not been studied before. In the present study firstly, rats were evaluated for the effects of different doses of the PDE4 inhibitor rolipram and the PDE5 inhibitor sildenafil on spatial memory consolidation in the water maze task. Rolipram or sildenafil was daily administered intraperitoneally 3 or 0 h after the last trial of training, respectively. Then in a separate related experiment the effect of the most efficient doses of rolipram or sildenafil accompanied by an intrahippocampally injected protein kinase A (PKA) or protein kinase G (PKG) inhibitor, respectively, was examined. Finally for determination of the hippocampal cholinergic activity the protein expression of hippocampal vesicular acetylcholine transporter (VAChT) and cholineacetyltransferase (ChAT) was measured. Rolipram at 0.03 mg/kg as well as sildenafil at 3 mg/kg increased spatial memory and their enhancing effect was completely blocked following inhibition of PKA and PKG, respectively. Furthermore, none of the treatments had a significant effect on the hippocampal ChAT and VAChT levels. Our data showed that rolipram and sildenafil enhanced spatial memory consolidation in an inverted U-shaped dose-response curve. This effect is dependent on the activity of cAMP/PKA- and cGMP/PKG-mediated pathways, respectively in the hippocampus. However, we did not find evidence for a chronic increase of cholinergic activity in the observed PDE inhibitor-induced memory improvement.

Cholinergic vulnerability, characterized by loss of acetylcholine (ACh), is one of the hallmarks of Alzheimer9s disease (AD). Previous work has suggested that decreased ACh activity in AD may contribute to pathological changes through global alterations in alternative splicing. This occurs, at least partially, via the regulation of the expression of a critical protein family in RNA processing, heterogeneous nuclear ribonucleoprotein (hnRNP) A/B proteins. These proteins regulate several steps of RNA metabolism, including alternative splicing, RNA trafficking, miRNA export, and gene expression, providing multilevel surveillance in RNA functions. To investigate the mechanism by which cholinergic tone regulates hnRNPA2/B1 expression, we used a combination of genetic mouse models and in vivo and in vitro techniques. Decreasing cholinergic tone reduced levels of hnRNPA2/B1, whereas increasing cholinergic signaling in vivo increased expression of hnRNPA2/B1. This effect was not due to decreased hnRNPA2/B1 mRNA expression, increased aggregation, or degradation of the protein, but rather to decreased mRNA translation by nonsense-mediated decay regulation of translation. Cell culture and knock-out mice experiments demonstrated that M1 muscarinic signaling is critical for cholinergic control of hnRNPA2/B1 protein levels. Our experiments suggest an intricate regulation of hnRNPA2/B1 levels by cholinergic activity that interferes with alternative splicing in targeted neurons mimicking deficits found in AD. SIGNIFICANCE STATEMENT In Alzheimer9s disease, degeneration of basal forebrain cholinergic neurons is an early event. These neurons communicate with target cells and regulate their long-term activity by poorly understood mechanisms. Recently, the splicing factor hnRNPA2/B, which is decreased in Alzheimer9s disease, was implicated as a potential mediator of long-term cholinergic regulation. Here, we demonstrate a mechanism by which cholinergic signaling controls the translation of hnRNPA2/B1 mRNA by activation of M1 muscarinic type receptors. Loss of cholinergic activity can have profound effects in target cells by modulating hnRNPA2/B1 levels.

CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior Após a aplicação de solução hipertônica nas sinapses, ocorre aumento duradouro na liberação de neurotransmissores, evidenciado pelo aumento no tamanho dos potenciais de placa em miniatura (MEPPs). O aumento no tamanho dos quanta foi interpretado como sendo devido à maior incorporação de acetilcolina mediada pelo transportador vesicular de acetilcolina (VAChT) nas vesículas prontamente disponíveis para liberação, num processo chamado de segundo estágio de preenchimento vesicular. Nosso objetivo foi testar esta hipótese sem a utilização de fármacos, utilizando animais geneticamente deficientes do VAChT, no intuito de pesquisar o papel deste transportador nestas modificações duradouras na neurotransmissão. Nós utilizamos preparações neuromusculares de camundongos C57BL machos, que apresentam reduzida expressão de VAChT (KDVAChT). Para avaliar o desenvolvimento temporal do segundo estágio de preenchimento vesicular, nós utilizamos animais em diferentes idades. Para medida dos MEPPs, utilizamos a técnica de current clamp. Foram registrados cerca de 100 MEPPs durante 300 segundos, em cinco fibras diferentes de cada animal, antes e após o tratamento com solução hipertônica (234 mM de NaCl, 521 mOsmol/kg). Para medida das correntes de placa em miniatura (MEPCs), utilizamos a técnica de voltage clamp e um protocolo semelhante de amostragem. Nós não observamos diferenças significativas no tamanho dos MEPPs dos camundongos KDVAChT quando comparados aos selvagens (WT). Nos animais de 3 meses, após o tratamento hipertônico, a amplitude nos WT passou de 1,13 ± 0,19 mV para 1,55 ± 0,13 mV (n=9) e nos KDVAChT passou de 0,79 ± 0,09 mV para 1,21 ± 0,11 mV (n=5). Nos animais de 12 meses, após o tratamento hipertônico, a amplitude nos WT passou de 1,29 ± 0,14 mV para 1,69 ± 0,15 mV (n=11) e nos KDVAChT passou de 0,98 ± 0,14 mV para 1,75 ± 0,15 mV (n=9). As diferenças entre as medições pré e pós tratamento hipertônico foram estatisticamente significativas nos grupos WT e KDVAChT. Entretanto não houve diferença no aumento dos MEPPs entre estes grupos. Também não observamos diferenças significativas no tamanho das MEPCs entre os grupos WT e KDVAChT sem o tratamento hipertônico. O tamanho das MEPCs nos animais WT foi de 3,02 ± 0,19 nA (n=3), e nos animais KDVAChT foi de 2.57 ± 0.23 nA (n=4). Nós concluímos que a diminuição do VAChT não alterou o segundo estágio de preenchimento vesicular de acetilcolina, o que sugere que o VAChT pode não ser o alvo deste processo, ou que possam existir mecanismos ainda desconhecidos capazes de suprir a redução desta proteína no transporte vesicular. After application of hypertonic solution to synapses, there is a long lasting increase in neurotransmitter release, as evidenced by an increase in the size of miniature endplate potentials (MEPPs). The increase in quantal size was interpreted as being due to increased incorporation of the acetylcholine into readily available to release vesicles in a process dependent of the vesicle acethylcholine transporter (VAChT). This process was called vesicle second stage loading. Our goal was to test this hypothesis using non pharmacological tools, in order to study the participation of VAChT in this long lasting changes in neurotransmission. We used neuromuscular preparations from genetically modified C57BL male mice with reduced VAChT expression (KDVAChT). We used animals at two different ages to evaluate the temporal development of second stage loading. To measure MEPPs, we used the current clamp technique. We recorded about 100 MEPPs during 300 seconds at five different fibers of each animal, before and after treatment with hypertonic solution (NaCl 234mM). To measure the MEPCs, we used the voltage clamp technique and a similar sampling protocol. We did not detect significant differences between WT and KDVAChT in MEPP size before and after treatment with hypertonic solution. In WT 3 months old animals hypertonic treatment increased MEPPs from 1.13 ± 0.19 to 1.55 ± 0.13 mV (n = 9) and in KDVAChT it increased from 0.79 ± 0, 09 mV to 1.21 ± 0.11 mV (n = 5). In 12 months old animals hypertonic treatment increased MEPP amplitude from 1.29 ± 0.14 mV to 1.69 ± 0.15 mV (n = 11) and in KDVAChT it increased from 0.98 ± 0, 14 mV to 1.75 ± 0.15 mV (n = 9). We did not observe significant differences in the size of MEPCs between WT and KDVAChT mice, when recorded before hypertonic stimulation. MEPCs size in WT animals was 3.02 ± 0.19 nA, n = 3, while in KDVAChT animals it was 2:57 ± 0:23 nA, n = 4. We conclude that reducing VAChT did not change second stage loading, which suggests that VAChT is not the target of hypertonic solution effect, or there may be unknown mechanisms to compensate vesicular filling in reduced VAChT animals.

Autonomic dysfunction is a characteristic of cardiac disease and decreased vagal activity is observed in heart failure. Rodent cardiomyocytes produce de novo ACh, which is critical in maintaining cardiac homeostasis. We report that this nonneuronal cholinergic system is also found in human cardiomyocytes, which expressed choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter (VAChT). Furthermore, VAChT expression was increased 3- and 1.5-fold at the mRNA and protein level, respectively, in ventricular tissue from patients with heart failure, suggesting increased ACh secretion in disease. We used mice with genetic deletion of cardiomyocyte-specific VAChT or ChAT and mice overexpressing VAChT to test the functional significance of cholinergic signaling. Mice deficient for VAChT displayed an 8% decrease in fractional shortening and 13% decrease in ejection fraction compared with angiotensin II (Ang II)–treated control animals, suggesting enhanced ventricular dysfunction and pathologic remodeling in response to Ang II. Similar results were observed in ChAT-deficient mice. Conversely, no decline in ventricular function was observed in Ang II–treated VAChT overexpressors. Furthermore, the fibrotic area was significantly greater (P < 0.05) in Ang II–treated VAChT-deficient mice (3.61 ± 0.64%) compared with wild-type animals (2.24 ± 0.11%). In contrast, VAChT overexpressing mice did not display an increase in collagen deposition. Our results provide new insight into cholinergic regulation of cardiac function, suggesting that a compensatory increase in cardiomyocyte VAChT levels may help offset cardiac remodeling in heart failure.—Roy, A., Dakroub, M., Tezini, G. C. S. V., Liu, Y., Guatimosim, S., Feng, Q., Salgado, H. C., Prado, V. F., Prado, M. A. M., Gros, R. Cardiac acetylcholine inhibits ventricular remodeling and dysfunction under pathologic conditions.

Sympathetic neurons have the capability to segregate their neurotransmitters (NTs) and co-transmitters to separate varicosities of single axons; furthermore, in culture, these neurons can even segregate classical transmitters. In vivo sympathetic neurons employ acetylcholine (ACh) and other classical NTs such as gamma aminobutyric acid (GABA). Herein, we explore whether these neurons in vivo segregate these classical NTs in the superior cervical ganglia of the rat. We determined the topographical distribution of GABAergic varicosities, somatic GABAA receptor, as well as the regional distribution of the segregation of ACh and GABA. We evaluated possible regional differences in efficacy of ganglionic synaptic transmission, in the sensitivity of GABAA receptor to GABA and to the competitive antagonist picrotoxin (PTX). We found that sympathetic preganglionic neurons in vivo do segregate ACh and GABA. GABAergic varicosities and GABAA receptor expression showed a rostro-caudal gradient along ganglia; in contrast, segregation exhibited a caudo-rostral gradient. These uneven regional distributions in expression of GABA, GABAA receptors, and level of segregation correlate with stronger synaptic transmission found in the caudal region. Accordingly, GABAA receptors of rostral region showed larger sensitivity to GABA and PTX. These results suggest the presence of different types of GABAA receptors in each region that result in a different regional levels of endogenous GABA inhibition. Finally, we discuss a possible correlation of these different levels of GABA modulation and the function of the target organs innervated by rostral and caudal ganglionic neurons.

The mouse gastro-intestinal and biliary tract mucosal epithelia harbor choline acetyltransferase (ChAT)-positive brush cells with taste cell-like traits. With the aid of two transgenic mouse lines that express green fluorescent protein (EGFP) under the control of the ChAT promoter (EGFP\(^{ChAT}\)) and by using in situ hybridization and immunohistochemistry we found that EGFP\(^{ChAT}\) cells were clustered in the epithelium lining the gastric groove. EGFP\(^{ChAT}\) cells were numerous in the gall bladder and bile duct, and found scattered as solitary cells along the small and large intestine. While all EGFP\(^{ChAT}\) cells were also ChAT-positive, expression of the high-affinity choline transporter (ChT1) was never detected. Except for the proximal colon, EGFP\(^{ChAT}\) cells also lacked detectable expression of the vesicular acetylcholine transporter (VAChT). EGFP\(^{ChAT}\) cells were found to be separate from enteroendocrine cells, however they were all immunoreactive for cytokeratin 18 (CK18), transient receptor potential melastatin-like subtype 5 channel (TRPM5), and for cyclooxygenases 1 (COX1) and 2 (COX2). The ex vivo stimulation of colonic EGFP\(^{ChAT}\) cells with the bitter substance denatonium resulted in a strong increase in intracellular calcium, while in other epithelial cells such an increase was significantly weaker and also timely delayed. Subsequent stimulation with cycloheximide was ineffective in both cell populations. Given their chemical coding and chemosensory properties, EGFP\(^{ChAT}\) brush cells thus may have integrative functions and participate in induction of protective reflexes and inflammatory events by utilizing ACh and prostaglandins for paracrine signaling.

The role of striatal acetylcholine in cognition is currently unknown. A genetic mouse model has been developed which disables the cholinergic system in the striatum by targeting the Vesicular Acetylcholine Transporter (VAChT). Striatal VAChT mice were tested to determine the role of striatal ACh in cognition. Results from the present study indicate that VAChT mice are impaired at an object recognition paradigm when the memory retention delay is 15-minutes, whereas recognition is preserved with a 3-hour delay. Male mice show impairment in short-term social recognition. Mice showed normal performance on a spatial task. When assessed on the 5-choice serial reaction time task, it was found that VAChT mice had reduced attention performance. VAChT mice have impairments in behavioural flexibility when given a large amount of visual discrimination training. The present research suggests that striatal ACh is involved in aspects of cognition.

To optimize the semi-preparative separation of a 18F-labeled PET radiotracer from its nitro precursor in a recently developed radiosynthesis, we performed an analytical HPLC study using the unlabeled reference compound and the corresponding nitro precursor. Several RP phases as well as a bare silica column were investigated with ACN and MeOH as organic modifiers and aqueous NH4OAc because of the basic character of the analytes. Four types of separation were observed based on different interaction mechanisms. When ACN/20 mM NH4OAc aq. was used mainly cationic-exchange and hydrophobic interactions contribute to the retention. A reversal of elution order could be observed starting from 95% ACN and subsequent increasing of the water content. This phenomenon was observed for all RP phases and seems to be independent of the different spacers bound to the silica. By contrast, using MeOH/20 mM NH4OAc aq. the elution order depends on the phase material. Two columns with the potential to perform π-π interactions showed different separation behavior compared to the other RP phases.

This letter describes the synthesis of two regioisomers of a new class of vesamicol analogs as possible ligands for imaging the vesicular acetylcholine transporter in future PET studies. The reaction mechanism of the synthesis of these two pyrrolovesamicols was studied by HPLC and the molecular structures were determined by X-Ray structure analysis. Binding affinities to VAChT were evaluated by competitive binding analysis using a cell line stably transfected with ratVAChT.

Radiolabeled vesamicol analogs are promising candidates as ligands for the vesicular acetylcholine transporter (VAChT) to enable in vivo imaging of early cholinergic degenerations in brain. The 4-fluorobenzoyl-substituted azaspirovesamicol derivative FBASV is one out of six novel vesamicol analogs and demonstrated most appropriate in vitro binding data. 18F-radiolabeling was performed by microwave-assisted nucleophilic aromatic substitution of the corresponding nitro precursor and two methods were developed for the purification of [18F]FBASV. Utilizing method A, the remaining nitro precursor was reduced to its corresponding amine, which was separated via semi-preparative HPLC on a conventional RP column. In method B a phenyl column was used for the direct separation of [18F]FBASV and its nitro precursor, resulting in a change of the elution order and better separation parameters. Thus, [18F]FBASV was synthesized with a RCY of 16–18%, a specific activity >300 GBq/µmol, and a radiochemical purity of >99.5% suitable for future in vivo studies. Copyright © 2011 John Wiley & Sons, Ltd.

Located in presynaptic cholinergic nerve terminals, the vesicular acetylcholine transporter (VAChT) represents a potential target for quantitative visualization of early degeneration of cholinergic neurons in Alzheimer's disease using PET. Benzovesamicol derivatives are proposed as radioligands for this purpose. We report QSAR studies of vesamicol and benzovesamicol derivatives taking into account the stereoselectivity of the VAChT binding site. Use of different data sets and different models in this study revealed that both enantiomers of 5-fluoro-3-(4-phenyl-piperidin-1-yl)-1,2,3,4-tetrahydro-naphthalen-2-ol (5-FBVM) are promising candidates, with predicted VAChT affinities between 6.1 and 0.05 nM. The synthesis of enantiopure (R,R)- and (S,S)-5-FBVM and their corresponding triazene precursors for future radiofluorination is reported. Both enantiomers exhibited high in vitro affinity for VAChT [(+)-5-FBVM: K(i)=6.95 nM and (-)-5-FBVM: K(i)=3.68 nM] and were selective for σ(2) receptors (∼70-fold), only (+)-5-FBVM is selective for σ(1) receptors (∼fivefold). These initial results suggest that (+)-(S,S)-5-FBVM warrants further investigation as a potential radioligand for in vivo PET imaging of cholinergic nerve terminals.

Different RP-HPLC columns (phenyl, conventional ODS, cross-linked C18 and special end-capped C8 and C18 phases) were used to investigate the separation of four basic ionizable isomers. Using ACN/20 mM NH4OAc aq., a separation was observed exclusively on RP columns with higher silanol activity at unusual high ACN concentration, indicating cation-exchange as main retention mechanism. Using MeOH/20 mM NH4OAc aq., another separation at low MeOH concentrations was observed on both, RP columns with higher as well as RP columns with lower silanol activity, which is mainly based on hydrophobic interactions. The isomers were also separated on a bare silica column at higher MeOH content using NH4OAc as buffer. Since cation-exchange governs this retention, the elution order was different compared to the RP phases. A strong retention on the silica column was observed in ACN, which could be attributed to partition processes as additional retention mechanism.

The 3,4-disubstituted piperidine framework plays an important role in many fields of pharmaceutical research[1] The ring opening of a conformationally rigid oxirane, annelleted to an N-protected six membered piperidine by an amine nucleophile, is a powerful method for establishing a trans-beta-aminoalcohol at the 3,4-position. Compounds derived from azavesamicol (trozamicol), which have been reported as usefulligands for the vesicular acetylcholine transporter (VAChT),[2] are displaying a 3-amino-piperidin-4-ol as a consistent structural feature. As part of our efforts in the search for new VAChT ligands for F18-PET (positron emission tomography) we have found a regioselective ring opening approach applying 3-(4-fluorobenzyl)-7-oxa-3-azabicyclo[4.1.0]heptane, which was utilized for a new 4-step synthesis of 4-fluorobenzyltrozamicol. In contrast to former methodology, processing a common acyl[3] or carbamoyl[4] protective group at the piperidine nitrogen, we choose an N-benzyl protection. Starting from pyridine, which was converted to an N-4-fluorobenzylpyridinium salt, we obtained the corresponding N-protected 1,2,3,6-tetrahydropyridine via a standard hydrogenation using NaBH4 in EtOH. It was possible to epoxidize the olefine in high yield without interfering the basic tertiary amine, if applying trifluoroacetic acid in combination with its anhydride and H2O2-urea. As expected, the LiCIO4 assisted reaction of 1-aralkyl-3,4-epoxypiperidines with a series of aliphatic and aromatic amines in acetonitril led to a regioselective attack at position 4 of the piperidine to obtain 1-aralkyl-4-amino-piperidin-3-ol.[5] However, if applying EtOH as a protic solvent, without a coordinating Li-cation as additive, the inverse reg ioselectivity was observed. In this case, 1-aralkyl-3-aminopiperidin-4-oIs were derived as main products from the cleavage of the epoxy C-O bond at position 3. Following these protocols the aminoalcohol products were obtained as pure compounds by simple work-up without the need of chromatographic separation. In summary, we present an efficient method for the preparation of 3-amino-4-hydroxypiperidines and its regioisomers in 4-steps. The highly VAChT affine 4-fluorobenzyltrozamicol was obtained in an overall yield of 35%.

The basic helix–loop–helix transcription factor Hand2 is essential for the proliferation and noradrenergic differentiation of sympathetic neuron precursors during development. Here we address the function of Hand2 in postmitotic, differentiated sympathetic neurons. Knockdown of endogenous Hand2 in cultured E12 chick sympathetic neurons by siRNA results in a significant (about 60%) decrease in the expression of the noradrenergic marker genes dopamine-β-hydroxylase (DBH) and tyrosine hydroxylase (TH). In contrast, expression of the pan-neuronal genes TuJ1, HuC and SCG10 was not affected. To analyze the in vivo role of Hand2 in differentiated sympathetic neurons we used mice harboring a conditional Hand2-null allele and excised the gene by expression of Cre recombinase under control of the DBH promotor. Mouse embryos homozygous for Hand2 gene deletion showed decreased sympathetic neuron number and TH expression was strongly reduced in the residual neuron population. The in vitro Hand2 knockdown also enhances the CNTF-induced expression of the cholinergic marker genes vesicular acetylcholine transporter (VAChT) and choline acetyltransferase (ChAT). Taken together, these findings demonstrate that the Hand2 transcription factor plays a key role in maintaining noradrenergic properties in differentiated neurons.

O transportador vesicular de acetilcolina (VAChT) é responsável por armazenar acetilcolina em vesículas sinápticas, passo importante para a manutenção da transmissão colinérgica. Com o objetivo de estudar o papel do VAChT na manutenção do tônus colinérgico, geramos diferentes linhagens de animais em que esse gene foi alterado através de recombinação homóloga. Uma estratégia para interferir com a expressão do gene do VAChT foi a inserção de um cassete contendo o gene de resistência a neomicina na região 5 não traduzida do VAChT. Esta modificação genética gerou camundongos com diminuição HET HOM e KD da expressão do VAChT (VAChT Knockdown). Os animais KD apresentam redução de aproximadamente 45 e 65% da expressão de VAChT, respectivamente. Observamos acúmulo de acetilcolina não liberada no cérebro dos camundongos KD e isso não parece ser devido a alterações de expressão da choline acetyltransferase ou do transportador de alta afinidade de colina. Ensaios de eletrofisiologia na junção neuromuscular mostraram que existe uma diminuição no HET HOM tamanho do quanta nos camundongos KD e KD sendo a diminuição maior nos HOM HETcamundongos KD que nos KD . Nós também produzimos camundongos em que o gene do VAChT foi Flox/Floxflanqueado por seqüências LoxP (camundongos VAChT ) a fim de utilizá-los para o acasalamento com camundongos que expressam a enzima Cre em diferentes regiões do cérebro. Os camundongos gerados, portanto, poderiam apresentar deleção do gene do VAChT em tecidos espeficos. Nossa caracterização inicial dos camundongos Flox/Flox VAChT mostrou que eles apresentam redução em torno de 50% na expressão do mRNA de VAChT, além de um aumento em torno de 100% na expressão do mRNA da enzima ChAT e um aumento de 6 vezes nos níveis de acetilcolina no cérebro quando WT/WT comparados com os animais VAChT . A amplitude dos potencias miniatura Flox/Flox (MEEPs) de placa motora está aumentada nos camundongos VAChT , sem Flox/Flox alterações na freqüência. Apesar dessas alterações, os camundongos VAChT são fenotipicamente normais. Flox/WT Flox/Flox Camundongos VAChT ou VAChT foram acasalados com camundongos que expressam Cre sob o controle do promotor CAMKII. Os Flox/Flox, cre+ camundongos VAChT gerados são morfologicamente normais ao nascimento, entretanto, após cinco dias de vida tornam-se menos ativos e exibem menor crescimento que os animais controle da mesma ninhada. Além disso, os camundongos Flox/Flox VAChT apresentam fraqueza muscular generalizada, desenvolvimento anormal da coluna espinhal acarretando em cifose e sobrevivem apenas cerca de 8 semanas. Nós também geramos uma linhagem de camundongos com a deleção de um dos alelos do Flox/Del, cre+ VAChT. Os camundongos VAChT, apresentaram os mesmos fenótipos dos Flox/Flox, cre+, Flox/Del, cre+ camundongos VAChT porem os camundongos VAChT sobrevivem apenas cerca de 20 dias. Flox/Flox,cre- Flox/Del, cre- Surpreendentemente, os animais VAChT e VAChT embora não expressem a Cre, apresentam ausência de VAChT em diversas regiões do SNC. Além disso, os níveis teciduais de acetilcolina também estão aumentados em torno de Flox/Del, cre-. doze vezes nos camundongos VAChT Interessantemente, nenhum dos estudos comportamentais e eletrofisiológicos realizados sugerem que o VAChT esteja alterado, já que os animais são fenotipicamente normais. Como os camundongos Flox/Flox,cre- Flox/Del, cre- VAChT e VAChT apresentam mRNA para o VAChT, é possível que exista um mecanismo regulatório da expressão do transportador ainda desconhecido. Nossos resultados confirmam a importância de VAChT na regulação da liberação de acetilcolina e, consequentemente, na manutenção das funções fisiológicas normais do sistema nervoso central e periférico. The vesicular acetylcholine transporter (VACht) is responsible for acetylcholine (Ach) storage in synaptic vesicles, an important step for cholinergic transmission. Our goal was to study the role of VAChT in cholinergic tonus maintenance through the generation of mice presenting alterations in VAChT gene by homologous recombination.. One of used strategies was the insertion of a neomycin resistance cassette in untranslated 5 region of VAChT gene. This genetic modification generated mice HET HOM presenting reduced expression of VAChT (VAChT Knockdown). KD and KD present a 45% and 65% decrease in VAChT protein expression, respectively. Accumulation of not released Ach in brain of KD was observed and it is not related with increased expression or activity of ChAT (choline acetyltransferase) or CHT1 (High affinity choline transporter). Electrophysiological analysis at neuromuscular HET HOM and KD junction showed a decrease in quantal size distribution for both, KD specially in the second one. We produced mice in which VAChT gene was flanqued by LoxP sites to Flox/Flox generate VAChT mice. These mice were bred with mice expressing Cre enzyme in different regions of brain. The lineage generated would present deletion of VAChT Flox/Flox mice showed that gene in specific tissues. Our initial characterization of VAChT they presented decrease of 50% in VAChT mRNA expression, an increase of 100% in ChAT mRNA expression and an increase around 6 fold in brain Ach when compared to WT/WT VAChT animals. The amplitude, but not frequency, of miniature potentials Flox/Flox (MEEPs) in neuromuscular junction is increased in VAChT mice. However, Flox/Flox VAChT mice are phenotipically normals. Flox/WT Flox/Flox VAChT or VAChT animals were bred with mice expressing Cre Flox/Flox,cre+ under CAMKII promoter control. The generated VAChT are morphologicaly normals in birth, however, after five days of live they look smaller and Flox/Flox,cre+ not as active as the control littermates animals . Besides this, the VAChT mice present general muscular weakness, anormal development of spinal cord represented by a kyphosis and survive just 8 weeks. Another mice lineage was generated due to VAChT allele deletion: Flox/Del, cre+ Flox/Flox, cre+ VAChT . These mice present similar phenotype to VAChT mice; however this genotype present a reducted time life to approximately 20 days. Flox/Flox,cre- Flox/Del,cre- Even not shown Cre, VAChT and VAChT animals surprisely, present abolishment of VAChT in different regions of CNS. Besides this, ACh tissue Flox/Del, cre- WT/WT is 12 fold higher than in VAChT animals. content in VAChT Interestingly, comportamental and eletrophisiological studies, do not suggest alteration of VAChT, since the Cre- animals are phenotypically normals. Similarly to Flox/Del, cre+ Flox/Flox,cre- Flox/Del, cre- VAChT animals, VAChT and VAChT mice present VAChT mRNA, suggesting an unknown regulatory mechanism of VAChT expression. Our results confirm the important role of VAChT in cholinergic transmission through Ach releasing regulation and, in consequence, in maintaining normal physiological function of central and peripheral nervous system.

In Nederland waren tot op heden geen proeven in bedrijfsverband gedaan met het wel of niet scheren van ooien tijdens de dracht. Het PR is daarom in 1990 gestart met een onderzoek naar het al dan niet scheren van ooien op ca. 8 weken voor het lammen.

In Nederland waren tot op heden geen proeven in bedrijfsverband gedaan met het wel of niet scheren van ooien tijdens de dracht. Het PR is daarom in 1990 gestart met een onderzoek naar het al dan niet scheren van ooien op ca. 8 weken voor het lammen.

Uit praktijkproeven blijkt dat ooien, als ze geschoren worden op ongeveer 8 weken voor het werpen, zwaardere lammeren brengen.

Uit praktijkproeven blijkt dat ooien, als ze geschoren worden op ongeveer 8 weken voor het werpen, zwaardere lammeren brengen.

Voederproeven zijn uitgevoerd om het effect van scheren op ongeveer 8 weken voor het werpen bij Flevolander ooien te onderzoeken, die in maart of mei werpen. Hierbij waren de ruwvoeropname, het geboortegewicht van de lammeren, het gewicht van de ooien en een aantal bloedparameters de voornaamste kenmerken

Voederproeven zijn uitgevoerd om het effect van scheren op ongeveer 8 weken voor het werpen bij Flevolander ooien te onderzoeken, die in maart of mei werpen. Hierbij waren de ruwvoeropname, het geboortegewicht van de lammeren, het gewicht van de ooien en een aantal bloedparameters de voornaamste kenmerken

Background: Cholinergic dysfunction occurs during aging and in a variety of diseases, including amyotrophic lateral sclerosis (ALS). However, it remains unknown whether changes in cholinergic transmission contributes to age-and disease-related degeneration of the motor system. Here we investigated the effect of moderately increasing levels of synaptic acetylcholine (ACh) on the neuromuscular junction (NMJ), muscle fibers, and motor neurons during development and aging and in a mouse model for amyotrophic lateral sclerosis (ALS). Methods: Chat-ChR2-EYFP (VAChTHyp) mice containing multiple copies of the vesicular acetylcholine transporter (VAChT), mutant superoxide dismutase 1 (SOD1G93A), and Chat-IRES-Cre and tdTomato transgenic mice were used in this study. NMJs, muscle fibers, and a-motor neurons' somata and their axons were examined using a light microscope. Transcripts for select genes in muscles and spinal cords were assessed using real-time quantitative PCR. Motor function tests were carried out using an inverted wire mesh and a rotarod. Electrophysiological recordings were collected to examine miniature endplate potentials (MEPP) in muscles. Results: We show that VAChT is elevated in the spinal cord and at NMJs of VAChTHyp mice. We also show that the amplitude of MEPPs is significantly higher in VAChTHyp muscles, indicating that more ACh is loaded into synaptic vesicles and released into the synaptic cleft at NMJs of VAChTHyp mice compared to control mice. While the development of NMJs was not affected in VAChTHyp mice, NMJs prematurely acquired age-related structural alterations in adult VAChTHyp mice. These structural changes at NMJs were accompanied by motor deficits in VAChTHyp mice. However, cellular features of muscle fibers and levels of molecules with critical functions at the NMJ and in muscle fibers were largely unchanged in VAChTHyp mice. In the SOD1G93A mouse model for ALS, increasing synaptic ACh accelerated degeneration of NMJs caused motor deficits and resulted in premature death specifically in male mice. Conclusions: The data presented in this manuscript demonstrate that increasing levels of ACh at the synaptic cleft promote degeneration of adult NMJs, contributing to age-and disease-related motor deficits. We thus propose that maintaining normal cholinergic signaling in muscles will slow degeneration of NMJs and attenuate loss of motor function caused by aging and neuromuscular diseases.